Graduate Theses and Dissertations Iowa State University Capstones, Theses andDissertations

2019

Therapeutic efficacy of antiviral compounds in theneonatal lamb model of respiratory syncytial virusinfection of infants with pathogenesis of secondaryStreptococcus pneumoniae infectionSarhad AlnajjarIowa State University

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Recommended CitationAlnajjar, Sarhad, "Therapeutic efficacy of antiviral compounds in the neonatal lamb model of respiratory syncytial virus infection ofinfants with pathogenesis of secondary Streptococcus pneumoniae infection" (2019). Graduate Theses and Dissertations. 16959.https://lib.dr.iastate.edu/etd/16959

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Therapeutic efficacy of antiviral compounds in the neonatal lamb model

of respiratory syncytial virus infection of infants with pathogenesis of

secondary Streptococcus pneumoniae infection

by

Sarhad S.A. Alnajjar

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Veterinary Pathology

Program of Study Committee:

Mark R. Ackermann, Co-major Professor

Jodi D. Smith, Co-major Professor

Jesse M Hostetter

Brett A Sponseller

Mitchell Van Palmer

The student author, whose presentation of the scholarship herein was approved by the

program of study committee, is solely responsible for the content of this dissertation. The

Graduate College will ensure this dissertation is globally accessible and will not permit

alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2019

Copyright © Sarhad S.A. Alnajjar, 2019. All rights reserved.

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DEDICATION

This dissertation is dedicated to:

My wife

My family

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TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... vii

LIST OF TABLES .......................................................................................................... viii

ACKNOWLEDGMENTS ................................................................................................ ix

ABSTRACT ...................................................................................................................... x

CHAPTER 1: GENERAL INTRODUCTION ...................................................................... 1

Statement of the Problem .................................................................................................. 1

Specific Aims .................................................................................................................... 2

Dissertation Organization .................................................................................................. 2

Literature Review .............................................................................................................. 3

Respiratory Syncytial Virus Structure and Strains ........................................................ 3

Disease Burden .............................................................................................................. 5

Animal Models of RSV Infection .................................................................................. 8

Immune Response to RSV Infection: .......................................................................... 11

Secondary Bacterial Infection ...................................................................................... 16

RSV Therapeutics ........................................................................................................ 18

References .................................................................................................................... 20

: STREPTOCOCCUS PNEUMONIAE INFECTION IN RESPIRATORY

SYNCYTIAL VIRUS INFECTED NEONATAL LAMBS ............................................... 35

Abstract ............................................................................................................................ 35

Introduction ..................................................................................................................... 36

Material and Methods ...................................................................................................... 38

Experimental Design .................................................................................................... 38

Infectious Agents ......................................................................................................... 39

Immunohistochemistry (IHC) ...................................................................................... 40

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) ............ 40

Hematoxylin-Eosin Staining and Histological Scoring of Lung Sections ................... 41

Statistical Analysis ....................................................................................................... 41

Results ............................................................................................................................. 42

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Infected Lambs Showed Marked RSV and Spn Titer................................................... 42

RSV and Spn Induce a Well-recognized Macroscopic and Microscopic Lesion ......... 42

Discussion ........................................................................................................................ 44

References ........................................................................................................................ 48

Figures and Legends......................................................................................................... 52

: THERAPEUTIC EFFICACY OF JNJ-49214698, AN RSV FUSION

INHIBITOR, IN RSV-INFECTED NEONATAL LAMBS ................................................ 56

Authors Contributions .................................................................................................. 56

Acknowledgments ............................................................................................................ 57

Conflict of Interests ...................................................................................................... 57

Funding ......................................................................................................................... 57

Abstract ............................................................................................................................ 58

Key Words .................................................................................................................... 58

Introduction ...................................................................................................................... 58

Materials and Methods ..................................................................................................... 61

Compound and Dosing ................................................................................................. 61

Animals ......................................................................................................................... 61

Experimental Design .................................................................................................... 62

RSV Infection ............................................................................................................... 62

Assessed Parameters ..................................................................................................... 63

Statistical Analysis ....................................................................................................... 64

Results .............................................................................................................................. 64

JNJ-49214698 Efficiently Distributes to Different Lung Compartments of

Neonatal Lambs ............................................................................................................ 64

Treatment with JNJ-49214698 Reduces Incidence and Duration of

RSV-Associated Symptoms ......................................................................................... 65

Treatment with JNJ-49214698 Inhibits a Multicyclic RSV Infection in Neonatal

Lambs ........................................................................................................................... 66

Treatment with JNJ-49214698 Inhibits RSV-Induced Lung Pathology and Cellular

Immune Response of Neonatal Lambs ......................................................................... 67

Discussion ........................................................................................................................ 69

References ........................................................................................................................ 71

Figures and Legends......................................................................................................... 77

Page

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Supplementary Data ........................................................................................................ 83

Materials and Methods ................................................................................................. 83

Compound and Dosing ................................................................................................ 83

Animals ........................................................................................................................ 83

Experimental Design .................................................................................................... 84

RSV Infection .............................................................................................................. 84

Animal Monitoring for Appearance of Clinical Signs and the Clinical Score ............ 85

Blood sampling for PK Analysis ................................................................................. 85

Lung Collection and Processing .................................................................................. 85

BALF Collection .......................................................................................................... 86

Quantification of JNJ-49214698 Exposure .................................................................. 87

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) ............ 88

Hematoxylin-Eosin Staining and Histological Scoring of Lung Sections ................... 89

Immunohistochemistry (IHC) of Lung Sections .......................................................... 90

RNAscope .................................................................................................................... 91

Infectious Focus-Forming Unit (IFFU) Assay ............................................................. 92

Statistical Analysis ....................................................................................................... 93

References ....................................................................................................................... 97

:THERAPEUTIC EFFICACY OF RESPIRATORY SYNCYTIAL

VIRUS NON-FUSION INHIBITOR (RSV-NFI) IN NEONATAL LAMBS

INFECTED WITH A HUMAN STRAIN OF RSV ............................................................ 99

Summary .......................................................................................................................... 99

Introduction ................................................................................................................... 100

Materials and Methods .................................................................................................. 101

Experimental Design .................................................................................................. 101

Tissue and Sample Collection .................................................................................... 102

Infectious Focus-Forming Unit (IFFU) Assay ........................................................... 102

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) .......... 103

Histologic Evaluation ................................................................................................. 103

Statistics ..................................................................................................................... 105

Results ........................................................................................................................... 105

Pharmacokinetics of Different Doses of RSV-NFI in the Neonatal Lambs .............. 105

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vi

Viral Load in BALF and Lavaged-Lung Samples of Neonatal Lambs ...................... 106

Treatment with RSV-NFI Significantly Reduced RSV-Induced Lung Lesion .......... 107

Treatment with RSV-NFI Significantly Decrease Inflammatory Chemokines .......... 108

Discussion ...................................................................................................................... 109

References ...................................................................................................................... 111

Figures and Legends....................................................................................................... 116

CHAPTER 5: GENERAL CONCLUSION ....................................................................... 125

Secondary Bacterial Co-Infection .................................................................................. 125

Efficacy of Anti-RSV Fusion Inhibitor .......................................................................... 125

Efficacy of Anti-RSV Non-Fusion Inhibitor .................................................................. 126

Future Directions ............................................................................................................ 127

References ...................................................................................................................... 128

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LIST OF FIGURES

Figure 2-1:RSV and Spn titer in lung tissue and blood ....................................................... 52

Figure 2-2:Percent of lung tissue associated with RSV and/or Spn infection. ................... 53

Figure 2-3:Histologic lesions associated with RSV, Spn, and RSV-Spn

combined infection. ...................................................................................................... 54

Figure 2-4:Immunohistochemistry staining of RSV and Spn in FFPE lung tissue

sections. ........................................................................................................................ 55

Figure 3-1:Exposure of JNJ-49214698 in different body compartments of

neonatal lambs. ............................................................................................................ 77

Figure 3-2: Clinical signs score in different groups. ........................................................... 78

Figure 3-3:Effect of JNJ-49214698 on viral titers in BALF and lung tissue at

Day 6 p.i. ...................................................................................................................... 79

Figure 3-4: Effect of JNJ-49214698 on viral antigen and RNA in lung tissue at

Day 6 p.i. ...................................................................................................................... 80

Figure 3-5: Effect of JNJ-49214698 on development of gross lung lesions at

Day 6 p.i. ...................................................................................................................... 81

Figure 3-6:Effect of JNJ-49214698 on RSV-induced accumulated lung histopathology at

Day 6 p.i. ...................................................................................................................... 82

Figure 3-7: Effect of JNJ-49214698 on RSV-induced lung histopathology at

Day 6 p.i. ...................................................................................................................... 95

Figure 3-8:Effect of JNJ-49214698 on RSV-induced lung histopathology at

Day 6 p.i. ...................................................................................................................... 96

Figure 4-1:Experimental design. ....................................................................................... 116

Figure 4-2: Exposure of RSV-NFI in different body compartments. ............................... 117

Figure 4-3: Effect of RSV-NFI on viral titer in BALF and lung tissue

homogenates at day 6 p.i. ........................................................................................... 118

Figure 4-4: Effect of RSV-NFI on the development of RSV associated gross lesion. ..... 119

Figure 4-5: Effect of RSV-NFI on the development of RSV associated histologic

lesion. ......................................................................................................................... 120

Figure 4-6:Effect of RSV-NFI on RSV antigen. ............................................................... 121

Figure 4-7: Effect of RSV-NFI on RSV M37 RNA expression. ...................................... 122

Figure 4-8: Effect of RSV-NFI on the development of RSV associated histologic

lesion. ......................................................................................................................... 123

Figure 4-9: Effect of RSV-NFI inflammatory chemokine RNA expression in lung

tissue. ......................................................................................................................... 124

Page

viii

LIST OF TABLES

Table 1: Clinical signs observed in lambs in different groups throughout the study

period……………………………………………………………………………………94

Page

ix

ACKNOWLEDGMENTS

First of all I would like to thank God, the Most Gracious, the Most Merciful for helping

me throughout my life.

I would like to thank the Higher Committee for Education Development (HCED) in

Iraq for their support and funding of my study.

I would like to thank my major professor Dr. Mark Ackermann for his unwavering

guidance and support throughout my study program. Mark, your efforts are greatly

appreciated.

I would like to thank Dr. Jodi Smith, Dr. Jesse Hostetter, Dr. Brett Sponseller, and Dr.

Mitchell Palmer for serving in my program of study committee and for their constructive

comments and suggestions.

A special thank you to Jack Gallup. Thank you for sharing your expertise and help.

Thank you to Dr. Albert Van Geelen for your advice and mentoring in the laboratory. A

great thank you to our collaborators Drs. Dirk Roymans and Peter Rigaux for sharing their

scientific expertise and help in constructing, editing the scientific manuscripts. Great thanks

to Dr. David Verhoeven for his thoughts, ideas, and his constructive feedback.

A big thank you to my colleagues in the Veterinary Pathology, Immunobiology

programs for making my life enjoyable and for providing their help when asked especially

Dr. Panchan Sitthicharoenchai my friend, and lab mate, and also Dr. Saleh Albarrak thank

you for sharing your ideas, thoughts, and time.

A big thank you tothe Veterinary Pathology faculty and staff, who did not hesitate to

help and provide their expertise. Also, thank you for the laboratory animal veterinarian and

animal caretakers from Iowa State University Laboratory Animal Resources for their help

in ensuring the welfare of the animals and success of the research.

Dr. Mary Sauer, thank you for all your thoughts, time, and advice. My life would not

be this way without you. Debbie and Kurtis Younkin, what a great help you are. I cannot

find words that fulfill what you have done for my family and me during my study.

Finally, I would like to thank the wonderful Ames community, international family

(Stonebrook Church). Thank you for making us feel that we are home.

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ABSTRACT

Respiratory Syncytial Virus (RSV)is one of the most common causes of acute lower

respiratory tract infection in humans that can cause severe infections in infants and the

elderly. It is estimated that annually 33 million lower respiratory tract infections in children

were associated with RSV worldwide. However, in the US alone there were 75000-125,000

hospitalizations due to RSV in infants per year. Although most RSV infections of the lower

respiratory tract are caused by RSV alone, occasionally, secondary bacterial infection further

increases lung damage and disease severity. Despite the widespread infections by RSV

throughout the world, till now there are no approved vaccines or therapeutic compounds to

treat RSV infection other than Ribavirin and prophylactic administration of Palivizumab.

There are several animal models of RSV infection, but neonatal lambs infected with RSV

have several advantages for modeling RSV infection in infants such as similarity in

pulmonary architecture, immune response, and respiratory tract size, viral replication, and

lesion development.

To further extend the neonatal lamb model to evaluate secondary bacterial infection

associated with RSV, lambs coinfected with RSV and Streptococcus pneumoniae were used

to determine feasibility, susceptibility and disease development. Lambs developed severe

disease when coinfected with both microorganisms with more severe suppurative bronchitis

and pneumonia. Streptococcus pneumonia infection enhanced the severity of RSV in lambs

when lambs were coinfected with both microorganisms.

To evaluate the efficacy of some antiviral compounds, two antiviral compounds

efficacy were tested in the neonatal lamb model of RSV infection. First, three regimens of

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JNJ-49214698, a small molecule RSV fusion protein inhibitor were tested (prophylactic,

early treatment, late treatment). JNJ-49214698 prevented RSV infection when given before

infection and reduced RSV induced lung lesion when used to treated established infection.

Additionally, late treatment at day 3 post RSV infection had a wide window for RSV

treatment. Secondly, 3 doses of RSV-NFI, an RSV non-fusion inhibitor were tested for

efficacy. RSV-NFI was well tolerated and reduced RSV induced lung lesions and viral titer.

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CHAPTER 1: GENERAL INTRODUCTION

Statement of the Problem

Respiratory Syncytial Virus (RSV) is one of the leading cause of acute lower respiratory tract

infection in infants. Each year there are about 33 million lower respiratory tract infection cases in

children under the age of five around the world and these often lead to significant hospital

admission and mortality[1]. Additionally, RSV is one of the leading cause of pneumonia in older

adults and high-risk (e.g., immunosuppressed) individuals [2,3]. RSV infections can occur year

round but peaks in the winter months [4,5]. RSV has similar hospitalization rate and disease

burden to influenza in elderly[3]. Secondary to RSV infection, about 40% of RSV infected children

develop co-infections with bacteria or at increased risk of secondary bacterial pneumonia [6].

Furthermore, RSV infection in the early childhood may have delayed sequelae such as airways

hyperreactivity, wheezing, and asthma in later childhood [7,8]. There is no vaccine or antiviral

agent available clinically against RSV except for prophylactic humanized monoclonal antibody

Palivizumab which has questionable efficacy and is expensive and Ribavirin [9,10].

There are many investigators developing new vaccines and therapeutic compounds for RSV

and many of these are in advanced human clinical trials. Some newly developed vaccines under

investigation provide an acceptable level of protection against RSV [11,12]. However, formalin-

inactivated vaccines developed in the 1960’s led to enhanced disease and even death upon RSV

infection in infants. Therefore, there are fears of adverse side effects of any new RSV vaccination

regimen [13]. Newly developed anti-RSV therapeutic compounds that have reached advanced

clinical trials include those targeting several critical viral components such as RSV fusion protein,

N protein, L protein, SH protein, and M2-1 protein, and are from four chemical classes including:

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anti-RSV immunoglobulins, siRNA-interference, fusion inhibitors, and small molecule inhibitors

[14,15]. These therapeutic strategies require animal models to evaluate their efficacy.

Creating animal models for RSV investigations is challenging. There are several animal

models used to evaluate RSV infection, pathogenesis, and therapeutic efficacy and these include

but are not limited to: chimpanzees, baboons, sheep, cotton rats, mice, ferrets, and cattle [16]. Of

these, lambs have several features advantageous as a model of RSV infection of infants including

susceptibility to the human strains of RSV, lung development, structure and cellular morphology,

and lesion development and lesion composition [17,18].

Specific Aims

The goals of the studies done in this dissertation are to determine the extent to which RSV

infection can alter the susceptibility of the lung to Streptococcus pneumoniae (Spn), and test the

efficacy of novel anti-RSV compounds. The hypothesis is that RSV infection enhances Spn

infection and/or vice versa by lung damage induced by either pathogen and that anti-RSV (fusion

and non-fusion inhibitors) can reduce RSV infection and disease severity. This hypothesis was

tested by: 1) a study in which lambs were infected with Spn 3 day after initial RSV infection

(Chapter 2). 2) a study to test the efficacy of anti-RSV small molecule fusion protein inhibitor

(Chapter 3). 3) a study to test the efficacy of anti-RSV non-fusion inhibitor (Chapter 4).

Dissertation Organization

This dissertation describes the pathogenesis of secondary bacterial pneumonia in the neonatal

lamb model of RSV infection and two therapeutic approaches to RSV infection. The dissertation

composed of five chapters with the 1st one as general introduction and literature review. Chapters

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2, 3, and 4 are composed of the three individual manuscript prepared for the peer review journal

submission, with the final chapter (Chapter 5) as the conclusion and future direction.

The first paper Spn infection in RSV infected neonatal lambs (Chapter 2) were submitted to

the journal Emerging Microbes and Infection. The second paper, Therapeutic efficacy of JNJ-

49214698, an RSV fusion inhibitor, in RSV-infected neonatal lambs, were partially published in

the journal Nature Communications, and the full manuscript was submitted to Frontiers in

Microbiology. The third manuscript, therapeutic efficacy of RSV-NFI in RSV infected lambs will

be submitted to a journal publishes work on antiviral compounds at a later time.

Literature Review

Respiratory Syncytial Virus Structure and Strains

RSV is an enveloped virus with non-segmented negative-sense single-stranded RNA within

the family of Pneumoviridae, genus Orthopneumovirus, [19,20]. The viral RNA is composed of

10 genes that encode 11 protein two of them non-structural protein. In general, Orthopneumovirus

have three externally protruded glycoproteins, the attachment (G), fusion (F), and small

hydrophobic (SH). These glycoproteins pin the outer lipid envelope obtained from the host cell

plasma membrane and attached to the underlining matrix (M) protein. The G protein has carboxy-

terminal located to the outside of the virion and is variable among RSV strains. The G protein

mediates viral attachment to cellular membrane. The F protein has amino-terminus oriented to the

outside of the viral envelope and is less variable between strains than the G protein and responsible

for the viral fusion and entry into the target cell. These two proteins are critical for the viral

replication and infection, although virions lacking G protein can penetrate and replicate in cell

culture, but less efficiently than virion having both G and F protein[21]. The G protein is heavily

glycosylated and attaches to glycosaminoglycans on the host cell surface[22]. Most neutralizing

4

antibodies are directed against G protein. Therefore, RSV has an immune evasion strategy by

which a secreted form of G protein (sG) is produced in order to avoid inhibition by neutralizing

antibody [23–25]. On the other hand, F protein is highly conserved, and upon attachment and

activation, F protein undergoes conformational changes leading to fusion of the virus to the target

cell membrane, which subsequently induce internalization of the virus into the cell cytoplasm [26–

28]. Interestingly, one study demonstrated that RSV internalized intact into the epithelial cell by

macropinocytosis initiated by the RSV attachment. This process leads to RSV virion to be present

within intracytoplasmic fluid-filled macropinocytosome, then followed by F protein cleavage and

viral fusion that leads to internalization of RSV to the cytoplasm and infection [29]. SH protein

function and location is not fully characterized [30,31]. However, SH protein was demonstrated

to have an antiapoptotic activity by inhibiting TNF-α signaling [32]. Another study demonstrated

that SH protein is a small hydrophobic protein (∼100 amino acid) called Viroporin, which enhance

infected cell permeability by forming a hydrophilic pore in the infected cell membrane [33]. The

non-segmented single-stranded RNA associated with five structural proteins, the first 3 are

nucleoprotein (N), large (L) protein, and phosphoprotein (P) to form the helical nucleocapsid. The

L protein considered the RNA dependent RNA polymerase used in viral genome replication.

Additionally, L protein responsible for the transcription of the positive sense viral mRNA, and

possess capping enzyme activity at the 5’end and polyadenylation at the 3’ end of the viral mRNA

[34]. N Protein forms a complex with the RSV RNA called ribonucleoprotein complex that acts

as a template for the L protein, while P protein serves as a link between L and N protein to facilitate

efficient and specific recognition of ribonucleoprotein complex by L protein [35]. The other two

proteins associated with the nucleocapsid are the M2-1 and M2-2 proteins which are transcriptional

5

enhancer proteins [36]. These proteins bind to the RNA and P protein to prevent premature

termination of transcription [37,38].

There are two RSV strains: an A and a B strain which differ substantially in the G protein

and noncoding portion of the genome but have less variability in the other structural

proteins[39,40]. These two RSV strains co-circulate clinically throughout the year with the

dominance of A strain. Furthermore, RSV group A causes more severe infection than group B

RSV [41–44].

Disease Burden

RSV is a common respiratory disease affecting all ages and especially children worldwide

[1,45,46]. In most infected individuals, RSV causes mild to moderate upper respiratory tract

infection characterized by fever, nasal congestion, cough and rhinorrhea that persists for several

days [47]. However, RSV can lead to severe acute lower respiratory tract infection (LRTI) in

infants, elderly and immunocompromised individuals [48–50].

All infants eventually become infected by RSV, and it is estimated that there were 33.8

million cases of RSV associated pneumonia worldwide in children under the age of five in 2005

[1]. Furthermore, about 3.4 million RSV associated pneumonia cases required hospital admission

with 66,000-199,000 resulted in mortality. Most cases of mortality occur in developing countries

[1]. Based on US National viral surveillance data from 1990-1999, RSV associated deaths in

infants was nine times the number of deaths related to influenza infection in the United States and

RSV was the second leading viral cause of death after influenza in children age 1-5 years and older

adults [51]. While the rate of RSV associated hospitalization is 30 per 1000 in the US, Japan

reported 60 hospital admissions per 1000 [47]. RSV seasonality is consistent throughout Europe

which accounts for 42-45% hospital admission due do LRTI [47]. In Belgium alone and during

6

2000 season, RSV was associated with 63% of acute LRTI in children under the age of 5 [52].

Another study in England and Wales concluded that RSV was associated with 60-80% mortality

more than that of influenza in a total of 15 winters from January 1975 to December 1990 [53]. In

Australia, a retrospective study of 3 RSV seasons in 1997-1999, concluded that RSV is a

significant cause of morbidity and low mortality with 11.4% of infants required admission to

intensive care unit [54]. In general, temperate reigns experience RSV year round with a peak in

winter months, while a specific RSV seasons were seen in tropical regions [55]. RSV considered

an important nosocomial agent since RSV aerosolized particles persist for an extended time in the

urgent care clinic and can be inhaled and infect other patients [56]. However, several risk factors

determine the severity of RSV infection in infants.

RSV infection has considerable morbidity and mortality in infants less than 3 months and

those having one or more risk factors [57]. There are several risk factors associated with the host

include not limited to premature birth, congenital heart diseases, and chronic lung diseases [58,59].

However, a high proportion of infants hospitalized due to RSV are healthy without any

involvement of any of these risk factors [60]. Other risk factors related to the host or the

environment are gender (males), young age (less than 6 months), number of siblings, daycare

attendance, and exposure to tobacco smoke[61–64]. Other risk factors that can contribute to the

severity of RSV infection are related to the virus itself. There is a significant association between

viral load the severity of infection [65–67]. In addition to the viral load, RSV type A is associated

with more severe respiratory illness in compare to type B RSV [41,68,69]. Considering all the

mentioned risk factors, RSV disease severity is an association of all these risk factors, and despite

the massive burden of RSV disease, there are no fully satisfactory treatment or vaccine strategies

available for RSV infection.

7

Only two therapeutic agents, Ribavirin and Palivizumab, were approved against RSV

infection but both have limitations in efficacy. Ribavirin, a nucleoside inhibitor, developed in 1972

as a virostatic agent for both RNA and DNA viruses[70]. Even though Ribavirin had a positive

impact in high-risk patients such as transplant and immune compromised patients [71,72], only

marginal clinical benefits were seen in RSV lower respiratory tract infection in general patients.

Also, Ribavirin has a degree of toxicity. Thus, Ribavirin is no longer recommended as anti-RSV

treatment[73–75]. Palivizumab is a humanized monoclonal antibody against RSV F protein and

thereby prevents/blocks virus fusion to the host cell as well as cell to cell fusion [76]. Palivizumab

use is restricted to premature infants 32-35 weeks gestation due to high cost and is used as

prophylactic treatment [77]. Adding to Palivizumab’s high cost, a Palivizumab-resistant variant

of RSV have been isolated from Palivizumab treated patients [78]. Vaccination and vaccine

development has been held back by the severe RSV infection which occurred in a 1960 vaccination

trial in which majority of vaccinated infants had a vaccine-enhanced disease that require

hospitalization with several fatalities [79]. Animal models for RSV infection is an essential step

in the investigation of RSV pathogenesis and the search for new therapeutics and vaccines.

RSV causes respiratory tract lesions that include severe bronchiolitis characterized by

epithelial cell degeneration and necrosis with areas of hyperplasia in response to the cell damage

along with syncytial cell formation. The bronchiolar lumen becomes partially occluded by

neutrophils, occasional macrophages, seroproteinaceous fluid, mucin, and cell debris.

Lymphocytes infiltrate the airway adventitia. The virus also infects ciliated epithelial cells of the

upper respiratory tract and bronchi. With time after infection, lesions can resolve. However, there

can be increases in Goblet cells and increased residual mast cells and eosinophils.

8

Animal Models of RSV Infection

Animal models are needed to study RSV pathogenesis and evaluate new therapeutics and

vaccines candidates. Animal models are considered the middle stage between tissue and cell

culture studies in vitro and human clinical trials. However, developing animal models for RSV

infections is very challenging due to the high degree of specificity of the hRSV to its natural host

and lack of virulence in other species. The specificity of hRSV is due to F protein, while lack of

virulence in other species is due to the inability to block interferon response [80,81]. Ideal RSV

models need to mimic several RSV disease aspects such as clinical signs and symptoms, viral

replication, upper and lower respiratory pathology, and immune response. Several animal models

have been developed to address some of these aspects if not all, but each model has its strengths

and limitations. In general, RSV animal models are either heterologous or cognate host-virus

models. hRSV can infect and replicate in heterologous host-virus models such as chimpanzees,

baboons sheep, Cotton rats, ferrets and mice, while related Orthopneumovirus specific to the

model was used in the cognate host-virus models such as murine pneumonia virus in mice model

and bovine RSV (bRSV) in calves [16]. In this section, several animal models of RSV infection

will be discussed concluding with the neonatal lamb model.

Chimpanzees seem ideal as RSV animal model due to anatomical similarity to human and

since RSV isolated originally from chimpanzees with respiratory tract infection in 1956 [82].

hRSV able to infect and replicate in the nasal sinuses and upper respiratory tract epithelium, and

induce disease symptoms similar to that found in human RSV associated upper respiratory tract

infection [83,84]. The drawback of this model is that chimpanzees rarely develop RSV LRTI.

There are also concerns with the substantial economic, ethical and emotional burden associated

with the use of chimpanzees. However, vaccine studies have benefited from chimpanzees since

9

chimpanzees tend to develop anti-RSV neutralizing antibody and their immune response similar

to that found in human [85,86]. Baboons have been used as well and are being bred in large

numbers at Oklahoma State University for use in RSV studies and other disease conditions [87,88].

Although rodents are an excellent animal model for experimental studies, they are considered

semi-permissive for hRSV replication, and need a large inoculum to induce mild to moderate RSV

disease[89,90]. Clinical signs are difficult to interpret in rodents, and RSV induces only mild to

moderate bronchiolitis and pneumonia[91,92]. Cotton rats are more permissive than mice for RSV

replication and considered the standard model for testing RSV therapeutics[93]. Mice, however,

have the advantage of wide variety of transgenetic mice and the availability of molecular markers.

Furthermore, mice can be used as a cognate host-virus model by usingmurine pneumonia virus,

which develop a disease in mice similar to RSV disease in human[94]. Murine pneumonia virus

targets bronchiolar epithelium and lead to severe disease with marked respiratory disease correlate

positively with the viral inoculum[94,95]. The drawback of murine pneumonia virus model of

hRSV is the far phylogenetic distance between the two viruses [96]. The critical disadvantage of

rodents as a model for RSV disease is the difference in lung anatomy, histology and immune

response between human and rodents that subsequently question the translations of studies done

in these models to human.

Another cognate host-virus model for hRSV is the bRSV in calves. bRSV induce a

respiratory disease in calves similar to what seen in human RSV. bRSV is more closely related to

hRSV than other non-RSV viruses and share about 38-41% homology on nucleotide level[97].

bRSV induces upper and lower respiratory tract infection in calves[98]. Furthermore, calves have

similar lung anatomy and histology, i.e., the presence of pharyngeal and nasopharyngeal tonsils,

the presence of ciliated pseudostratified epithelium and submucosal glands, and similar innate and

10

adaptive immune response to human [99,100]. The drawback of the calves bRSV model is the

large size of calves that need special housing and handling. Also, calves are not susceptible to

human strains of RSV. Thus, bRSV and hRSV are two distinct viruses that induce similar disease

process in their natural target host.

Neonatal lambs infected with RSV has several similarities to a human infant advantageous

for comparison. The most important criterion is that lambs are naturally susceptible to human,

ovine and bovine strains of RSV. hRSV replicates mostly in the lower part of the respiratory tract

of lamb lungs (bronchioles and bronchi) which models well bronchiolitis in infants and reduces

airflow to alveoli. hRSV replicates well in neonatal lamb respiratory tract airways with a peak of

viral replication at day 6 after intratracheal inoculation with A2 hRSV strain then declines with

time[17,18]. However, another study using hRSV M37 strain showed that peak viral replication

was at day 3 and persisted until day 6 post nebulization[101]. As in human infants, lambs have

variable clinical signs associated with hRSV infection. Clinical symptoms vary from mild such as

nausea, fever, reluctant to move, and reduce milk consumption to moderate and severe such as

cough, wheezing, expiratory efforts. Signs of infection appear as early as 2 days post infection

and progress till day 6 [17,101,102]. hRSV infected lambs develop lower respiratory tract

infection characterized by moderate to severe bronchiolitis and interstitial pneumonia. There is

modest thickening of alveolar septae due to edema, type II cell hyperplasia and, leukocytes

infiltration in the alveolar wall, and neutrophils and macrophages infiltration into the alveolar and

bronchiolar lumens. In addition, hRSV incites epithelial cell necrosis, hyperplasia of nearby

epithelial cells and syncytial cell formation in lambs [103] as in infants.

Additionally, immunological response to hRSV in neonatal lambs were characterized by the

Th1 proinflammatory response as it characterized by the elevated level of IFN-γ and TNFα

11

cytokine and decrease in the level of TGF-β and IL-10. Leukocyte recruitment to the lung were

associated with IL-8, MCP-1, and MIP-1α increase in the lung [18]. IL-10 increased to the highest

level in lambs lung at day 3 post-infection while other cytokine increase later in the course of the

disease [101,104]. Lambs have many similarities to human infant that make it ideal for studying

infant respiratory diseases. Lung development is similar between lambs and infants in terms of

alveolarization which occurs prenatally, similar airways size and branching (dichotomous

branching), presence of submucosal glands, and similar percent of club cells in the airways[105–

108]. Lambs can be born preterm similar to human. Since there is no transfer of immunoglobulin

from ewes to their lambs in utero, lambs can be colostrum deprived to avoid transmission of

maternal immunoglobulin to the lambs. Finally, the size of lambs allows for better evaluation of

the clinical signs, sampling, and lung evaluation.

Immune Response to RSV Infection:

Cell-mediated immune response guided by the proliferation of cytotoxic T cell is the

preferred response to clear many viral infections, although both cell-mediated and humoral

response needed for the ideal antiviral response. The innate immune response, however, has a huge

effect in limiting RSV replication and respiratory infection in infants.

The innate immune response is a nonspecific response aiming to prevent and reduce initial

infection to allow time for more specific acquired immune responses to develop. Innate barriers in

the respiratory tract such as mucociliary system act to prevent attachment of RSV to the airway

epithelial cell, which is the target cell for RSV. Additionally, in response to infection, infected or

neighboring cell upregulate genes, and produce cytokines and antimicrobial peptides and proteins,

which limit the microbial proliferation. There are several common cells in the bronchoalveolar

tree: ciliated bronchial epithelial cells, club cells, non-ciliated cells of the bronchiolar epithelium,

12

and pneumocytes (Type II and Type I). These cells sense the presence of RSV through pattern

recognition receptor most importantly TLR-2, TLR-4, TLR-6, TLR-7, TLR-8, Retinoic acid-

inducible gene I-liKe receptor, and MDA-5[109,110]. Activation of the TLRs eventually leads to

the production of cytokines that shape up the immune response and attract more immune cells to

the lung. Such cytokines include IL-8, IL-10, and IL-6 that induced by TLR4 activation, RANTES,

which produced in response to TLR3 activation, and IFN-α, IFN-β and IP10 production in response

to RIG-I activation[109,111–114]. IL-8, which is a neutrophils chemoattractant, is produced in

the respiratory tract during RSV infection and higher levels of IL-8 is associated with more severe

RSV bronchiolitis in human[115,116]. IL-10 and IL-6 are associated with the Th2 response.

RANTES is a chemoattractant for T cells and eosinophils and associated with severe RSV

bronchiolitis and induction of allergic cellular response [117–119]. IL-10 is an anti-inflammatory

cytokine produced by inflammatory cells mainly macrophages and elevated during acute RSV

infection[120,121]. IFN-α and IFN-β are type I interferons that act on reducing viral replication

and promote MHC type I upregulation and subsequently killing of viral infected cells. Although

IFN-α is elevated in RSV infected infants, its level is markedly lower than influenza-infected

patients[122–124]. That is may be due to the ability of RSV to resist Interferon through the

function of NS1 and NS2 proteins [125–127]. IFNγ, which is type II interferon, is associated with

Th1 response, and it is produced in part by natural killer and macrophages. Low level of IFNγ is

associated with severe RSV lower respiratory tract infection [128]. On the other hand, high level

of type III interferon, IFN-λ, is associated with increased RSV severity. Type III interferon has

similar activity to type I IFN[129].

Mucus and fluid covering the respiratory epithelium contain other innate antimicrobial

substances that act on deactivating microorganisms such as secretion of the submucosal glands

13

and antimicrobial peptides. Submucosal glands, which are present in both human and ruminant

lung airways, secrete lactoperoxidase, lactoferrin, and lysozyme into the mucosal surface.

Lactoperoxidase acts on thiocyanate and hydrogen peroxide present on the mucosal surface to

produce oxythiocyanate which has antimicrobial activity against bacteria and viruses[130,131].

Antimicrobial peptides present in the respiratory system include alpha and beta-defensins, and

cathelicidin. Significant antimicrobial proteins include surfactant protein A and D. Both SP- A

and D are produced by type II pneumocytes and club cells into the airways lumen. Surfactant

protein A and D are globular mannose-binding C-type lectins that bind to RSV F protein and

enhance clearance[132–134]. A higher level of SP-A and SP-D are associated with severe RSV

LRTI[135]. Beta-defensins are antimicrobial peptides secreted by respiratory epithelial cells either

constitutively such as HBD1 or inducible upon infection of the cell such as HBD2, 3, and 4.

Inducible HBD bind to RSV envelop preventing the virus from infecting the cell[136]. Sheep

produce Sheep beta defensins 1 and 2. In contrast, the antimicrobial peptide Cathelicidin is

secreted by leukocytes and stored in neutrophils and can be upregulated during viral

infection[137]. All these innate immune responses are directed towards carbohydrate or other

moieties rather than specific antigens and some are upregulated with RSV lower respiratory tract

infection. In addition to the ability to deactivate RSV, some of these molecules act as a

chemoattractant for other inflammatory and immune cells.

Several inflammatory cells are associated with severe RSV lower respiratory tract infection.

However, neutrophils are the predominant inflammatory cell that increased with the RSV both in

peripheral blood and in the respiratory system [138,139], and the increased number of neutrophils

in blood correlated with the severity of the disease and the peak of the viral load[140]. These

neutrophils are activated and producing neutrophil elastase[141], with neutrophils apoptosis and

14

NETosis to trap virions and prevent further spread of virions[142]. In contrast, eosinophils also

are activated during RSV infection, but they are associated with the healing process[143].

Eosinophils are increased following some RSV infections and contribute to asthma development

later in life and peripheral eosinophils are increased in children hospitalized for RSV LRTI along

with an increase in the Leukotriene C4, eosinophil-derived neurotoxin, and eosinophil cationic

protein in the respiratory tract [85,144,145]. Other inflammatory/ innate immune response

associated cells are macrophages, Natural killer cells, and Dendritic cells.

Although there is a predominance of neutrophil in the airway lavage fluid obtained from

severe RSV infected infant, changes in other cell type can be associated with the disease

outcomes[146,147]. Alveolar macrophages have an essential role in phagocytizing foreign bodies

and microorganisms leading to the subsequent microbial killing and antigen presentation. In

addition to the expression of RSV glycoproteins, alveolar macrophages associated with RSV

infection express immune modulatory molecules such as HLA-DR, interleukin-1β, and TNF as a

response to lung injury[148,149]. RSV immunoreactive staining were seen in alveolar

macrophages in lambs infected with hRSV[150]. Similar to macrophages, dendritic cells (DC)

internalize proteins, process and present antigen to other immune cells, and both conventional and

plasmacytoid DC are recruited to the respiratory system early in RSV infection. These DC are

activated and express a proinflammatory phenotype[151–153]. Most of these cells are nonspecific

and respond to RSV infection to either eliminate the virus or stimulate other type of immune cells

such as lymphocytes.

Lymphocytes are critical for RSV infection since they determine the magnitude and type of

the acquired immune response. However, T lymphocyte number in the blood is decreased in

patients with RSV associated LRTI, and the reduction in T lymphocytes number correlated with

15

the severity of RSV infection [138,154]. In contrast, there is an increase in the B lymphocytes

number in circulation during severe RSV bronchiolitis [155]. Both T and B lymphocytes are

needed to resolve RSV disease.

During RSV infection, there is a decrease in the number of all T lymphocyte population in

the blood, which is more pronounced in infants, and these peripheral lymphocytes are not activated

as is shown by the low level of expression of CD11a and CTL-4 markers[138,156,157]. Although

there is a predominance of CD4 T cell in the BALF obtained from early severe RSV infected

infants, there is a higher expansion of effector CD8 T cell with the course of infection[146,158].

Most research in RSV immunobiology suggests an imbalance in Th1/Th2 response, which

determines the severity of RSV infection. While Th1 responses associated with cytotoxic T

lymphocytes activation and IgG type immunoglobulin production result in resolution of RSV

infection, Th2 shifted responses are associated with increased mucus secretion, cellular infiltration

and atopic type reaction characterized by eosinophilia and eosinophil infiltration in the lung. Th1

response characterized by elevation of IFN-γ, IL-1, IL-2, IL-12, IL-18, and TNF-α, while TH2

response associated with the increase in IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13[159]. Since TH1

and Th2 mutually inhibit each other by IFN-γ to suppress Th2 and IL-4 to inhibit Th1, INF-γ/IL-

4 ratio is used as an indicator for the Th2 bias[160]. There is lower IFN-γ and higher IL-4 leading

to higher IFN-γ/IL-4 ratio in RSV associated bronchiolitis, in addition to the higher IL-10/IL-12

ratio[161]. This high IFN-γ/IL-4 ratio indicates either poor Th1 response or enhanced Th2

response. The fact that IFNγ (Th1 cytokine) level in RSV infected infants is lower than what found

in Pneumovirus infected infants and that the IL-4 level (Th2 cytokine) is higher in RSV infected

infants, it suggests that theTh2 response is enhanced in RSV infected infants[162]. Another study

16

in RSV infected infant indicted the predominance of the Th2 cytokines in the nasopharyngeal fluid

[163]. Thus, Th2 enhanced response might be associated with the severe RSV bronchiolitis.

There is an increase in B-lymphocytes in the blood of RSV lower respiratory tract infected

infants. Humoral immune response to RSV infection includes IgM, IgA, and IgG production

within 5-10 days post infection. However, lower immunoglobulin responses are detected in

children under 6 months old. Both free and cell bounded anti-RSV IgA is present in the

nasopharyngeal secretion of RSV infected patients. Anti-RSV IgG is increased in RSV infected

patients in both IgG1 and IgG3 subclasses. Antibodies directed to F protein crossreact with

different RSV strains, while anti-G protein antibodies are strain specific [164,165]. Although both

anti-RSV IgG and IgA are associated with protection against RSV infection, nasal IgA is more

protective than serum neutralizing IgG antibody[166,167]. A similar finding was seen in children

where high IgA level seemed to be associated with recovery[168]. Another study indicated that

lack of IgA RSV-specific memory B cells in the blood of experimentally infected adults and this

may explain the susceptibility to recurrent infection with RSV [169].

Secondary Bacterial Infection

Secondary bacterial infection is a challenging potential sequel to viral pneumonia. There are

few reports of bacterial infections secondary to RSV infections due to the limitation in the

diagnosis of such infections i.e., the bacterial infection masked by the pathological changes

induced by the virus with difficulty in obtaining noninvasive specific diagnostic samples[170].

Thus, there are few reports on the magnitude/extent of bacterial infection secondary to RSV

infection. One study indicates approximately 40% of patients hospitalized due to viral respiratory

tract infection were associated with bacterial co-infection[171], while another study showed that

40% of children infected with RSV have bacterial coinfection or in high risk of bacterial

17

pneumonia [6]. The most common bacteria associated with secondary bacterial pneumonia are

Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, and Haemophilus

influenza[172,6,173,174]. Because of a high percent of viral-bacterial coinfections, a synergistic

relationship between the two microbial agents that leads to enhanced host susceptibility to each

pathogen has been proposed.

Several in vitro and in vivo models investigated the relationship between viral and bacterial

respiratory infection and how these two microorganisms enhance virulence and persistence of both

pathogens. One study determined that IFN elevation induced by RSV infection enhanced

Pseudomonas aeruginosa biofilm formation in human bronchial epithelial cell culture along with

an increase in iron and Iron transport protein (Transferrin) secretion in the apical epithelium

promoting P. aeruginosa biofilm both in vitro and in vivo [175]. Another study with influenza

infected mice followed by Streptococcus pneumoniae (Spn) infected challenge revealed increases

in both viral and bacterial titers. It is thought that Spn leads to enhanced viral release from infected

cells, and alveolar macrophages impairment resulting in increased Spn [176]. An In vitro study

determined a 2-10 fold increase in the adherence of Spn to RSV infected respiratory cell line in

comparison to non-infected cells[177], while another study showed that 2-2.2 fold increase in

Haemophilus influenza and Streptococcus pneumoniae attachment to cells expressing RSV G

glycoprotein than cells infected with vector only. Furthermore, this bacterial adherence was

reduced to 78-84% when cells were incubated with anti-RSV G antibody [178]. In addition to

viral-specific characteristics that enhances bacterial coinfection, the viral infection itself lead to

alterations in host susceptibility to infection.

There are several potential mechanisms by which viruses predispose to secondary bacterial

infection. The most critical factor is epithelial necrosis and damage, which give access for

18

pathogenic bacterial to its receptors and to obtain iron and other micronutrients [179].

Additionally, viral infection leads to disabled innate barriers such as loss of mucociliary clearance

mechanisms leading to increased bacterial colonization and increased numbers of bacteria reaching

deeper location within the respiratory tree[180]. Viral infection creates a microenvironment

suitable for bacterial proliferation such as aggregation of fibrin, mucus, and necrotic cells, and the

occlusion of small airways leading to reduced O2 and CO2 concentration[181,182]. Furthermore,

bacterial coinfection increases viral titer and prolong the course of infection with some reports

demonstrating that bacterial infection increases susceptibility to viral infection [180,183].

RSV Therapeutics

RSV is a global burden and contributes significantly to increased hospital admission rates

and mortality due to severe bronchiolitis and pneumonia, and there is no effective specific

therapeutics for RSV infection. There are only two approved treatments, Ribavirin, and

palivizumab, although Ribavirin no longer being recommended for treatment and Palivizumab

recommended as a prophylactic treatment only in high-risk individuals. Additionally,

corticosteroids and bronchodilator have limited effects and benefits. The only therapeutic options

available in clinical settings are supportive cares such as oxygenation and intravenous fluid[184].

There is an increased interest from pharmaceutical companies to develop direct antiviral

compound suitable for treatment of RSV infection especially for infants and immunocompromised

individuals. Several compounds that differ in their characteristics and molecular targets have been

evaluated, and some have reach advanced clinical trials.

Four classes of anti-RSV therapeutics been developed and investigated: immunoglobulins,

siRNA-interference, fusion inhibitors, and small molecule inhibitors. Several types of

Immunoglobulins have been developed and most of them targeting RSV F protein. Both

19

polyclonal and monoclonal antibodies have been utilized as therapies with monoclonal showing

the higher neutralizing effect and lower adverse effects. siRNA is used to interfere with RSV-

directed protein synthesis. Anti-RSV therapeutic been developed target five different RSV

proteins[15].

Since RSV F protein is critical for RSV entry and spread to adjacent cells, most of the anti-

RSV therapeutics target F protein. Several F protein inhibitors are either neutralizing antibody

fragments or small molecules. Two compounds in this category (ALX-0171, REGN-2222) have

reached advanced clinical trials (phase 3 human clinical trials). ALX-0171 is trimeric Nanobody

(camelia antibody ) that binds the antigenic site II of RSV F protein [185], while REGN-2222 is a

fully human monoclonal antibody[186]. Both antibodies bind to prefusion F protein preventing

entry and multicyclic RSV infection. Other small molecules that target F protein have reached

advanced clinical trials. These anti F protein compounds bind to F protein preventing its functional

activity and transformation to post-fusion conformation. Since the transformation of the metastable

pre-fusion to the 6-helix bundle post-fusion conformation is critical for RSV entry to the cell, these

anti-F small molecule inhibitors deactivate RSV and limit the infection. Several molecules in this

category reached advanced clinical trails such as GS-5806, JNJ-53718678, and AK0529). One

downside for F protein inhibitor is the development of resistant viral mutation (escape mutations)

[187]. Another RSV target is the L-protein, which is the RNA-dependent- RNA polymerase.

Previously recommended anti-RSV compound Ribavirin is in this class. ALS-008176, a

nucleoside analog chain terminator, is a newly developed RSV polymerase inhibitor developed by

Alios Janssen and now in advanced clinical trials[188]. Other target proteins are N protein, SH

protein, and M2-1 protein. Several anti-RSV molecules developed targeting these proteins, and

20

they are progressing toward clinical trails [189–191]. Another possible anti-RSV infection

therapeutic is to target host immune response to infection.

The severe inflammatory and cellular infiltration associated with severe RSV infection lead

to the belief of bidirectional approach in developing anti-RSV lower respiratory tract infection

therapeutic, i.e., specific anti-RSV molecules and immunomodulatory therapeutic such as

chemokines and anti-leukotriene[192]. Blocking either of CCL3 (MIP-1α) or CCL5 (RANTES) in

RSV infected animal model showed a significant reduction in the recruitment of inflammatory

cells and increased survival[193,194]. Anti-leukotriene is also investigated since leukotriene is

prominent during RSV bronchiolitis and showed that anti-leukotriene administration reduces

inflammatory cellular infiltrate, reduced airway blockage, and reduced bronchiolitis in RSV

infected mice and infants [195,196]. Other immunomodulating agents such as surfactant protein,

vascular endothelial growth factor are also investigated and reduced RSV-associated pathology

[197].

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35

: STREPTOCOCCUS PNEUMONIAE INFECTION IN

RESPIRATORY SYNCYTIAL VIRUS INFECTED NEONATAL LAMBS

A paper submitted to Emerging Microbes and Infections

Sarhad Alnajjar1,2,3, Panchan Sitthicharoenchai1, Jack Gallup1, Mark Ackermann3,5, David

Verhoeven4*

1Departments of Veterinary Pathology and 4Veterinary Microbiology and Preventative

Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa

2Department of Veterinary Pathology, College of Veterinary Medicine, Baghdad

University, Baghdad, Iraq

3Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon

State University

5LambCure, LLC, Corvallis, Oregon

Abstract

Background: Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis

resulting in hospitalization and the most frequent cause of secondary respiratory bacterial infection

especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated

enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting

results. Moreover, less is known about the pathogenesis of Spn serotype 22F and especially the

co-pathologies between RSV and Spn dual infections.

36

Objective: The objective of this study was to determine interactions and mechanisms that

contribute to co-pathogen-induced morbidity using a neonatal lamb model naturally permissive to

infection by both pathogens.

Methods: Colostrum deprived lambs (aged 3-5 days) were randomly divided into four

groups. Two of the groups were nebulized with RSV M37 (1.27 x 107 IFFU/mL), and the other

two groups nebulized with cell–conditioned mock media. At day 3 post-infection, one RSV group

(RSV/Spn) and one mock-nebulized group (Spn only) were infected with (2x106 cfu of Spn)

intratracheally. At day 6 post-infection all lambs were humanely euthanized, and bacterial/viral

pathogeneses were assessed by culture, viral infectious focus forming unit assay, RT-qPCR,

immunohistochemistry, and histopathology.

Results: Lambs dually infected with RSV and Spn had higher RSV titers, but lower Spn

than the other comparable groups. Additionally, lung lesions were observed to be more intense in

the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by

neutrophil and lymphocyte infiltration.

Conclusions: Despite lower Spn in lungs, lambs co-infected with RSV demonstrated greater

morbidity and tissue histopathology. Thus, the perception of enhanced disease severity may be

due to observed lesion development rather than elevated bacterial pathogenesis.

Introduction

Respiratory Syncytial Virus is one of the leading cause of severe lower respiratory infection

in infants under the age of five leading to 600,000 deaths worldwide [1]. RSV is a member of the

pneumoviridae family that infects all infants by the age of two years [2]. Although mild to

37

moderate upper respiratory tract infection is the most common form of infection, severe lower

respiratory tract can develop leading to severe pneumonia and/or bronchiolitis that leads to

hospitalization and sometimes death [3–5]. Lower respiratory tract infection increases the

susceptibility to secondary bacterial infection(s) leading to severe and life-threatening pneumonia

[6]. Streptococcus pneumoniae (Spn) is one of the most common bacterial infections that occurs

concurrently with respiratory viruses such as influenza and RSV [7].

Spn is a Gram-positive facultative anaerobic bacterial pathogen that causes invasive disease

including sepsis, meningitis, and pneumonia. Similar to RSV, Spn cause severe illness and presents

with higher incidence in both children and the elderly worldwide [8]. Pneumococcal pneumonia

is one of the leading causes of bacterial pneumonia in children worldwide, responsible for about

11% of all deaths in children under the age of five (700,000-1 million every year). Most of these

deaths occur in developing countries [9]. Spn vaccines are effective in reducing the incidence of

pneumonia caused by the serotypes contained in the vaccine [10]. However, the emergence of

non-vaccine serotypes and persistence of antibiotic-resistant Spn such as serotype 19A highlights

the importance of more investigation into Spn pathogenesis and therapy. Since Spn has an essential

role in secondary bacterial infections following viral pneumonia or viral-bacterial co-infection

[7,11], animal modeling for the purpose of understanding viral-bacterial co-infections are crucial

to investigating therapeutics that combat both. Moreover, most studies have concentrated on

influenza and Spn co-infections but mainly in murine models with few mechanistic studies done

in humans other than calculation of frequencies of co-infections with these two pathogens. Despite

the importance of RSV/Spn co-infections, far fewer studies have been done in this area as

compared to influenza/Spn. Furthermore, less is known about emergent serotype 22F pathogenesis

[12,13]. We have extensively used a neonatal lamb model to mimic RSV lower respiratory tract

38

infection in infants as a preclinical model to evaluate the efficacy of new therapeutics [14,15] and

to understand RSV pathogenesis [16–18]. Sheep are also permissive to Spn infection and have

served as a model of Spn sepsis that appears to manifest clinical signs similar to human infection

[19,20]. Thus, in the present study, we investigated a co-infection of neonatal lambs using

RSV/Spn as an alternative large animal model to better understand viral/bacterial co-infections in

the young with the objective of gaining more insight into serotype 22F pathogenesis in the lung.

Material and Methods

Experimental Design

Animals: A total of 20, 2-3 day-old, colostrum-deprived lambs, were randomly divided into

four groups with 5 animals per group: RSV only, RSV-Spn co-infection, Spn only, and uninfected

control. Animal use was approved by the Institutional Animal Care and Use Committee of Iowa

State University. Two groups were nebulized with RSV M37 (1.27x107 IFFU/mL) on day 0. One

of the RSV infected groups was injected intratracheally with 2 ml normal saline as a mock Spn

infection (RSV group), while the second RSV-infected group was injected intratracheally with 2

ml solution containing Spn serotype 22F (2x106 CFU/ml) 3 days post-RSV nebulization (RSV-

Spn group). The other two groups were nebulized with cell-conditioned mock media containing

20% sucrose at day 0 and injected intratracheally with either normal saline (control group) or

solution containing Spn (2x106 CFU/ml) at day 3 post nebulization (Spn group). At day 6 post-

RSV infection, all lambs were humanely euthanized. Autopsy was performed to evaluate the

macroscopic lung lesions. Lung samples were collected including sterile lung tissue for bacterial

isolation, frozen lung sample for RT-qPCR, bronchioalveolar lavage fluid (BALF) from right

caudal lung lobe for RSV infectious focus forming unit (IFFU) assay and RT-qPCR, and lung

39

pieces from different lobes were fixed in 10% neutral buffered formalin for histological

assessment.

Infectious Agents

Lambs were infected with RSV strain M37, purchased from Meridian BioSciences

(Memphis, TN, USA). This strain is a wild type A RSV isolated from the respiratory secretions of

an infant hospitalized for bronchiolitis [21,22]. M37 was grown in HELA cells and stored at -80°C

in media containing 20% sucrose [23]. 6 mL of 1.27 x 107 IFFU/mL in media containing 20%

sucrose or cell-conditioned mock media (also containing 20% sucrose) was nebulized using PARI

LC Sprint™ nebulizers to each lamb over the course of 25-30 minutes resulting in the total

inhalation of about 3 mL by each lamb [23]. Spn serotype 22F was grown overnight at 37°C in

Todd Hewitt media containing 2% yeast extract, 50 g/ml of gentamicin, and 10% bovine serum.

Colony forming units (CFUs) were calculated by OD600 with confirmation by dilution plating on

Tryptic Soy Agar (TSA) plates with 5% sheep blood containing gentamicin.

Lung RSV viral and Spn bacterial titers

BALF collected from right caudal lobe at necropsy was used to evaluate RSV IFFU. BALF

were spun for 5 minutes at 3,000g to pellet large debris. Supernatants were then spun through 0.45

am Costar SPIN-X filters (microcentrifuge 15,600g) for 5 minutes. The resulting BALF samples

were applied to HELA cells grown to 70% confluence in 12-well culture plates (Fisher Scientific,

Hanover Park, IL) at full strength, and three serial dilutions (1:10, 1:100, and 1:1000); all samples

were tested in triplicate to determine the viral titer. Lung tissue samples were used to determine

Spn titer. Lung tissue samples were placed in 500 μl of sterile PBS and were mechanically

homogenized. Lung homogenates were pelleted at 100x g, for 5 minutes. Supernatants were

serially diluted and applied to 5% sheep blood TSA plates containing gentamycin.

40

Immunohistochemistry (IHC)

Formalin-fixed paraffin-embedded tissue sections were used for IHC which was performed

according to a previously published protocol in our laboratory [18,24]. Briefly, after

deparaffinization and rehydration, antigen retrieval was performed in 10mM TRIZMA base (pH

9.0), 1mM EDTA buffer, and 0.05% Tween 20 with boiling under pressure for up to 15 minutes.

Polyclonal goat anti-RSV antibody (Millipore/Chemicon, Temecula, CA; Cat. No. AB1128) was

used as the primary antibody after two blocking steps. The first blocking was with 3% bovine

serum albumin in Tris-buffered saline+0.05% Tween 20 (TBS-T) and the second was 20% normal

swine serum in TBS-T for 15 minutes each. Primary antibody was followed by application of

biotinylated rabbit anti-goat secondary antibody (KP&L; Cat. No. 16-13-06). Signal development

was accomplished using a 1:200 dilution of streptavidin-horseradish peroxidase (Invitrogen; Cat.

No. 43-4323) for 30 minutes followed by incubation with Nova Red chromagen solution (Vector;

Cat. No. SK-4800). Positive signal was quantified in both bronchioles and alveoli for each tissue

section, and a score of 0-4 was assigned according to an integer-based scale of: 0=no positive

alveoli/bronchioles, 1=1-10 positive alveoli/bronchioles, 2=11-39 positive alveoli/bronchioles,

3=40-99 positive alveoli/bronchioles, 4=>100 positive alveoli/bronchioles. IHC for Spn was

performed using rabbit anti-Streptococcus pneumoniae polyclonal antibody (Thermo Fisher

scientific cat. # PA-7259) followed by biotin-labeled goat anti-rabbit IgG antibody (Thermo Fisher

Scientific Cat.#: 65-6140). Five random images were taken for each tissue section that were then

analyzed by the quantitative Halo program.

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

BALF and lung tissue homogenates in Trizol were used to assess RSV mRNA expression by RT-

qPCR. The assay was performed as published previously in our laboratory [18,24,25]. Briefly,

41

RNA isolation from lung tissue and BALF was performed using the TRIzol method followed by

standard DNase treatment. RT-qPCR was carried out using One-Step Fast qRT-PCR Kit master

mix (Quanta, BioScience, Gaithersburg, MD) in a StepOnePlus™ qPCR machine (Applied

Biosystems, Carlsbad, CA) in conjunction with PREXCEL-Q assay-optimizing calculations.

Primers and probe for RSV M37 nucleoprotein were designed based on RSV accession number

M74568. Forward primer: 5′-GCTCTTAGCAAAGTCAAGTTGAACGA; reverse primer: 5′-

TGCTCCGTTGGATGGTGTATT; hydrolysis probe: 5′-6FAM-

ACACTCAACAAAGATCAACTTCTGTCATCCAGC-TAMRA.

Hematoxylin-Eosin Staining and Histological Scoring of Lung Sections

hematoxylin-eosin stained sections were examined via light microscope. An integer-based score

of 0-4 was assigned for each parameter (bronchiolitis, syncytial cells, epithelial necrosis, epithelial

hyperplasia, alveolar septal thickening, neutrophils in bronchial lumen, neutrophils in alveolar

lumen, alveolar macrophages, peribronchial lymphocytic infiltration, perivascular lymphocytic

infiltration, lymphocytes in alveolar septa, fibrosis), with 4 as the highest score. A final score was

calculated by adding up all measured scores to form a 0-48 score, with 48 as the highest, which is

called the accumulative histopathological lesion score.

Statistical Analysis

Statistical analysis used the Wilcoxon signed-rank test for non-parametric parameters such as

accumulative microscopic lesion scoring, followed by nonparametric comparisons for each pair

also using the Wilcoxon method. One-way ANOVA was followed by all pairs comparison by the

Tukey-Kramer HSD method for gross lesion scores and viral titer analyses by RT-qPCR and IFFU

assays.

42

Results

Infected Lambs Showed Marked RSV and Spn Titer

RSV titers and Spn colony-forming units were measured in this study to evaluate the degree

of infection by each pathogen and to investigate the possible effect(s) of co-infection in the

combined RSV-Spn group on the replication of each infectious agent. As measured by IFFU,

infectious RSV was detected in both RSV and RSV-Spn groups (P<0.0001) (Figure 1a). RSV titer

trended (not significant) 0.15 fold higher in the RSV-Spn group than RSV alone. A similar trend

was observed when assessing RSV mRNA detected in BALF by RT-qPCR. RSV mRNA was

elevated in both RSV and RSV-Spn groups (7.28 and 7.31 virion/ml) (Figure 1b). Furthermore,

similar to the viable virus titer increase, RSV virions measured by RT-qPCR in the lung of the

RSV-Spn group were 0.6 fold higher than the RSV-only group (Figure 1c). In contrast, Spn was

isolated in the lung tissue of both the Spn only and the RSV-Spn groups (56902.75 and 5456

CFU/ g, respectively) (p<0.0001) (Figure 1d). In contrast to the RSV/Spn group, the bacterial

titer was 9.4 fold higher in the Spn only infected group (p<0.05) (Figure 1d). Interestingly, Spn

titers in lambs that died before the end of the study were the highest of their groups. Lamb number

22 that was found dead 36hr after Spn infection in the RSV-Spn group had 59,302 CFU/ g, while

lamb number 9 that was euthanized 48 hr after Spn infection in the Spn group had a titer of

9,302,325 CFU/ g (Figure 1d). Spn was detected in the blood in both Spn infected groups -

indicating bacteremia/sepsis (Figure 1e).

RSV and Spn Induce a Well-recognized Macroscopic and Microscopic Lesion

Percent of lung tissue with gross lesions related to either infectious agent was determined at

necropsy coupled with post-necropsy retrospective qualitative analyses. Both RSV and Spn-related

lesions were found scattered across the lung surface in all lung lobes. Pinpoint dark red areas of

43

lung consolidation characterized RSV lesions. These areas were obvious in RSV and RSV-Spn

groups (P<0.001, and p<0.005, respectively). There were no significant differences in the

percentage of lung RSV macroscopic lesions detected between RSV and RSV-Spn groups (Figure

2a). Spn gross lesions are characterized by larger sizes of lung consolidation with bright red color

- which was seen to a lesser extent when compared to RSV lesions (Figure 2b). When combining

lesions observed associated with both RSV and Spn, there was a significant increase in observed

lesions within both RSV-infected groups in comparison to the Spn only group (p<0.001) (Figure

2c).

Microscopic lesions observed within the lung tissue reflected the infectious agent used and

contradicted our initial expectations (i.e. microscopic lesions caused by RSV infection were

multifocal areas of interstitial pneumonia and bronchiolitis scattered randomly and

homogeneously throughout the lung tissue). However, Spn induced diffuse homogenous and subtle

pathological changes in the lung tissue. Infection with either Spn, RSV, or both, markedly increase

microscopic lesions (accumulative microscopic lesion score) associated with the disease in

comparison to the control group (p<0.05) (Figure 3a-f). Additionally, the combined RSV-Spn

infection significantly increased the severity of microscopic lesions in comparison to the Spn only

group (p<0.05). Lesions varied among lambs, and RSV lesions consisted of thickening of the

interalveolar wall with inflammatory cellular infiltrates in the airway adventitia and lamina propria

(lymphocytes and plasma cells), alveolar lumen (alveolar macrophages and neutrophils), and

bronchiolar lumen (neutrophils). With RSV, overall, there was a varied degree of epithelial

necrosis and syncytial cell formation. On the other hand, Spn lesions consisted of moderate

interalveolar wall thickening with inflammatory cellular infiltrate mainly in the alveolar septae.

Most of the microscopic lesions seen with RSV overlapped with Spn-induced injury. However,

44

congestion of the interalveolar wall capillaries and hemorrhage was seen only in Spn-infected

lambs.

Immunohistochemistry was used to identify and localize RSV and Spn in tissue sections.

RSV were present multifocally throughout the sections with bronchial and peribronchial

distribution (Figure 4c). Therefore, RSV expression was evaluated in bronchioles and alveoli

separately. There were marked increases in RSV expression in bronchioles in both RSV only and

RSV-Spn groups (p<0.005 and p<0.001, respectively) with high RSV expression in the alveoli of

the RSV-Spn group (p<0.01) (Figure 4a). There were no significant differences between the RSV

only and RSV-Spn groups in the degree of RSV expression in lung tissue sections. Spn was random

and homogenously scattered throughout the lung sections with more intense signal in interalveolar

walls and blood capillaries (Figure 4d). Although not significant, there was a 0.5 fold increase in

Spn in the Spn only group when compared with the RSV-Spn group (Figure 4b).

Discussion

There is a critical need for an animal model to study bacterial pneumonia secondary to an

initial viral infection in the lung in order to study the mechanisms of viral-bacterial co-infection

and to evaluate therapeutic interventions. There are significant advantages of using lambs to model

RSV infection as a correlate for human infants - including the ability to use human viral strains

without adaptation and the similarity of the pathological sequelae [16,17]. In this study, we

demonstrate that Spn readily infects the lungs of lambs and establishes active bacterial pneumonia.

A previous study revealed that the peak of RSV titer and infection in lambs is around day 3 post-

viral nebulization, and we used this time-frame to model early human co-infection [24]. The

results of this study demonstrate consistency in the infection rate of both RSV and Spn, as well as

a good relation to the lesion development induced by either of the infectious agents. Although we

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used 2 x 106 CFU of Spn for infection, murine studies typically use 5x105 to 107 CFU to establish

productive infections. Moreover, the lung volume of lambs is significantly larger than mice which

suggests that our inoculating dose may be more dispersed throughout the lungs than murine

studies. We believe that we may also be able to reduce the infection dosage to a lower CFU or

potentially use a colonization model to examine co-infection and pneumonia development.

The RSV titer in our study was increased by 0.5 fold in the RSV-Spn group, but bacterial

presence was reduced by 9.4 fold when compared to the RSV and Spn groups, respectively. These

data are consistent with prior studies in mice with influenza/Spn co-infections demonstrating

higher viral loads [11,26], although our observed viral (RSV) increase was quite modest in

comparison. Influenza co-infection studies also predict higher Spn burdens in the lungs due to

damaged epithelial cells serving as anchor points for the opportunistic bacteria. In other studies,

RSV with Spn in mice or cell culture predict that the RSV G protein on the infected epithelial

surface could also serve as an anchor point for Spn in the lungs [27]. In contrast to these murine

models, we found lower bacterial loads in the co-infection group over the Spn only group. These

findings suggest that the immune response might control Spn in the lungs of lambs better than

mice. Importantly, in human clinical studies of co-infection, show in increase in nasal

colonization numbers of Spn upon viral infection but this does not translate into higher invasive

lung disease [28]; suggesting that higher bacterial burdens could be a murine artifact rather than a

mechanism enhancing disease. In human studies of high Spn colonization, RSV disease appeared

less severe [29] suggesting that further using the lamb model to explore mechanistic differences

between Spn colonization and pneumonia during RSV.

The only deaths that occurred in the present study were in the Spn-infected groups, and both

lambs (lamb 11 in the Spn only group, and lamb 23 in the RSV-Spn group) had high lung Spn

46

colony-forming units/gram tissue. These could represent a failure to control bacterial division and

subsequent septicemia.

Lesion severity was consistent with the RSV titer and Spn burden as is shown by the

significant increase in the percent of lung tissue involved by gross lesions, and the increase in the

evaluated histological parameters. Gross lesions were multifocal lesions scattered randomly in all

lung lobes - which is the typical lesion distribution induced by RSV nebulization [18,24].

However, presentation of Spn gross lesions contradicted what was expected by the apparent

development of lesions in all lobes - including the caudal lung lobe, which is not typical for

bacterial pneumonia in lambs. However, the diffuse bacterial lesions and the presence of Spn

lesions in the caudal lobe may be due to the inoculation technique used for Spn infection. For Spn

infection, lambs were held vertically by one person and injected intratracheally by the second

person leading to a fall of inoculum through the bronchial tree into the caudal lobe, which in this

case, was favorable since it gives a bronchopneumonic distribution similar to that found in humans.

It is also possible that Spn spreads across lung lobes after inoculation either by airflow or vascular

flow. RSV-induced microscopic lesions were more prominent in comparison to Spn-induced

lesions and subsequently lead to significant differences between the RSV-Spn and Spn only

groups’ accumulative histologic lesion scores. RSV was more prominent in the bronchioles, while

Spn was diffuse throughout the lung sections.

Although mechanisms are being assessed, the higher observed morbidity in the RSV/Spn

group may derive from an enhanced neutrophil response present in the lungs. Evidence for this is

supported by the histopathology and the lower Spn burdens in these animals. Likely, RSV

infection served as a first activating response to neutrophils that could have then better controlled

the secondary bacterial infection. It is also possible that alveolar macrophages were activated by

47

RSV that, in turn, secreted inflammatory mediators that enhance neutrophil activation. Enhanced

neutrophil/leukocyte activation contrasts with studies in influenza co-infections in mice – which

suggests innate immune exhaustion [30]. While the time of inoculation could be a reason for the

observed differences, another could be the mere difference between influenza and RSV

pathogenesis. In either case, the results suggest further avenues of study using this model. We are

currently evaluating the neutrophil responses in these animals to ascertain how the innate antiviral

response may have played a role in better limiting the bacterial infection. The observed higher

RSV infection rate in the co-infection could also derive from the greater number of neutrophils in

the lungs in this group. There is evidence that RSV can infect neutrophils in humans [31] including

our unpublished data. Thus, if dual infection with Spn leads to enhanced neutrophil recruitment to

the lungs over RSV alone, those cells could become infected and contribute to the higher viral titer

we observed in the dual infection group. The effects of RSV infection on neutrophilic antibacterial

responses would be an interesting further study.

In this study, we have developed an animal model of co-infection for RSV and Spn. We

have determined enhanced disease with co-infection of both pathogens that correlates with human

and murine influenza studies, but this may all be due to a complex enhanced inflammatory/immune

response to co-infection rather than direct damage by either pathogen alone, although we cannot

rule the culpability of co-infection causation out. Future studies will plan to utilize other serotypes

of Spn to determine whether the results we observed are unique to serotype 22F or whether they

are a trend for all Spn strains in general. Additional studies will allow refinement of this model

and will include variations in inoculum volume/concentration, time between infections, and kinetic

analyses.

48

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52

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53

Percent of lung tissue associated with RSV lesions (a), Spn (b), and both RSV and Spn (c); with

photographic representative of each. All show average and SEM. Lambs were either infected with

mock media (control), RSV, Spn, or RSV followed by Spn (RSV-Spn). *P<0.05, **P<0.01,

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54

(a) accumulative histologic lesion associated with RSV and Spn infection shown as average +

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stain (b), H&E stained tissue section of control (c), RSV only (d), Spn only (e), combined

RSV-Spn (f). Lambs were either infected with mock media (control), RSV, Spn, or RSV

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55

Number of bronchioles and alveoli express the RSV positive signal (a), surface area (mm2)

occupied by Spn IHC positive staining (b), all shown as average + SEM. (c) and (d) show a photo

representation of RSV (c) and Spn (d) IHC positive staining. Animals were either infected with

mock media (control), RSV, Spn, or RSV followed by Spn (RSV-Spn). * P<0.05, **P<0.01,

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56

THERAPEUTIC EFFICACY OF JNJ-49214698, AN RSV

FUSION INHIBITOR, IN RSV-INFECTED NEONATAL LAMBS

A paper submitted to Frontiers in Microbiology Journal

Sarhad S. Alnajjar1,2,3, Alejandro Larios-Mora1, Albert Van-Geelen4, Jack M. Gallup1,

Anil Koul5, Peter Rigaux5, Dirk Roymans5, Mark R. Ackermann3

1Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State

University, Ames, Iowa USA

2Department of Veterinary Pathology, College of Veterinary Medicine, Baghdad

University, Baghdad, Iraq

3Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State

University

4 Agricultural Research Service, United States Department of Agriculture, Ames, Iowa

5Respiratory Infections Discovery, Janssen Infectious Diseases, Beerse, Belgium

Authors Contributions

S.S.A., A. L., J.M.G, and M.R.A carried out several aspects of the neonatal lamb infection

experiment including viral inoculation, treatment deliveries, blood draws, animal monitoring and

assessment of RSV clinical signs, necropsy, IFFU, qRT-PCR, histological, IHC, and RNAscope

assays. A.V. assisted with viral preparation and growth, and IFFU assays. D.R., P.R., and J.M.G

performed data processing. D.R., P.R., and A.K. provided the JNJ-49214698 compound, assayed

the compound for distribution in different body compartments and performed global statistical

analysis of all data. D.R., P.R., A.K., S.S.A., J.M.G, and M.R.A assisted with experimental

design and preparation of this manuscript.

57

ACKNOWLEDGMENTS

The authors graciously thank Laboratory Animal Resources (LAR) veterinarians Dr.

Mary Sauer and Dr. Kathleen Mullin for their expertise with medical intervention and critical

animal care. We also thank Livestock Infectious Disease Isolation Facility (LIDIF)

staff: Diane McDonald, Michelle Tye, Angelia Troutwine, Lori Bonnell, Alan Elsberry, and

Dean Isaacson for animal acquisition and superb daily animal care and data collection. As

well, we thank members of the Histology Laboratory staff: Jennifer Groeltz-Thrush, Toni

Christofferson, Diane Gerjets, and Ronisue Aust for their expert help with preparing sections

for histology and immunohistochemistry. We also kindly thank Dr. Panchan

Sitthicharoenchai for her excellent input in the writing of this manuscript. Finally, we thank

the professional student help we received for this project from Lauren Rowan, Meghann

McCaull, Marissa Kleve, Nicole Lacy, Alanna Hennen, and Drew Grosz.

Conflict of Interests

Sarhad S. Alnajjar, Alejandro Larios-Mora, Albert Van-Geelen, and Jack M. Gallup

worked at Iowa State University for this study and have no disclosures or conflicts. Mark R

Ackermann also worked at Iowa State Univesity for this study served as principal

investigator who received funds from Janssen Pharmaceutica to conduct this study. Anil

Koul, Peter Rigaux, and Dirk Roymans are employees of Janssen Pharmaceutica NV.

Funding

This study was funded by a grant from Janssen Pharmaceutica NV ( Iowa State Univesity

grant # MA14002).

58

Abstract

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection, hospitalization,

and death in infants worldwide. No fully effective RSV therapy using direct antivirals is marketed.

Since clinical efficacy data from naturally infected patients for such antivirals are not available

yet, studies in animals are indispensable to predict if therapeutic intervention. Here we report the

impact of an RSV fusion inhibitor, JNJ-49214698, on severe RSV-associated acute lower

respiratory tract infection (ALRTI) in neonatal lambs. Randomized animals were treated once-

daily with 25 mg/kg JNJ-49214698, starting either before RSV infection, 1 day post-infection, or

as late as peak lung viral load on Day 3 post-infection. The efficacy of treatment was assessed by

scoring clinical signs of illness, development of RSV-induced gross and microscopic lung lesions,

and measuring virus titers in the lungs. Treatment with JNJ-49214698 was very effective in all

treatment groups. Even in animals for which treatment was delayed until peak viral load was

reached, a reduced amount and severity of gross and microscopic lesions as well as RSV titers and

RNA levels were found. These results strongly suggest that treatment with small-molecule fusion

inhibitors is an effective strategy to treat patients who are diagnosed with an RSV-induced ALRTI.

Key Words

Respiratory Syncytial Virus, small molecule fusion inhibitor, RSV Fusion inhibitor,

antiviral, neonatal lamb model, acute lower respiratory tract infection

Introduction

Respiratory syncytial virus (RSV) is one of the leading causes of acute lower

respiratory tract infection (ALRTI). Globally, it is estimated that 33.8 million cases of

59

ALRTI associated with RSV infection occur yearly in children under the age of five.

Subsequently, RSV-associated ALRTI is a major cause of hospital admissions with an

estimated 3.4 million cases annually; and RSV-associated mortality is estimated to be

66,000-199,000 in children under the age of five occurring mostly in developing countries

[1]. Furthermore, post-bronchiolitic development of wheezing syndrome is sometimes

observed, and although still under debate, it was suggested that the development of asthma

could be associated with RSV infection [2,3]. RSV is also one of the leading causes of

respiratory infection in the elderly and high risk adults [4,5]. A recent study showed that 3%

of pneumonia cases in human adults is caused by RSV [6] and with the widespread use of

influenza vaccination, RSV has a similar disease burden in the elderly [4].

Despite decades of investigation, no market-approved vaccine exists [7]. Seasonal

prophylaxis with the monoclonal antibody palivizumab, restricted to high-risk infants in

developed countries, is the only specific antiviral strategy available [8]. The disease

preventive effects of palivizumab in these patients were demonstrated by a significant

reduction in hospitalization rate, hospital residence time, admission to intensive care unit and

days requiring oxygen therapy [9]. However, the therapeutic efficacy of antibodies like

palivizumab in infants hospitalized with an established RSV-associated ALRTI is

questionable at best [10–12]. Other drugs like bronchodilators, corticosteroids, and

antibiotics, often used but not indicated to treat RSV-associated bronchiolitis, do not seem

to confer sufficient therapeutic benefit either, leaving supportive care essentially as the major

treatment option [13,14].

New treatments are clearly needed to decrease the medical consequences related to

RSV-associated ALRTI. Data on the impact of small-molecule direct antivirals on RSV

60

disease manifestation in naturally infected hospitalized patients or outpatients are not

available, and as such, reliable modeling of human RSV disease remains a necessary step in

the search for novel therapies.

The development of an RSV experimental human infection model has provided a new

tool to obtain human proof-of-concept efficacy data of RSV direct antivirals early in the

clinical development pipeline [15–17]. However, volunteers are often young, healthy adults

and infection is studied only in the upper respiratory tract, limiting the information about the

efficacy of therapeutics to treat ALRTI. Therefore, the recent development of a fully-

replicative neonatal lamb model for RSV has presented a significant step forward to generate

reliable preclinical efficacy data and to improve the selection of the proper clinical candidate

molecules. Neonatal lambs are susceptible to human as well as ovine and bovine strains.

Similarities between the pulmonary and immunological systems between lambs and humans

make the neonatal lambs an excellent model to study human RSV disease and the impact of

new potential RSV therapeutics[18,19]. Moreover, neonatal lambs infected with RSV

develop clinical symptoms including but not limited to fever, tachypnea or increased

expiratory effort (wheeze), lethargy, and develop mild to moderate bronchiolitis and

pneumonia. Innate and adaptive immune responses by neonatal lambs are very similar to

those of human infants [20,21].

A promising approach to inhibit RSV is by targeting the viral fusion (F) protein [22,23].

A few small-molecule RSV fusion inhibitors are currently being evaluated in early-stage

clinical trials, but their clinical impact on RSV-associated ALRTI in hospitalized infants is

yet unknown [24–26]. The purpose of this study is therefore to evaluate the therapeutic

61

impact of an experimental RSV fusion inhibitor, JNJ-49214698, on RSV-associated ALRTI

using a lamb model of RSV infection.

Materials and Methods

(see online Supplementary Data for detailed methods)

Compound and Dosing

JNJ-49214698 was discovered and synthesized by Janssen Infectious Diseases (Beerse,

Belgium). The compound was formulated in 10% acidified hydroxypropyl-β-cyclodextrin

[10% HP-β-CD + HCl, pH 2 (vehicle)] at 6.25 mg/mL prior to dosing and stored throughout

the study at 4 °C. The compound was dosed orally by catheter-mediated orogastric gavage

at 4 mL/kg body weight (25 mg/kg) once daily. Dose selection in this study aimed for

reaching the highest possible, safe exposure in the animals in order to maximize the

likelihood to obtain efficacy while avoiding toxic side effects. A daily oral dose of 25 mg/kg

was selected based on the antiviral activity of JNJ-49214698 [EC50 = 0.4 ng/mL (0.8 nM)

and EC90 = 2.4 ng/mL (4.8 nM)], JNJ-49214698 exposure levels obtained in neonatal lambs

at different doses during a separate pharmacokinetic (PK) study and an observed lack of

toxicity of JNJ-49214698 at least until Cmax = 17,167 ng/mL and AUC0-24h = 139,993

ng.h/mL in a 5-day repeated dose rat tolerance study.

Animals

Twenty-one colostrum-deprived neonatal lambs (Suffolk, Polypay, Dorsett cross) aged

1-3 days and 2-7 kg body weights were obtained for this experiment. Animal use was

approved by the Institutional Animal Care and Use Committee of Iowa State University.

RSV-infected lambs were kept in a separate room from the non-infected animals in the

Livestock Infectious Disease Isolation Facility (LIDIF). Lambs were fed iodide-free lamb

62

milk replacer diet (Milk Products Inc., Chilton, WI, USA) [27], and were treated with Naxcel

(Ceftiofur sodium, Pfizer) intramuscular once daily to reduce/prevent secondary bacterial

infections.

Experimental Design

Lambs were randomly assigned to five different groups. Three groups (Px, Tx-1 and

Tx-2) were infected with RSV and treated with JNJ-49214698. The first group (Px, n = 4)

was treated prophylactically, 1 day before RSV challenge and then daily afterward up until

Day 5 post infection (p.i.). The second (Tx-1, n = 5) and the third (Tx-2, n = 5) groups were

treated one day and three days after viral challenge and daily afterward up until Day 5 p.i.,

respectively. The vehicle group (n = 4), serving as a positive RSV control group, was infected

with RSV, but received treatment with vehicle only. The No RSV group (n = 3), served as

negative RSV control group, and was aerosolized with RSV-free, HEp-2 cell-conditioned

media and also received vehicle. All lambs were euthanized at Day 6 p.i. and all endpoints

measured after euthanasia.

RSV Infection

Lambs were infected with RSV strain M37, purchased from Meridian BioSciences

(Memphis, TN, USA). This strain is a wild type A RSV isolated from the respiratory

secretions of an infant hospitalized for bronchiolitis [28,29]. In our laboratory, M37 was

grown in HEp-2 cells and stored at -80°C in media containing 20% sucrose [30]. Six mL of

1.27 x 107 Infectious Forming Unit (IFFU)/mL in media containing 20% sucrose or cell-

conditioned mock media (also containing 20% sucrose) was nebulized using PARI LC

Sprint™ nebulizers to each lamb over the course of 25-30 minutes resulting in the total

inhalation of about 3 mL by each lamb [31].

63

Assessed Parameters

JNJ-49214698 exposure in plasma, BALF, and lung homogenate samples was assessed

using a method based on Protein precipitation and HPLC/MS/MS analysis. Quantitative

reverse transcription polymerase chain reaction (qRT-PCR) was done to measure the RSV

mRNA expression in BALF and lung homogenate samples as previously done in our lab

[30,32,33]. Infectious focus-forming unit (IFFU) assay was used to determine the viable RSV

in BALF samples as previously described [32]. Chlincal score was calculated by the number

of respiratory associated signs present in each animal. For the pathological study, percent of

lung parenchyma involved with RSV induced lesion were measured to evaluate the RSV

gross lesion. Histologically, Hematoxylin-eosin stained sections were examined via light

microscope as described previously [32] with some modification. Lung lesions were scored

according to an integer-based score of 0-4 for each parameter (bronchiolitis, syncytial cells,

epithelial necrosis, epithelial hyperplasia, peribronchial lymphocytic infiltration,

perivascular lymphocytic infiltration, neutrophils), with 4 as the highest score. Then a final

score (accumulative histo lesion score) was assigned by adding up the scores from the seven

individual parameters resulting in final accumulative scores ranging from 0-28 representing

the total RSV-associated lesion in each tissue section. For further evaluation of the JNJ-

49214698 therapeutic efficacy, Immunohistochemical staining was performed to evaluate

RSV antigen expression in lung tissue section as previously described in our laboratory

[32,34,35]. Formalin-fixed paraffin-embedded tissue sections were used for the RNAscope

detection of RSV mRNA in situ. A probe designed to the hRSV M37 nucleoprotein gene

(accession number KM360090) was used (Probe-V-RSV-NP, Advance Cell Diagnostic,

Catalog number 439866). The assay was performed according to the manufacturer’s manual

(user manual document number 320497; RNAscope® 2.0 HD Detection Kit (BROWN) User

64

Manual PART 2). Sections were examined under light microscope, and the number of

bronchioles and alveoli containing the positive signal were counted. The number of positive

bronchioles and alveoli per tissue section was then assigned a score according to the simple

integer-based scale of 0 = no positive alveoli/bronchioles, 1 = 1-10 positive

alveoli/bronchioles, 2 = 11-39 positive alveoli/bronchioles, 3 = 40-99 positive

alveoli/bronchioles, 4 = >100 positive alveoli/bronchioles.

Statistical Analysis

Statistical analysis was completed by using the Kruskall-Wallis test for non-parametric

parameters such as accumulative microscopic lesion scoring, immunohistochemistry and

RNAscope integer-based scores, followed by Dunn’s post-hoc test for multiple comparisons.

One-way ANOVA followed by Dunnett multiple comparisons test was used to compare the

treated groups to the RSV-infected non-treated control group for gross lesion scores and viral

titer analyses by qRT-PCR.

Results

JNJ-49214698 Efficiently Distributes to Different Lung Compartments of Neonatal

Lambs

During a multicyclic RSV infection, small-molecule fusion inhibitors are thought to

inhibit each new infection event at the time of viral fusion. The concentration of JNJ-

49214698 was measured in BALF as well as in homogenized lavaged-lung tissue. To assess

the potential of JNJ-49214698 to distribute from the blood to the lungs of the neonatal lambs,

we also measured its level in plasma. JNJ-49214698 was absorbed very quickly into the

circulation and tended to slightly accumulate over time during the study period. The average

steady-state concentration of JNJ-49214698 in plasma was in the range of 1770 ± 435 and

65

3112 ± 1441 ng/mL across the different treatment groups and was reached within 24-48 hr

after the first dose (Figure 1a). Twenty-four hour after the final dose, the level of JNJ-

49214698 in the BALF of the group receiving prophylactic treatment (Px), or groups in

which compound administration was started one (Tx-1) or three (Tx-2) days after viral

inoculation was 2044 ± 648, 3289 ± 977 and 1109 ± 252 ng/mL, respectively (Figure 1b).

The average lavaged-lung concentrations measured were 2867 ± 403, 5211 ± 841 and 1976

± 175, in Px, Tx-1, and Tx-2 treatment groups, respectively, resulting in an approximate

lung/plasma ratio of 1.4 to 1.8 and a good distribution of JNJ-49214698 in the BALF (Figure

1c). Together, these results indicate a good distribution of JNJ-49214698 to different lung

compartments.

Treatment with JNJ-49214698 Reduces Incidence and Duration of RSV-Associated

Symptoms

Clinical signs of RSV infection in lambs, as in human infants, can be variable and

sometimes difficult to assess. Despite this, RSV-associated respiratory distress can be

observed in the animals as early as one day p.i., developing further in some animals into clear

external signs of RSV-associated illness such as nasal discharge, wheezing or lethargy. By

Day 1 p.i., most of the lambs in this study, except for the animals in the non-infected group,

had already become lethargic as characterized by decreased activity (lower frequency of

getting up and movement in general). The behavior and respiratory-related symptoms in

lambs of the vehicle-treated group (RSV and no treatment) steadily worsened during the

following days (Figure 2a). One lamb started to produce nasal discharge and displayed

episodes of wheezing as from Day 3 p.i. onwards, while its respiratory rate was clearly

increased by Day 6. One lamb on Day 3 p.i. died and was found during post-mortem analysis

with severe gross lesions consistent with RSV infection in its lungs. The other lambs

66

survived until Day 6 when they were euthanized, but by Day 4, all 4 remaining animals from

this group were lethargic.

In contrast, while by Day3 post-infection 3 lambs in the Px group showed fever, the

remaining lambs in Px and Tx-1 groups were free of symptoms by Day 3 post-infection

(Figure 2b and c). Although less prominently, the incidence and duration of RSV-associated

symptoms was also reduced in the lambs of the Tx-2 group as compared to the vehicle-only

treated animals (Figure 2d).

In summary, these findings suggest that treatment with JNJ-49214698 has the potential

to reduce the incidence and duration of the clinical manifestation of RSV disease in lambs.

Treatment with JNJ-49214698 Inhibits a Multicyclic RSV Infection in Neonatal Lambs

The presence of nucleoprotein RNA in BALF and lung samples was quantified by qRT-

PCR. In BALF, The Px group showed a significant 3.4 log10 (p<0.0001) reduction of the

viral RNA level in comparison to the RSV-infected vehicle group (Figure 3a), while the viral

RNA in the Tx-1 and Tx-2 groups was reduced less by approximately 7- and a 3-fold as

compared to the vehicle only treated animals. We could not detect viral RNA in the lung

tissue of prophylactically treated animals, and profound reductions in RSV RNA of

approximately 23-fold in Tx-1 group (p<0.02), and 10-fold in Tx-2 in compared to the

vehicle group (Figure 3b). The infectious viral titer in BALF were done and partially reported

in a separate manuscript [23] in which IFFU were significantly reduced in all treated groups

with no infective virus detected in Px group that receive prophylaxis with JNJ-49214698

similar to what we found in RSV mRNA in lung tissue. There were 34- and a 93-log

reduction in the infectious titer as compared to the RSV-infected control group, was observed

67

in the Tx-1 and Tx-2 groups, respectively (p<0.001) (Figure 3c). To confirm the quantified

viral titers in BALF and lavaged-lung tissue, we determined RSV (M37) antigen and RNA

by immunohistochemical and RNAscope analysis, respectively. RSV antigen was present in

infected airway epithelial cells of bronchi, bronchioles, and alveoli and occasionally in

alveolar macrophages. However, staining of RSV antigen was markedly reduced in all drug-

treated groups in comparison to the RSV-infected control group (Figure 4a, c). RSV antigen

was highly decreased in the Tx-2 group as compared to the vehicle-treated group, and again,

almost no RSV antigen was detected in both Px (p<0.01) and Tx-1 (p<0.05) treatment groups

(Figure 4a, c). When RSV RNA (M37 nucleoprotein) expression in lungs was determined by

RNAscope, concomitant with the RNA levels as determined by qRT-PCR, there was a

marked reduction of RSV RNA expression in all the compound-treated groups in both

bronchioles and alveoli (Figure 4b, d). Heavy brown staining of RSV RNA in RSV-infected

non-treated lung sections was observed which was concentrated in the bronchioles and

alveoli in the consolidated part of the lung (Figure 4d). In contrast, only minimal staining

was seen in the Px (p<0.05) and Tx-1 groups (Figure 4d), aligning with

immunohistochemical and qRT-PCR data.

Treatment with JNJ-49214698 Inhibits RSV-Induced Lung Pathology and Cellular

Immune Response of Neonatal Lambs

Approximately 43% of the lung surface of infected vehicle-treated animals had RSV

associated macroscopic lesions which were multifocal dark red pinpoint foci of consolidation

scattered randomly over the lung surface and deeply throughout the lung tissue (Figure 5a,

b). Moreover, gross lung lesions were evenly distributed over the different left and right lung

lobes. Prophylaxis (Px) or treatment starting twenty-four hours after infection (Tx-1)

completely abolished the formation of RSV associated gross lung lesion (Figure 5a, b). When

68

treatment was delayed even as late as three days after infection (Tx-2), a significant reduction

in the RSV associated gross lesions was observed; dropping from 43 to 16% (p<0.0001)

(Figure 5b). As a negative control, no lesions were observed in the lungs of non-infected,

vehicle-treated animals (Figure 5b).

To investigate the effect of JNJ-49214698 on lung tissue microscopically, we assessed

the impact of the compound on the development of bronchiolitis, syncytial cell formation,

epithelial necrosis and hyperplasia, and inflammatory cell infiltration in lungs. An

accumulative histological lesion score was assigned by combining all the assessed

parameters. Significant reductions of the accumulative score were seen across

prophylactically treated animals or animals that received early treatment starting 1 day p.i.

with JNJ-49214698, (p<0.01 and p<0.05, respectively) (Figure 6a). Prophylaxis with JNJ-

49214698 completely prevented the formation of microscopic lung lesions after RSV

infection (Figure 6a, b). Although the effect on the accumulative histologic score was least

prominent in animals that received treatment with JNJ-49214698 as late as 3 days after

infection, a prominent reduction as compared to the vehicle-treated group was still observed,

with some animals displaying almost no microscopic lesions at all.

Typical RSV lesions that were present in the RSV-infected control group were

multifocal interstitial pneumonia and bronchiolitis. There was thickening of the inter-

alveolar wall due to type II pneumocyte hyperplasia and lymphocyte infiltration (Figure 6b).

There was neutrophil infiltration in the bronchial and bronchiolar lumens and the alveolar

lumen with multifocal and segmental areas of necrosis and sloughing of the epithelial cells

lining bronchioles. Some bronchioles had modest thickening of epithelium due to

hyperplasia. In addition, there were occasional multinucleated syncytial cells present

69

throughout the lung sections. In the lesions formed in the latter Tx-1 group, the formation of

syncytial cells, epithelial necrosis or hyperplasia and infiltrating neutrophils was absent,

while the other parameters assessed were markedly reduced, resulting in a prominent

decrease of the accumulative histological lesion scores.

Discussion

Currently, there are no effective direct antivirals available for the treatment of RSV-

associated ALRTI. Even though a number of RSV inhibitors have reached early-stage

clinical evaluation, no data are available yet demonstrating their clinical benefit in naturally

infected patients suffering from severe RSV-associated ALRTI, and so uncertainty remains

about the therapeutic treatment window and the impact of such molecules on severe RSV

disease. Attempting to minimize the risk for intended late-stage clinical development, we

therefore evaluated an experimental small-molecule RSV fusion inhibitor, JNJ-49214698, in

neonatal lambs, a fully-replicative animal model of RSV infection closely mimicking infant

RSV disease.

The three treatment regimens chosen in the study all reflect realistic potential clinical

drug administration regimens. The first group (Px) began to receive JNJ-49214698 one day

before viral nebulization to test the pre-exposure prophylactic effect of the compound, while

the Day 1 p.i. treatment group (Tx-1) was used to test the effect of very early post-exposure

treatment of an established infection. Previous RSV viral kinetic studies in our laboratory

demonstrated that RSV replication in neonatal lambs is highest between viral inoculation and

Day 3 p.i., reaching peak viral titer at Day 3, while RSV-associated lung pathology peaks at

Day 6 p.i. [32]. Therefore, the Day 3 post viral challenge treatment group (Tx-2) was used

70

to test the effect of JNJ-49214698 on RSV-associated ALRTI when treatment is started close

to peak viral titer, essentially the time patients seek first-line medical assistance and on

average 1 day before they present to hospital [36–40].

The overall assessments demonstrate a strong pre-exposure protective effect of JNJ-

49214698 against RSV infection. The prophylaxis (Px) group showed no gross/microscopic

lung lesions and no viral antigen by IHC. In addition, viral RNA levels significantly reduced

close to undetectable levels and were mirrored by no detectable IFFU[23]. Only very

occasional infection of individual lung cells, as demonstrated by the presence of viral RNA

(by RNAscope) in these cells was observed, consistent with earlier studies which showed

that fusion inhibitors are effective in inhibiting syncytia formation by preventing

transmission of the virus via cell-cell spreading [38,39]. Our data are also consistent with the

prophylactic efficacy of Synagis®, a humanized monoclonal antibody inhibiting the fusion

protein of RSV [8]. In contrast to Synagis®, which lacks therapeutic efficacy[8], early (Tx-

1) and late (Tx-2) therapeutic administration regimens with JNJ-49214698 displayed

unambiguous evidence of efficacy in the neonatal lambs. Administration of JNJ-49214698

after the establishment of a multi-cyclic ALRTI strongly reduced the severity of the infection

as evidenced by the strong and statistically significant reduction of gross and microscopic

changes in the lung, and decreased infectious titer in BALF as compared to vehicle-only

animals [23]. Consistent with these results, a considerable reduction of the viral RNA

expression in lung tissue was measured. However, the significant reduction of infectious

virus in BALF of therapeutically treated animals was not mirrored by a reduction of the viral

RNA to the same extent. This seeming discrepancy between viral RNA expression and

infectious viral titer in the BALF may be explained by the ability of JNJ-49214698 to bind

71

tightly to prefusion RSV F present on the envelope of the virus, resulting in neutralization of

the infectivity of virus particles present in the airways [22]. Although early administration of

JNJ-49214698, starting one day after RSV infection resulted in better reduction and clearing

of RSV infection as compared to the group that received late treatment starting at peak viral

load, the efficacy results obtained in the latter group were clearly noticeable.

Together, the results of this study demonstrate that pre-exposure prophylaxis and

treatment of an established RSV infection with a small-molecule RSV fusion inhibitor results

in significant reduction of viral replication as well as improvement of RSV-associated lung

pathology. Moreover, our data suggest a favorable window within which to treat RSV

infections in a community or hospital setting and contribute to the de-risking of late-stage

clinical compound development pathways.

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77

Figures and Legends

a) shows the daily plasma concentration (ng/mL) 24 hr after the first dose and daily just before the next

dose (Ctrough) to the end of the study. The compound reached a steady state concentration in the plasma

within 24 h of treatment and maintained that level throughout the study. b), c) show JNJ-49214698 exposure

in BALF (b) and lavaged lung (c) at necropsy (day 6 p.i.).

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

T im e (h )

Co

nc

en

tra

tio

n J

NJ

-49

21

46

98

(n

g/m

L)

Px

Tx-1

Tx-2

P la s m a

P x T x -1 T x -2

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

T r e a tm e n t g r o u p

Co

nc

en

tra

tio

n J

NJ

-49

21

46

98

(n

g/m

L) B A L F

P x T x -1 T x -2

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

T r e a tm e n t g r o u p

Co

nc

en

tra

tio

n J

NJ

-49

21

46

98

(n

g/g

lu

ng

)

L a va g e d -lu n g

a c b

Figure 3-1:Exposure of JNJ-49214698 in different body compartments of neonatal lambs.

78

1 2 3 4 5

0 .0

0 .5

1 .0

1 .5

D a y p o s t in fe c tio n

Cli

nic

al

sc

ore

V e h ic le a n d N o R S V G ro u p

a

V e h ic le

1 2 3 4 5

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

D a y p o s t in fe c tio n

Cli

nic

al

sc

ore

P x g ro u p

b

Px

1 2 3 4 5

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

D a y p o s t in fe c tio n

Cli

nic

al

sc

ore

T x 1 g ro u p

c

Tx1

1 2 3 4 5

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

D a y p o s t in fe c tio n

Cli

nic

al

sc

ore

T x 2 G ro u p

d

Tx2

Figure 3-2: Clinical signs score in different groups.

Data shown as average number of RSV-associated clinical signs observed in each group

(wheeze, increased respiratory rate and expiratory effort). Animals were infected with RSV

and received either vehicle only (Vehicle) and animal not infected with RSV (a), prophylaxis

(Px) (b) or therapeutic treatment with JNJ-49214698, starting one (Tx-1) (c) or three (Tx-2)

(d) days post infection. See online supplement for detailed table of the clinical signs

observed.

79

a) the viral RNA titer as measured by qRT-PCR in BALF, and b) the viral RNA titer as

measured by qRT-PCR in lung tissue, c) number of infectious particles as measured by IFFU

assay [23] with modification, all shown as average + SEM. Animals were infected with RSV

and received either vehicle only (Vehicle), prophylaxis (Px) or therapeutic treatment with

JNJ-49214698, starting one (Tx-1) or three (Tx-2) days post infection. Non-infected, vehicle-

treated animals were indicated as No RSV. JNJ-49214698 reduces the viable viral particles

in BALF, as well as RSV RNA in both BALF and lung tissue. Statistical analysis was

performed by one-way ANOVA, followed by Dunnett’s post-hoc test for multiple

comparison correction. *p-value < 0.02; **p-value < 0.002; ***p-value < 0.001; ****p-

value < 0.0001. LLOD = lower limit of detection.

N o R S V Ve h ic le P x T x -1 T x -2

0

1

2

3

4

5

Vir

al

tite

r (

log

10 F

FU

/mL

)

***

***

*****

****

L L O D

B A L Fc

N o R S V Ve h ic le P x T x -1 T x -2

2

3

4

5

6

7

8

Vir

al

lite

r (

log

10 R

NA

co

pie

s/m

l)

L L O D

****

****

B A L Fa

N o R S V Ve h ic le P x T x -1 T x -2

3

4

5

6

7

Vir

al

lite

r (

log

10 R

NA

co

pie

s/g

lu

ng

)L L O D

*

***

**

lungb

Figure 3-3:Effect of JNJ-49214698 on viral titers in BALF and lung tissue at Day 6 p.i.

80

a) integer-based scoring of M37 antigen in lung tissue detected by immunohistochemistry,

b) integer-based scoring of M37 RNA in lung tissue detected by RNAscope. Scoring on y-

axis represents the number of bronchioles or alveoli stained positive for M37 antigen (a) or

RNA (b) Bars represent the average score per group + SEM. Animals were infected with

RSV and received either vehicle only (Vehicle), prophylaxis (Px) or therapeutic treatment

with JNJ-49214689, starting one (Tx-1) or three (Tx-2) days post-infection. Non-infected,

vehicle-treated animals were indicated as No RSV. c) and d) show representative images of

M37 antigen (c) and M37 RNA (d) in the lung tissue of animals allocated to the different

treatment groups. JNJ-49214698 reduced the RSV M37 antigen and RNA in the lung of all

JNJ-49214698-treated animals. Statistical analysis was performed by Kruskall-Wallis non-

parametric test, followed by Dunn’s post-hoc test for multiple comparison correction. p-

value bars are representative for both bronchioles and alveoli. *p-value < 0.05; **p-value <

0.01.

N o R S V Ve h ic le P x T x -1 T x -2

0

1

2

3

4

M3

7 a

nti

ge

n s

co

re

B ro n c h io li

A lveo li

**

*

*

a

N o R S V Ve h ic le P x T x -1 T x -2

0

1

2

3

4

5

B ro n c h io li

A lveo li

M3

7 R

NA

sc

ore

*

**

b

RSV Px

Tx-1 Tx-2

RSV Px

Tx-1 Tx-2

c d

Figure 3-4: Effect of JNJ-49214698 on viral antigen and RNA in lung tissue at Day 6 p.i.

81

a) shows the average + SEM % of lung surface affected by RSV-induced gross lesions in the

different treatment groups. Animals were infected with RSV M37 and received either vehicle

only (Vehicle), prophylaxis (Px) or therapeutic treatment with JNJ-49214689, starting one

(Tx-1) or three (Tx-2) days post infection. Non-infected, vehicle-treated animals were

indicated as No RSV. JNJ-49214698 significantly reduces the RSV gross lesions in the

treated groups in comparison to the vehicle group. Statistical analysis was performed by one-

way ANOVA, followed by Dunnett’s post-hoc test for multiple comparison correction.

****p-value < 0.0001. b) shows representative images of lungs from an animal infected with

RSV receiving vehicle only, prophylaxis with JNJ-49214698, or therapeutic treatment,

starting one (Tx-1) or three (Tx-2) days post-infection and Non-infected, vehicle-treated

animals were indicated as Baseline. Yellow arrows indicate variably sized dark red

consolidated areas on the lung surface.

B a s e lin e Ve h ic le P x T x -1 T x -2

0

1 0

2 0

3 0

4 0

5 0

Av

era

ge

% o

f to

tal

lun

g w

ith

le

sio

ns

********

********

a

Baseline

Tx-1

Px Vehicle

Tx-2

b

Figure 3-5: Effect of JNJ-49214698 on development of gross lung lesions at Day 6 p.i.

82

a) shows the average + SEM of the accumulative RSV-induced microscopic lesions in the different

treatment groups. Animals were infected with RSV M37 and received either vehicle only

(Vehicle), prophylaxis (Px) or therapeutic treatment with JNJ-49214689, starting one (Tx-1) or

three (Tx-2) days post infection. Non-infected, vehicle only-treated animals were indicated as No

RSV. JNJ-49214698 reduces the pathological changes associated with RSV infection. Statistical

analysis was performed by Kruskall-Wallis non-parametric test, followed by Dunn’s post-hoc test

for multiple comparison correction. *p-value < 0.05; **p-value < 0.01. b) shows representative

images of the lung tissue sections from animals allocated to the different treatment groups.

Px

Tx-2

RSV

Tx-1

a b

N o R S V Ve h ic le P x T x -1 T x -2

0

2

4

6

8

1 0

ac

um

ila

tiv

e l

un

g l

es

ion

sc

ore

**

***

No RSV

Figure 3-6:Effect of JNJ-49214698 on RSV-induced accumulated lung histopathology at Day

6 p.i.

83

Supplementary Data

Materials and Methods

Compound and Dosing

JNJ-49214698 was discovered and synthesized by Janssen Infectious Diseases (Beerse,

Belgium). The compound was formulated in 10% acidified hydroxypropyl-β-cyclodextrin

[10% HP-β-CD + HCl, pH 2 (vehicle)] at 6.25 mg/mL prior to dosing and stored throughout

the study at 4°C. The compound was dosed orally by catheter-mediated orogastric gavage at

4 mL/kg body weight (25 mg/kg) once daily. Dose selection in this study aimed for reaching

the highest possible, safe exposure in the animals in order to maximize the likelihood to

obtain efficacy while avoiding toxic side effects. A daily oral dose of 25 mg/kg was selected

based on the antiviral activity of JNJ-49214698 [EC50 = 0.4 ng/mL (0.8 nM) and EC90 = 2.4

ng/mL (4.8 nM)], JNJ-49214698 exposure levels obtained in neonatal lambs at different

doses during a separate pharmacokinetic (PK) study and an observed lack of toxicity of JNJ-

49214698 at least until Cmax = 17,167 ng/mL and AUC0-24h = 139,993 ng.h/mL in a 5-day

repeated dose rat tolerance study.

Animals

Twenty-one colostrum-deprived neonatal lambs (Suffolk, Polypay, Dorsett cross) aged

1-3 days and 2-7 kg body weights were obtained for this experiment. Animal use was

approved by the Institutional Animal Care and Use Committee of Iowa State University.

RSV-infected lambs were kept in a separate room from the non-infected animals in the

Livestock Infectious Disease Isolation Facility (LIDIF). These rooms have separate

ventilation units as well as separate entrances and exits to avoid any cross-contamination

between infected and non-infected lambs. Lambs were fed iodide-free lamb milk replacer

84

diet (Milk Products Inc., Chilton, WI, USA) [1], and were treated with Naxcel (Ceftiofur

sodium, Pfizer) intramuscular once daily to reduce/prevent secondary bacterial infections.

Experimental Design

Lambs were randomly assigned to five different groups. Three groups (Px, Tx-1 and

Tx-2) were infected with RSV and treated with JNJ-49214698. The first group (Px, n = 4)

was treated prophylactically, 1 day before RSV challenge and then daily afterward up until

Day 5 post infection (p.i.). The second (Tx-1, n = 5) and the third (Tx-2, n = 5) groups were

treated one day and three days after viral challenge and daily afterward up until Day 5 p.i.,

respectively. The vehicle group (n = 4), serving as a positive RSV control group, was infected

with RSV, but received treatment with vehicle only. The No RSV group (n = 3), served as

negative RSV control group, and was aerosolized with RSV-free, HEp-2 cell-conditioned

media and also received vehicle. All lambs were euthanized at Day 6 p.i. and all endpoints

measured after euthanasia.

RSV Infection

Lambs were infected with RSV strain M37, purchased from Meridian BioSciences

(Memphis, TN, USA). This strain is a wild type A RSV isolated from the respiratory

secretions of an infant hospitalized for bronchiolitis [2,3]. In our laboratory, M37 was grown

in HEp-2 cells and stored at -80°C in media containing 20% sucrose [4]. PARI LC Sprint™

nebulizers were used to administer virus or cell-conditioned control media (lacking RSV) to

each lamb [5]. Six mL of 1.27 x 107 Infectious Forming Unit (IFFU)/mL in media containing

20% sucrose or cell-conditioned mock media (also containing 20% sucrose) was nebulized

to each lamb over the course of 25-30 minutes resulting in the total inhalation of about 3 mL

by each lamb.

85

Animal Monitoring for Appearance of Clinical Signs and the Clinical Score

Animals were monitored for clinical signs in the beginning of the study right before

viral inoculation (Day 0: all animals scored 0) and immediately after each administration of

vehicle or test article throughout the course of the study. In addition to the monitoring of

their behavior, respiratory associated clinical signs (respiratory rate, wheezing, expiratory

effort) were measured. Because clinical signs are variable in lambs and difficult to score in

terms of severity, an accumulative clinical score to summarize the overall distress per group

was applied by adding up the number of the scored clinical sings observed in each of the

individual lambs and then averaging the daily obtained sums for the respective groups to

normalize for group size.

Blood sampling for PK Analysis

Blood samples (1.5 - 2 mL) were collected from the jugular vein pre-dose (just before

the first dose) and at 24 h following each dose until 144 h for PK analyses. Blood was

dispensed into 3 mL blood collection tubes containing K2EDTA anticoagulant. Blood

samples were kept at room temperature prior to centrifugation. The blood samples were then

centrifuged at 1,600 x g for 10 min at 4°C to obtain the plasma. Plasma was stored in 2 mL

cryovials at -80°C.

Lung Collection and Processing

The thorax was opened, lungs removed and gross lesions were scored as performed

previously (40). The lungs were also photographed in situ and ex vivo. After removal,

percentage parenchymal involvement was scored for each lung lobe before the

bronchoalveolar lavage fluid (BALF) collection procedure. Left and right lungs were then

separated and each lobe excised. Tissue samples were collected from each lung lobe of all

86

animals. In brief, one sample from each lobe not destined for BALF collection (i.e. 4 lobes -

Right Cranial, Left Cranial, Left Middle and Left Caudal) were snap-frozen in liquid nitrogen

for qRT-PCR. Two samples from each of these lobes were placed in tissue cassettes and put

in 10% neutral-buffered formalin (NBF) for histological and immunohistochemical analysis.

Representative lung samples from each of these lobes were also placed into a cryovial and

immediately snap-frozen in liquid nitrogen, then transferred to -80°C for storage and shipped

on dry ice pellets for PK analysis of JNJ-53718678 compound at Janssen.

BALF Collection

BALF samples from each animal were collected immediately after euthanasia on Day

6 p.i. from right middle and right caudal lobes as performed previously in our laboratory [6].

Briefly, the excised lung lobes were instilled with 5 mL of cold DMIM (42.5% Iscove's

modified Dulbecco's medium, 7.5% glycerol, 1% heat-inactivated FBS, 49% DMEM, and 5

μg/ml kanamycin sulfate). 100 μL of the right caudal lobe BALF was added to 1 mL TRIzol

(Invitrogen) and kept in – 80 C for the qRT-PCR assay to assess RSV mRNA, and the rest

of BALF sample was placed on ice and used within 2 h for infectious focus-forming unit

(IFFU) assay to assess the infectious RSV titer. The right middle lobe BALF sample placed

-80°C for storage and shipped on dry ice pellets for determination of JNJ-49214698 and

blood urea nitrogen concentrations at Janssen.

87

Quantification of JNJ-49214698 Exposure

Samples of plasma (50 µL), lung homogenate (50 µL) and BALF (50 µL) were analyzed for

JNJ-49214698 using a method based on protein precipitation and HPLC/MS/MS analysis. To each

sample, DMSO (50 µL) and acetonitrile (500 µL) was added. Samples were mixed thoroughly

(mechanical shaking for 10 min), and then centrifuged (5000 g for 10 min at 15C). An aliquot

(400 µL) of the resulting supernatant was transferred to a 96-well plate and assayed for JNJ-

49214698 concentrations using HPLC/MS/MS employing positive-ion electrospray ionization

(Sciex API 4000) and a Waters ACQUITY UPLC C18 1.7um (50 x 2.1 mm i.d.) column. Elution

was achieved at a flow rate of 0.8 mL/min with a gradient of 0.1% FA and acetonitrile. The lower

limit of quantification was 1 ng/mL for plasma, 50 ng/g for lung and 1 ng/mL for BALF. The

assay was linear up to 20,000 ng/mL for plasma, 5,000 ng/mL for BALF and 100,000 ng/g for

lung. Samples of plasma and BALF were also analyzed for concentrations of urea in order to

calculate the dilution of BALF on sample collection. To a separate sample of plasma and BALF

(50 µL), 50 µL H2O/acetonitrile (50/50), 50 µL internal standard, 200 µl acetonitrile was added.

Samples were mixed thoroughly (mechanical shaking for 10 min), and then centrifuged (5000 g

for 10 min at 15C). An aliquot (200 µL) of the supernatant was transferred to a 96-well plate and

evaporated at 40C with nitrogen. An aliquot (200 µL) of camphanic chloride (1 mg/mL in

acetonitrile) was added for derivatization. Samples were mixed thoroughly (mechanical shaking

for 10 min) and incubated for 90 min at 37C. After incubation, 100 µL H2O was added and assayed

for urea concentrations using HPLC/MS/MS employing positive-ion electrospray ionization

(Sciex API 4000) and a Waters ACQUITY UPLC HSS T3 (50 x 2.1 mm i.d.) column. Elution

was achieved at a flow rate of 1 mL/min with a gradient of 0.01M Ammonium formate (pH = 3)

88

and acetonitrile. The lower limit of quantification was 2 µg/mL for plasma and BALF. The assay

was linear up to 10,000 µg/mL.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Tissue samples from right and left cranial, left middle and left caudal lung lobes (0.3–

0.4 g of each lobe) were homogenized in TRIzol (Invitrogen, Carlsbad , CA) for total RNA

isolation according to manufacturer’s instructions and as previously described [4,6,7].

Briefly, RNA isolation was followed by DNase treatment (Ambion, TURBO DNase, Austin,

TX) at 1:10 dilution in combination with RNaseOUT (Invitrogen) and nuclease-free water

(GIBCO/Life Technologies, Carlsbad , CA). Spectrometry (Beckman Scientific,

Indianapolis, IN) was used to assess each RNA sample isolate at a dilution of 1:50 to measure

sample purity and quantity (A260nm/A280nm all >1.95). Agilent Bioanalyzer 2100 analyses

of the RNA isolates gave RNA Integrity Number values > 8.0. qRT-PCR was carried out

using One-Step Fast qRT-PCR Kit master mix (Quanta, BioScience, Gaithersburg, MD) in

a StepOnePlus™ qPCR machine (Applied Biosystems, Carlsbad, CA) in conjunction with

PREXCEL-Q assay-optimizing calculations [8,9]. Primers and probe for RSV M37

nucleoprotein were designed based on RSV accession number M74568. Forward primer: 5′-

GCTCTTAGCAAAGTCAAGTTGAACGA; reverse primer: 5′-

TGCTCCGTTGGATGGTGTATT; hydrolysis probe: 5′-6FAM-

ACACTCAACAAAGATCAACTTCTGTCATCCAGC-TAMRA. Each 1:10-diluted total

RNA sample was further diluted so that each final qRT-PCR contained 0.784 ng total

RNA/µL; as determined to be optimal by PREXCEL-Q [10]. Thermocycling conditions were

5 minutes at 50°C; 30 seconds at 95°C; and 45 cycles of 3 seconds at 95°C and 30 seconds

at 60°C. Samples and standards were assessed in duplicate and each qRT-PCR quantification

89

cycle (Cq) was converted to a relative initial quantity (Xo) based on a standard curve using

the following equation: Xo =  EAMP(b-Cq), where  EAMP and b are the PCR exponential

amplification efficiency value and the y-intercept, respectively, from a sample mixture

derived standard curve for RSV M37 nucleoprotein mRNA. The efficiency-corrected delta

Cq (EAMPΔCq) method was used for qRT-PCR quantification calculations. Results were

normalized to total tissue RNA loaded per reaction (identical for all reactions). No-RT

control reactions proved negative for RSV M37. qRT-PCR was demonstrably free of

inhibition based on preliminary dilution threshold analyses as per the PREXCEL-Q method

for qPCR [10,11].

Hematoxylin-Eosin Staining and Histological Scoring of Lung Sections

Hematoxylin-eosin stained sections were examined via light microscope as described

previously [6] with some modification. Lung lesions were scored according to an integer-

based score of 0-4 for each parameter (bronchiolitis, syncytial cells, epithelial necrosis,

epithelial hyperplasia, peribronchial lymphocytic infiltration, perivascular lymphocytic

infiltration, neutrophils), with 4 as the highest score. Then a final score (accumulative histo

lesion score) was assigned by adding up the scores from the seven individual parameters

resulting in final accumulative scores ranging from 0-28 representing the total RSV-

associated lesion in each tissue section. The scale used to assess the pathological changes

briefly, for bronchiolitis scale: 0 = no remarkable lesions, 1 = minimal detectable lesion

(epithelial degeneration in one or a few bronchioles per 20 X field), 2 = epithelial

degeneration involving less than 10% of the airway lumen; minimal neutrophils and cell

debris; adventitial lymphocytes in multiple bronchioles, 3 = epithelial degeneration involving

more than 10-50% of the airway lumen with cell debris and neutrophils; adventitial

90

lymphocytes; multiple bronchioles, 4 = circumferential bronchiolitis with dense adventitial

lymphocytes; multiple bronchioles. For syncytial cells scale: 0 = none, 1 = one distinct

syncytial cell, 2 = up to three in three 20x fields, 3 = more than three in three fields, 4 =

numerous. Epithelial necrosis and epithelial hyperplasia scale: 0 = none, 1 = minimally

detectable in one or a few bronchioles per 20x per field, 2 = 10% of the bronchioles in

multiple airways per field, 3 = 10-50% of the bronchioles in multiple airways per field, 4 =

circumferential in multiple airways. Neutrophils scale (in bronchi, bronchioles or alveoli): 0

= none, 1 = minimally detectable, 2 = 10 or less neutrophils in one or a few airways/alveoli,

3 = 10 or more neutrophils in several airways/alveoli, 4 = 10 or more involving many or most

airways/alveoli. The same scale was used for peribronchiolar lymphocytic infiltrates and

perivascular lymphocytic infiltrates: 0 = none, 1 = earliest detectable lymphocytic infiltration

in the adventitia, 2 = segmental to circumferential infiltration, 3 = circumferential infiltrates

that expand more than three cells wide, 4 = circumferential infiltrates that form nodules.

Immunohistochemistry (IHC) of Lung Sections

IHC for determining the distribution of RSV antigen was performed as described

previously [6,12,13]. Briefly, after deparaffinization and rehydration of the formalin fixed

paraffin-embedded tissue sections, antigen retrieval was performed by placing the slides with

tissue sections in pH 9.0 10mM TRIZMA base, 1mM EDTA buffer and 0.05% Tween 20

and boiling under pressure for up to 15 minutes. Polyclonal goat anti-RSV antibody

(Millipore/Chemicon, Temecula, CA; Cat. No. AB1128) was used as the primary antibody

after two blocking steps, the first with 3% bovine serum albumin in Tris-buffered saline +

0.05% Tween 20 (TBS-T) and the second with 20% normal swine serum in TBS-T, 15

minutes each. Primary antibody was followed by biotinylated rabbit anti-goat secondary

91

antibody (KP&L; Cat. No. 16-13-06). Signal development was done by using 1:200 dilution

of streptavidin-horseradish peroxidase (Invitrogen; Cat. No. 43-4323) for 30 minutes

followed by incubation with Nova Red chromagen solution (Vector; Cat. No. SK-4800). The

positive signal was quantified in both bronchioles and alveoli for each tissue section, and a

score of 0-4 was assigned according to an integer-based scale of 0 = no positive

alveoli/bronchioles, 1 = 1-10 positive alveoli/bronchioles, 2 = 11-39 positive

alveoli/bronchioles, 3 = 40-99 positive alveoli/bronchioles, 4 = >100 positive

alveoli/bronchioles.

RNAscope

Formalin fixed paraffin embedded (FFPE) IHC tissue sections were used for the

RNAscope detection of RSV mRNA in situ. A probe designed to the hRSV M37

nucleoprotein gene was used (Probe-V-RSV-NP, Advance Cell Diagnostic, Catalog number

439866). This probe was designed to target the 8-1111 base region of the nucleoprotein gene

of accession number KM360090.1 CDS sequence (1114-2289). The assay was performed

according to the manufacturer’s manual (user manual document number 320497;

RNAscope® 2.0 HD Detection Kit (BROWN) User Manual PART 2). Sections were

examined under light microscope, and the number of bronchioles and alveoli containing the

positive signal were counted. The number of positive bronchioles and alveoli per tissue

section was then assigned a score according to the simple integer-based scale of 0 = no

positive alveoli/bronchioles, 1 = 1-10 positive alveoli/bronchioles, 2 = 11-39 positive

alveoli/bronchioles, 3 = 40-99 positive alveoli/bronchioles, 4 = >100 positive

alveoli/bronchioles.

92

Infectious Focus-Forming Unit (IFFU) Assay

Viral titers in BALF from the right caudal lobe were determined by IFFU assay [6].

BALF samples were spun down for 5 minutes in a centrifuge at 3,000 x g to pellet large

debris. Approximately 800–850 μL of each supernatant was collected and then spun through

850 μL-capacity 0.45 μm Costar SPIN-X filter (microcentrifuge 15,600 x g) for 5 minutes.

The resulting clear BALF samples were applied to HEp-2 cells grown to 70% confluence in

12-well culture plates (Fisher Scientific, Hanover Park, IL) at full strength and three serial

dilutions (1:10, 1:100,and 1:1000), all tested in triplicate. The BALF samples in the wells

were diluted with DMEM media (Mediatech, Inc., Manassas, VA) supplemented with 10%

with heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA) and 50

μg/mL kanamycin sulfate (Invitrogen/Life Technologies). Plates were incubated for 1 hr at

37°C, 5% CO2, then 1 mL of media was added to each well and plates were returned to the

incubator. After a 48 hr incubation, wells were fixed with cold 60% acetone/40% methanol

solution for 1 minute. Wells were then rehydrated with TBS-T, then blocked with 3% BSA

solution for 15 minutes followed by overnight incubation with primary polyclonal goat anti-

RSV (all antigens) antibody (EMD Millipore Corporation, Billerica, MA, USA). The next

day, plates were washed with TBS-T then incubated for 1 hr with secondary antibody (Alexa

Fluor® 488 F(ab’)2 fragment of rabbit anti-goat IgG (H+L) (Molecular Probes/Life

Technologies). Plates were rinsed and examined under inverted fluorescence microscopy

using the FITX/GFP filter (Olympus CKX41, Center Valley, PA), and total number of IFFUs

(which is defined here as 3 or more fluorescing cells) were counted for each well. IFFU/mL

calculations were obtained by multiplying the average number resulting from triplicate well

counts by the initial BALF sample dilution factor and multiplying that value by 5 to obtain

counts/mL since 1,000 μL/200 μl (the actual sample applied for each well) equals 5.

93

Statistical Analysis

Statistical analysis was completed by using the Kruskall-Wallis test for non-parametric

parameters such as accumulative microscopic lesion scoring, immunohistochemistry and

RNAscope integer-based scores, followed by Dunn’s post-hoc test for multiple comparisons.

One-way ANOVA followed by Dunnett multiple comparisons test was used to compare the

treated groups to the RSV-infected non-treated control group for gross lesion scores and viral

titer analyses by qRT-PCR and IFFU assays.

94

Table 1: clinical signs observed in lambs in different groups throughout the

study period

RSV-associated symptoms

G

roup

Animal ID Day 1 Day 2 Day 3 Day 4 Day 5

Vehicle

1 W L R

2 D

3 N

D

L

4 L

5 L

Px 6 R

7 R

8

9

Tx-1

1

1

W R

1

2

1

3

1

4

1

5

Tx-2

1

6

1

7

1

8

W

1

9

2

0

E

e

No

RSV

2

1

2

2

2

3

W = wheeze, L = lethargy, R = increased respiratory rate, Ee = expiratory effort, D = Dead,

ND = nasal discharge.

95

Panels show the average + SEM of integer-based scoring of the lung microscopic criteria

induced by RSV infection. Animals were infected with RSV and received either vehicle only

(Vehicle), prophylaxis (Px) or therapeutic treatment with JNJ-49214689, starting one (Tx-1)

or three (Tx-2) days post infection. Non-infected, vehicle-treated animals were indicated as

No RSV. Assessed criteria were a) bronchiolitis, b) syncytial cell formation, c) epithelial

necrosis, and d) neutrophils. (see supplementary data for Figure 8, which shows epithelial

hyperplasia, peribronchiolar nodules, and perivascular nodules. JNJ-49214698 greatly reduces

all assessed criteria in all treated lambs in comparison to the RSV-infected vehicle-treated lambs.

Statistical analysis was performed by Kruskall-Wallis non-parametric test, followed by

Dunn’s post-hoc test for multiple comparison correction. *p-value < 0.05; **p-value < 0.01;

***p-value < 0.001.

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Bro

nc

hio

liti

s s

co

re

**

**

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Sy

nc

yti

al

ce

ll s

co

re

**

**

***

***

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Ep

ith

eli

al

ne

cro

sis

sc

ore

**

**

**

*

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Ne

utr

op

hil

sc

ore

**

*

**

a

dc

b

Figure 3-7: Effect of JNJ-49214698 on RSV-induced lung histopathology at Day 6 p.i.

96

Panels show the average + SEM of integer-based scoring of the lung microscopic criteria

induced by RSV infection. Animals were infected with RSV and received either vehicle only

(Vehicle), prophylaxis (Px) or therapeutic treatment with JNJ-49214689, starting one (Tx-1)

or three (Tx-2) days post infection. Non-infected, vehicle-treated animals were indicated as

No RSV. Assessed criteria were a) epithelial hyperplasia, b) peribronchiolar nodules, and c)

perivascular nodules. JNJ-49214698 greatly reduces all assessed criteria in all treated lambs in

comparison to the RSV-infected vehicle-treated lambs. Statistical analysis was performed by

Kruskall-Wallis non-parametric test, followed by Dunn’s post-hoc test for multiple

comparison correction. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Ep

ith

eli

al

hy

pe

rp

las

ia s

co

re

**

**

**

*

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Pe

rib

ro

nc

hia

l ly

mp

ho

cy

tic

in

filt

ra

te s

co

re

**

**

*

N o R S V V e h ic le P x T x -1 T x -2

0 .0

0 .5

1 .0

1 .5

2 .0

Pe

riv

as

cu

lar l

ym

ph

oc

yti

c i

nfi

ltra

te s

co

re

**

**

a

c

b

Figure 3-8:Effect of JNJ-49214698 on RSV-induced lung histopathology at Day 6 p.i.

97

References

1. Derscheid RJ, Geelen A van, Berkebile AR, et al. Increased Concentration of Iodide in

Airway Secretions Is Associated with Reduced Respiratory Syncytial Virus Disease

Severity. Am J Respir Cell Mol Biol. 2013; 50(2):389–397.

2. DeVincenzo JP, Wilkinson T, Vaishnaw A, et al. Viral Load Drives Disease in Humans

Experimentally Infected with Respiratory Syncytial Virus. Am J Respir Crit Care Med.

2010; 182(10):1305–1314.

3. Kim Y-I, DeVincenzo JP, Jones BG, et al. Respiratory Syncytial Virus Human

Experimental Infection Model: Provenance, Production, and Sequence of Low-Passaged

Memphis-37 Challenge Virus. PLOS ONE. 2014; 9(11):e113100.

4. Derscheid RJ, Geelen A van, McGill JL, et al. Human Respiratory Syncytial Virus

Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus

Grown in Vero Cells. Viruses. 2013; 5(11):2881–2897.

5. Grosz DD, Geelen A van, Gallup JM, Hostetter SJ, Derscheid RJ, Ackermann MR.

Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and

experimental infection. BMC Res Notes. 2014; 7:158.

6. Larios Mora A, Detalle L, Van Geelen A, et al. Kinetics of Respiratory Syncytial Virus

(RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs

as an RSV Infection Model for Human Infants. PLoS ONE [Internet]. 2015 [cited 2016

Jan 7]; 10(12). Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671688/

7. Derscheid RJ, Gallup JM, Knudson CJ, et al. Effects of Formalin-Inactivated Respiratory

Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV. PLOS ONE. 2013;

8(12):e81472.

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8. Gallup JM, Ackermann MR. The ‘PREXCEL-Q Method’ for qPCR. Int J Biomed Sci

IJBS. 2008; 4(4):273–293.

9. Sow FB, Gallup JM, Sacco RE, Ackermann MR. Laser Capture Microdissection

Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR

Preparation Software (PREXCEL-Q). Int J Biomed Sci IJBS. 2009; 5(2):105–124.

10. Polack FP, Teng MN, L.Collins P, et al. A Role for Immune Complexes in Enhanced

Respiratory Syncytial Virus Disease. J Exp Med. 2002; 196(6):859–865.

11. Delgado MF, Coviello S, Monsalvo AC, et al. Lack of antibody affinity maturation due to

poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease.

Nat Med. 2009; 15(1):34–41.

12. Olivier A, Gallup J, De Macedo MMMA, Varga SM, Ackermann M. Human respiratory

syncytial virus A2 strain replicates and induces innate immune responses by respiratory

epithelia of neonatal lambs. Int J Exp Pathol. 2009; 90(4):431–438.

13. Meyerholz DK, Grubor B, Fach SJ, et al. Reduced clearance of respiratory syncytial virus

infection in a preterm lamb model. Microbes Infect. 2004; 6(14):1312–1319.

99

THERAPEUTIC EFFICACY OF RESPIRATORY

SYNCYTIAL VIRUS NON-FUSION INHIBITOR (RSV-NFI) IN

NEONATAL LAMBS INFECTED WITH A HUMAN STRAIN OF RSV

A paper will be submitted to virology journal.

Sarhad S. Alnajjar1,2, Panchan Sitthicharoenchai, Jack M. Gallup1, David Lançois3, Peter Rigaux3,

Dirk Roymans3, Mark R. Ackermann1

1Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University,

Ames, Iowa USA

2Department of Veterinary Pathology, College of Veterinary Medicine, Baghdad University,

Baghdad, Iraq

3Janssen Infectious Diseases, Janssen Pharmaceutica NV, Beerse, Belgium

Summary

Respiratory Syncytial Virus (RSV) infects individuals of all ages and can cause severe lower

respiratory tract infection in infants and elderly. Given the burden of RSV disease and the lack of

any vaccine or efficacious therapeutic intervention, the discovery of new antiviral compounds are

in critically need. Therefore, the efficacy of RSV-NFI, a non-fusion RSV inhibitor, was tested for

efficacy in this study in the neonatal lamb model. Five groups of lambs were used to test the

efficacy of this compound at different concentrations. Three groups, TX1, TX2, and TX3, were

treated with RSV-NFI 24hr after RSV inoculation and once daily with 16, 4, or 1 mg/kg body

weight, respectively. The two other groups were treated with vehicle only after nebulization with

RSV or mock media. All lambs were euthanized at day 6 post-infection, which corresponds to the

expression of severe disease features in untreated animals. Pharmacokinetic analysis indicated that

RSV-NFI reached a suitable plasma concentration within 24 hr after administration, and this level

persist throughout the study that helped relate the plasma level to the antiviral efficacy. All tested

100

doses drastically reduced the appearance of RSV-induced gross and microscopic lesions. There

was a significant reduction in the viral load in the lung in both TX1 and Tx2 groups. Together;

these results demonstrate efficacy of the RSV-NFI in reducing RSV infection and inhibiting RSV-

induced lung pathology.

Introduction

Respiratory Syncytial Virus (RSV) is a common respiratory virus in all ages and leads to

moderate upper respiratory tract infection. However, RSV can progress to severe acute lower

respiratory tract infection (ALRTI) in children under the age of five, older adults, and immune

compromised individuals[1,2]. A systematic review and meta-analysis of RSV infection

worldwide estimated that 2.4 million episodes of severe RSV associated ALRTI of 33.8 million

total RSV infection each year[3]. Currently, there is no fully effective treatment or vaccine

available for RSV infection.

Recently, several antiviral compounds showed promising antiviral activity against RSV

ALRTI and some of these have been approved for human clinical trials [4]. Only two approved

treatments are commercially available including Palivizumab, which is a monoclonal antibody

against RSV fusion protein administered as prophylaxis for preterm infants and high-risk

individuals, and Ribavirin, a nucleoside analog antiviral drug [5,6]. Both of these compounds have

limited indication and therapeutic efficacy [7–10]. Developing RSV antiviral drugs generally fall

into three different categories: nucleoside inhibitors, fusion inhibitors, and non-fusion non-

nucleoside inhibitors [4]. A recent study with a fusion protein inhibitor indicated the possibility of

treating existing RSV infection even after peak viral load and lung pathology and demonstrate a

wide therapeutic intervention window for RSV [11].

101

Several models for RSV infection have been developed, and of these the neonatal lamb

model has some characteristics similar to those in infants with RSV lower respiratory tract

infection [12]. Lambs are susceptible to the human strain of RSV that causes moderate lower

respiratory tract infection [13], have similar lung size, lung anatomy, and cellular composition of

human infants, with similarity in immune responses and lung development. In this study, we

evaluate the therapeutic efficacy of RSV-NFI, which is a non-fusion non-nucleoside RSV

inhibitor, against RSV lower respiratory tract infection in the neonatal lamb model of RSV

infection.

Materials and Methods

Experimental Design

This study was completed in two parts in an experimentally identical environment. Animal

use was approved by the Institutional Animal Care and Use Committee of Iowa State University.

In the first part of the study, 2 high doses of RSV-NFI were tested with 4 experimental groups:

RSV infected group; RSV (n=3) treated with vehicle only, TX1 (n=2) treated with RSV-NFI

16mg/kg body weight and TX2 (n=4) treated with RSV-NFI 4 mg/kg body weight. The fourth

group (n=4) infected with mock media and treated with vehicle to serve as a negative control

group. In the second part of the study, the efficacy of RSV-NFI low dose (1mg/kg body weight)

was tested. 21 lambs were divided into 3 groups. There were two RSV infected groups: RSV (n=6)

treated with vehicle only, and TX3 (n=7) treated with 1mg/kg RSV-NFI one day after viral

nebulization, and a third group with no RSV (n=8) were nebulized with mock media and treated

with vehicle only (Figure 1). Lambs were nebulized with 6 ml hRSV strain M37 (1.27 x 107

Infectious Forming Unit (IFFU)/mL) on day 0. M37 is wild-type RSV A strain isolated from

human infant and commercially provided by Meridian BioSciences (Memphis, TN, USA)[14,15].

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Treatments either with RSV-NFI or vehicle were started 24 hr after viral nebulization, then once

daily for 5 days. Lambs were monitored daily for the presence of clinical signs, and then all lambs

were humanely euthanized at day 6 post nebulization.

Tissue and Sample Collection

At necropsy (day 6 post nebulization), lambs were euthanized, and lungs were examined in

situ, removed, and the percent of the lung with lesion were determined. Lung tissues were collected

from right cranial, right middle, right caudal, and left cranial and placed in 10% neutral buffered

formalin for histologic evaluation, or snap frozen in liquid nitrogen for qRT-PCR and

pharmacokinetic study. Also, BALF samples were collected for left caudal lobe for infectious

focus forming unit assay (IFFU) and RT-qPCR assays, and from left middle lobe for compound

pharmacokinetic level assessment.

Infectious Focus-Forming Unit (IFFU) Assay

IFFU was completed on caudal lobe BALF, and homogenized lung tissue samples from the

4 collected lung lobes, and assays were performed as previously described in our laboratory [16].

Briefly, samples were pelleted and resultant supernate was spin again through a 0.45 μm Costar

SPIN-X filter (microcentrifuge 15,600 x g) for 5 minutes. The filtered samples were applied to

70% confluent HEp-2 cells in a 12-well culture plates (Fisher Scientific, Hanover Park, IL) at full

strength and three serial dilutions (1:10, 1:100, and 1:1000)in triplicate. After initial incubation at

37°C, 5% CO2 for 1 hr, 1 mL of media was added to each well and plates were returned to the

incubator and incubated for 48 hrs. plates, then, fixed with cold 60% acetone/40% methanol

solution for 1 minute followed by immunofluorescent staining. Primary and secondary antibody

were used. The primary was polyclonal goat anti-RSV (all antigens) antibody (EMD Millipore

Corporation, Billerica, MA, USA), and the secondary antibody was (Alexa Fluor® 488 F(ab’)2

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fragment of rabbit anti-goat IgG (H+L) (Molecular Probes/Life Technologies). Plates examined

under inverted fluorescence microscopy using the FITX/GFP filter (Olympus CKX41, Center

Valley, PA), and the total number of IFFUs (which is defined here as 3 or more fluorescing cells)

were counted for each well. IFFU/mL calculations were obtained by multiplying the average

number resulting from triplicate well counts by the initial BALF sample dilution factor and

multiplying that value by 5 to obtain counts/mL since 1,000 μL/200 μl (the actual sample applied

for each well) equals 5.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

RSV RT-qPCR were completed on BALF and lung tissue. Lung samples from the 4 collected

lung lobe were homogenized in TRIzol and extracted for total RNA according to the

manufacturer's instruction. RNA samples were used for qRT-PCR as described previously[13,16–

18] to detect RSV, IP-10, MCP-1, MIP-1α, PDL-1, IFN-λ, IL-13, and RANTES mRNA

expression.

Histologic Evaluation

Microscopic evaluation of the tissue section were completed with hematoxylin and eosin

stained tissue sections, slides stained RSV antigen by immunohistochemistry, and slides processed

by RSV RNAscope to detect RSV mRNA. In H and E sections, RSV lesions were scored on a

scale of 0-4. These changes include bronchiolitis, syncytial cells, epithelial cells necrosis,

epithelial cells hyperplasia, neutrophils, bronchiolar nodules, and perivascular nodules. Then an

accumulative histological lesion score was determined by adding up the individualized score

resulting in a score of 0-28 that represent the overall pathological changes in each tissue section

(FI paper). Immunohistochemistry was completed on formalin fixed paraffin embedded tissue

sections and as described previously [16,19,20]. Briefly, the slides were deparaffinized and

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rehydrated; then antigen retrieval step were completed using PH 9 10mM TRIZMA base, 1mM

EDTA buffer and 0.05 tween 20, then boiled and put in a steamer to maintain the temperature for

at least 30 minutes. Tissues were blocked with 3% BSA and 20% NSS for 15 minutes each,

followed by 1:500 dilution goat anti-RSV polyclonal antibody (Millipore/Chemicone Cat. No.

AB1128) and incubated in 4C overnight. The next day, tissue were washed 3 times with TBS-tw

followed by 1 hr incubation with 1:300 dilution of biotinylated Rabbit anti Goat secondary

antibody (KP&L Cat. No. 16-13-06). After 3 washes with TBS-tw, tissue were incubated with 3%

H2O2 for 25 minutes in room temperature followed by 2 TBS-tw washes and signal detection by

incubation with 1:200 dilution streptavidin-horseradish peroxidase (Invitrogen Cat. No. 43-4323)

followed by Nova Red staining solution. Slides were examined under light microscope and signal

were quantified in both alveoli and bronchioles. Score of 0-4 were applied according to an integer

based scale (of 0 = no positive alveoli/bronchioles, 1 = 1-10 positive alveoli/bronchioles, 2 = 11-

39 positive alveoli/bronchioles, 3 = 40-99 positive alveoli/bronchioles, 4 = >100 positive

alveoli/bronchioles). The RNAscope assay was also done on FFPE tissue sections for the detection

of RSV mRNA in situ by using a probe for RSV M37 nucleoprotein gene (Probe-V-RSV-NP,

Advance Cell Diagnostic, Catalog number 439866). The assay were done as described previously

( FI paper ) and according to the manufacturer protocol ((user manual document number 320497;

RNAscope® 2.0 HD Detection Kit (BROWN) User Manual PART 2). Microscopic tissue sections

were examined, and RNA signal were counted in both alveoli and bronchioles and applied for the

same integer based scale used to assess the IHC, which is 0 = no positive alveoli/bronchioles, 1 =

1-10 positive alveoli/bronchioles, 2 = 11-39 positive alveoli/bronchioles, 3 = 40-99 positive

alveoli/bronchioles, 4 = >100 positive alveoli/bronchioles.).

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Statistics

When appropriate and unless specified otherwise, due to the limited number of data

observations in some treatment groups, the non-parametric Kruskal-Wallis test is applied to test

whether there is difference between the compound groups and the vehicle group. P-values are

adjusted with Bonferroni's multiple comparisons method. For the histological analysis of RSV

antigen producing cells and for the histopathological analysis, treatment group at a value of zero

were remove from the analysis (on/off patterns cannot be appropriately analyzed by statistics.

However, this pattern indicates the presence of a treatment effect). In the case of the histological

analysis of RSV antigen producing cells, scores of at least 1 were recoded to 1 and logistic

regression with random effects was applied for Slide and Animal to compare differences between

treatment Tx3 and Vehicle. In the case of the histopathology analysis, scores of at least 1 are

recoded to 1 and logistic regression with random effects for Animal was used to compare

differences between treatments and Vehicle results

Results

Pharmacokinetics of Different Doses of RSV-NFI in the Neonatal Lambs

Antiviral compounds need to reach an adequate and consistent exposure in the lung tissue in

order to protect against multicyclic RSV infection. First, we assessed RSV-NFI levels in the

plasma, then the compound distribution to the lung was evaluated. Compound absorption allowed

a rapid increase in plasma exposure within 1 hr. Steady state was not reached at 24h probably due

to the long half-life and slow elimination/metabolism of the compound. RSV-NFI plasma level at

24 hr after the first dose were respectively :5902ng/ml (16 mg/kg group), 871ng/ml (4 mg/kg

group), and 265ng/ml (1 mg/kg group:) with more than dose proportional increase of 5.0 and 4.6

was observed when comparing 1 to 4 mg/kg doses and 4 to 16 mg/kg doses, respectively (Figure:

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2a). Once-daily treatment with 1, 4 or 16 mg/kg of RSV-NFI lead to an increase of Ctrough over

time across all different dose groups (3.6 fold at 1 mg/kg, 3.5 fold at 4 mg/kg, and 3.0 fold at 16

mg/kg). At necropsy, the RSV-NFI concentration was measured in both BALF representing the

airway lumen/ secretion, and in the whole lung homogenate representing lung tissue (cells,

interstitial tissue, and vasculature). The compound exposure in bronchoalveolar lavage fluid

(BALF) and homogenized-lungs tissue were the highest in the 16 mg/kg group (24431ng/ml,

36213ng/g, respectively). BALF exposure in 4 and 1 mg/kg group was (2091 ng/ml, 299 ng/ml,

respectively), and in the lung were (9740 ng/g, 2650 ng/g, respectively) (Figure 2 b & c).

Subsequently, lung/ plasma ratio of 1.3, 0.9, 0.9 in animal treated with 1, 4, and 16 mg/kg,

respectively, indicating a dose-dependent distribution of the compound to the lung compartments

and optimal conditions for investigating therapeutic exposure levels.

Viral Load in BALF and Lavaged-Lung Samples of Neonatal Lambs

The viral titer was determined at day 6 post-infection in BALF and homogenized lung tissue

samples from animals that were treated once-daily as from Day 1 post-infection with either vehicle

or different concentrations of RSV-NFI by the IFFU assay. There was a strong concentration-

dependent decrease of the production of infectious virions in BALF with 1.5 log10 reduction at 1

mg/kg, 4.2 log10 reduction at 4 mg/kg (p<0.01), and 4.5 log10 reduction at 16 mg/kg(p<0.01) (Figure

3 a). Concomitantly, lung titers of infectious virions were drastically reduced in all compound

treated groups. There was a 2.0 log10 reduction in the viral titer in 1 mg/kg treated group(p<0.01),

while full effect of 2.2 log10 reduction in groups treated with 4, and 16 mg/kg (p<0.01) & (p<0.05),

respectively (Figure 3b). To assess whether the reduction in the viable viral particle was

accompanied by a reduction in the viral RNA, a qRT-PCR was done on the same BALF samples

and lung tissues representative from four lung lobes. There was a great reduction in viral RNA

107

copies in both BALF and lung (Figure 3c and 2d). There was a significant 1.4 log10 decrease in

viral RNA at 1 mg/ml in BALF (p<0.0001) accompanied by 1 log10 reduction lung tissue. Superior

effects were observed at 4 mg/kg and 16 mg/kg treatment as is shown by 2.8 log10 reduction in

BALF(p<0.0001), accompanied by 4.4 log10 and 4 log10 reduction in lung tissue, respectively

((p<0.001) (p<0.0001) respectively). These results prove that the compound was efficient in

reducing the viral load in the treated lambs.

Treatment with RSV-NFI Significantly Reduced RSV-Induced Lung Lesion

Six days after infection, lungs from animals across different study groups were removed, and

the gross lesion developed by the lung upon RSV infection were scored. The RSV gross lesion

which was prominent in the RSV infected vehicle-treated group characterized by multifocal dark

red areas of consolidation on the lung surface and extend deep into the lung parenchyma in cut

section. In lungs of non-infected vehicle-treated animals, no gross lesions compatible with RSV

infection was seen. In contrast, in infected vehicle-treated animals, a significant level of the lung

surface area (32% on average) had gross lesions compatible with RSV infection (Figure 4 a and

b). Treatment with different doses of RSV-NFI drastically reduced the level of gross lesions

observed in all treated group when compared to the RSV infected vehicle-treated group. Gross

lesions were decreased to 0.76 % (p<0.05) or 0% (p<0.01) of lung surface when animals were

treated with 1 mg/kg or higher doses (4 and 16 mg/kg), respectively (Figure 4 a and b).

Microscopically, different concentrations of RSV-NFI resulted in a drastic reduction of all

assessed parameters and subsequently the accumulative histopathologic lesion score. The

histopathological lesion observed in the RSV infected vehicle-treated group was severe interstitial

pneumonia with bronchiolitis. There was inter-alveolar wall thickening due to inflammatory cell

infiltration, and epithelial cell hyperplasia with occasional necrotic epithelial cells and syncytia

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formation leading to an accumulative histopathologic score of 11 (figure 5 a and b and figure 8).

The compound treatments significantly reduce the accumulative histopathologic lesion score in all

treated group (P<0.01) (Figure 5 a and b). 16 mg/kg reduced the accumulative histopathologic

score to 0.12, while 4mg/k and 1mg/kg treatments reduced the accumulative histopathologic score

to 0.6 and 1.2 (Figure 5 a and b). To further evaluate the degree of RSV infection in the lung tissue,

RSV immunohistochemical stain was done on FFPE tissue sections. RSV M37 antigen

demonstrated a dose-dependent decrease in the density of M37 antigen in the different lung lobes.

No RSV antigen detected in animals treated with 4 and 16 mg/kg (P<0.01 and P<0.05,

respectively) (Figure 6 a). There was a significant decrease in the RSV antigen expression in

bronchioles of animal treated with the low dose RSV-NFI (1 mg/kg)(p<0.05), with a high

reduction in the alveoli (P=0.059) indicating the efficiency of treatment with the study compound

to reduce RSV dissemination. Similar to what found in qRT-PCR of the BALF and lung tissue,

there was a significant reduction in RNA expression in situ in all RSV-NFI treated groups (P<0.01)

(Figure 7 b) as tested by RNAscope technique. The compound treatment drastically reduces RSV

mRNA in all treated groups with the most reduction was in 16 and 4 mg/kg treatment. Hence, the

RSV-NFI showed a strong effect in reducing RSV antigen and mRNA and subsequently RSV

associated lung lesion.

Treatment with RSV-NFI Significantly Decrease Inflammatory Chemokines

By qRT-PCR in lung at day 6 p.i. RSV-NFI greatly reduce mRNA expression of IP-10, MCP-

1, MIP-1α, PDL-1, and IFN-λ in comparison to the RSV infected vehicle-treated group in which

the expression of these chemokines were elevated (Figure 9 a-e). There was no difference shown

in the endogenous levels of IL-13 and RANTES mRNA expression (figure 9 f, g).

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Discussion

Given the burden of RSV infection in infant and elderly, the development of active anti-RSV

therapeutic is urgently needed. Several antiviral drugs that target RSV have reached advanced

clinical trials. Most of these antiviral drugs are f monoclonal antibodies that inhibit fusion [4].

However, RSV resistant mutants can potentially form after few passages [21] and thus, additional

antiviral compounds with different mode of action that targets other viral structural component is

needed. In this study, we evaluate a non-fusion RSV inhibitor RSV-NFI, which is a non-

nucleoside replication inhibitor, in the neonatal lamb model. This study aims to assess the

therapeutic effect of different doses of RSV-NFI on established RSV associated acute lower

respiratory tract infection with treatment started one day after the viral nebulization and repeated

once daily up to 5 days. Our results demonstrated a potent antiviral activity as reflected by reduced

viral titer, mRNA expression, and RSV associated lesions in infected lambs.

Different concentrations of the compound resulted in different plasma exposure with more

than dose proportional when comparing each treatment with the one higher. However, the plasma

exposure was stable throughout the study with a tendency to increase over time. All the treatment

groups reached a concentration above the EC90 after 24 hr post-treatment except for 1mg/kg

treatment group which reached a concentration above the EC90 48hr after the first dose. This

plasma concentration leads to adequate lung exposure (at day 6 post-infection) which reflect the

concentration pattern seen in the plasma, i.e., high dose group produced a higher RSV-NFI

concentration in the lung tissue. Consistent RSV-NFI presence in the lung gives the compound the

ability to act on each new viral cycle reducing the RSV replication and cellular reinfection.

Lambs are permissive for hRSV and the human strains replicates well in lambs airways

causing acute lower respiratory tract infection [19,20,18,22]. Lambs nebulized with hRSV have

110

the peak viral load by day 3 of infection with the most lung lesions developing at day 4 to 6 post

nebulization [16]. RSV infected and vehicle-treated lambs in this study had high infective virus

and mRNA levels with distinct lesions in lung tissue characterized by interstitial pneumonia and

bronchiolitis. In contrast, treatment with different concentrations of RSV-NFI resulted in

consistent and significant reductions of all the assessed endpoints such as the reduction of lung

viral titers as measured by IFFU assay or qRT-PCR and reduced RSV associated lung lesions. The

results of both IFFU and qRT-PCR indicate the presence of viable RSV and whole viral load,

respectively, and were consistent and had the same pattern in BALF and lung tissue, and both had

a great reduction in the viral load with treatment that correlated with the RSV-NFI concentration.

Treatment with RSV-NFI as low as 1mg/kg caused a drastic reduction in the viral load in lung.

However, the most pronounced reduction occurred in 4 and 16 mg/kg groups. Several studies

indicated the positive relation between viral load and disease severity in infant [23–25]. Thus, the

observed reduction in the viral load in this study predicted to have a substantial effect in reducing

the pathological lesions associated with RSV by giving the lambs the ability to respond and repair

the tissue damage.

RSV associated gross and microscopic lesions were significantly decreased in all treated

animals. Almost no RSV lesions were seen at day 6 post-infection in the 4 and 16 mg/kg groups

with a great decline the lesions in the 1 mg/kg treated group. These results were consistent with

RSV immunohistochemistry and RNAscope results that had similar reduction trend in RSV

antigen and RSV RNA expression. Additionally, the increased inflammatory cell infiltration in

lung tissue of RSV infected vehicle treated lambs were accompanied by elevated level of MCP-1

and MIP-1α, and consistent with an earlier study in lamb indicated increase level of these

chemokines in RSV infected lambs at day 6 p.i. [18]. Several studies on RSV lower respiratory

111

tract infection in infants indicated that elevated levels of IP10, MIP-1α, and MCP-1 act as a

chemoattractant for inflammatory cells and subsequently associated with severe bronchiolitis and

hospitalization [26,27]. In contrast, an elevated level of PDL-1 in RSV infected vehicle treated

lambs may suggest a vital role in inhibiting lymphocytes antiviral response by modulating PDL-1

expression in infected epithelial cells [28,29]. The presence of minimal RSV antigen and mRNA

in the lung tissue section of the treated group indicate the ability of RSV-NFI to halt RSV

replication and infection of adjacent cells.

In summary, the compound under investigation had a strong effect on reducing RSV viral

load and lesions in all treated groups in comparison to the RSV infected vehicle-treated group.

This strong treatment effect occurred even when RSV-NFI was delivered to lambs one day after

viral nebulization, indicating the efficacy of the compound as a post infection therapeutic modality

to decrease the severity of lower respiratory tract infection after established RSV infection.

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Figures and Legends

Figure 4-1:Experimental design.

a) RSV-NFI dosing schedule. b) RSV nebulization, treatment, and blood draw timeline.

117

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

T im e (h r )

Pla

sm

a c

on

ce

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1 0 3

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1 0 5

R S V -N F I d o s e (m g /k g )

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R S V -N F I d o s e (m g /k g )

Lu

ng

co

nc

en

tra

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n (

ng

/g)

lu n gc

Figure 4-2: Exposure of RSV-NFI in different body compartments.

a) RSV-NFI daily plasma concentration (ng/ml) 24 hr post-treatment. RSV-NFI concentration in BALF

(b) and homogenized lung tissue (b) at day 6 post-infectionShownwn as average+ SEM. The compound

reached a steady state concentration in the plasma and accumulated over time with subsequent high

exposure in BALF and lung tissue after daily dosed with either 1, 4 or 16 mg/kg of RSV-NFI.

118

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Figure 4-3: Effect of RSV-NFI on viral titer in BALF and lung tissue homogenates at day 6

p.i.

a and b show the infectious viral titer as measured by IFFU assay in BALF (a) and in lung tissue (b). c and

d show the RSV mRNA titer as measured by qRT-PCR in BALF (c) and in lung tissue (d)All shown as

individual titer and mean. Treatment with RSV-NFI reduces the infectious viral titer and RNA in BALF

and Lung tissue. Animal infected with RSV and treated with either vehicle only (0) or different

concentration of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs are not infected and treated with vehicle only.

*p<0.05, **p<0.01, ***p<0.001,****p<0.0001. n.s.: not significant.

119

a) Show the average % of lung tissue parenchyma involved with RSV gross lesion shown as individual %

and mean. b) Shows picture representative of gross lesion observed in different treatment group. RSV-NFI

greatly prevent the development of RSV associated gross lesion. Animal infected with RSV and treated

with either vehicle only (0) or different concentration of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs are not

infected and treated with vehicle only.*p<0.05, **p<0.01.

Vehicle

4 mg/kg

1 mg/kg

16 mg/kg

Baseline

B a s e lin e 0 1 4 1 6

0

2 0

4 0

6 0

8 0

1 0 0

Pro

po

rtio

n o

f lu

ng

are

a

dis

pla

yin

g g

ro

ss

le

sio

ns

(%

)

R S V -N F I (m g /k g )

*

**

**

a b

Figure 4-4: Effect of RSV-NFI on the development of RSV associated gross lesion.

120

a) the accumulative histological lesion associated with RSV infection, which represents the sum of lesions

scored in RSV infected lung tissue which includes Bronchiolitis, Syncytial cells, Epithelial cells necrosis,

epithelial cells hyperplasia, neutrophils, Bronchiolar nodules, and perivascular nodules. b) a photo

representative for each treatment group. RSV-NFI greatly reduced the RSV associated microscopic lesion

in lung tissue. Animal infected with RSV and treated with either vehicle only (0) or different concentration

of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs are not infected and treated with vehicle only. *p<0.05,

**p<0.01.

b 0mg/kg

4 mg/kg 16 mg/kg

Baseline

1 mg/kg

B a s e lin e 0 1 4 1 6

0

5

1 0

1 5

2 0

Ac

cu

mu

lati

ve

lu

ng

le

sio

n s

co

re

R C r

L C r

LM

L C d

R S V -N F I (m g /k g )

****

****

****

a

Figure 4-5: Effect of RSV-NFI on the development of RSV associated histologic lesion.

121

a) integer-based scoring of RSV M37 antigen in lung tissue detected by IHC had as average + SEM.

Treatment with RSV-NFI greatly reduces RSV antigen expression in lung tissue. b) shows photo

representation antigen distribution from different treatment group. Lung from lamb infected with RSV and

treated with either vehicle only (0) or different concentration of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs

are not infected and treated with vehicle only. Estimated odds ratios: ***p<0.001. $: on/off pattern

indicating an obvious effect. n.s.: not significant.

0 mg/kg

Baseline

1 mg/kg

4 mg/kg

mg/kg

16 mg/kg

b

B a s e lin e 0 1 4 1 6

0

5 0

1 0 0

1 5 0

M3

7 a

ntig

en

sc

ore

R S V -N F I (m g /k g )

B ro n c h i

A lveo li

n .s .

* * *

$

$

$

$

a

Figure 4-6:Effect of RSV-NFI on RSV antigen.

122

.

a) integer-based scoring of RSV M37 RNA in lung tissue detected by RNAscope had as average + SEM.

Treatment with RSV-NFI greatly reduces RSV RNA expression in lung tissue. b) Picturesdepict findings

representation from different treatment group. Animal infected with RSV and treated with either vehicle

only (0) or different concentration of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs are not infected and treated

with vehicle only. *p<0.05, **p<0.01. n.s.: not significant.

0 mg/kg

Baseline

1 mg/kg

4 mg/kg 16 mg/kg

b

B a s e lin e 0 1 4 1 6

0

5 0 0

1 0 0 0

1 5 0 0

Vir

al

RN

A+ c

ell

s

(c

ou

nts

/ 3

52

mm

2)

R S V -N F I (m g /k g )

B ro n c h i

A lveo li

n .s .* *

*

n .s .

*n .s .

a

Figure 4-7: Effect of RSV-NFI on RSV M37 RNA expression.

123

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Bro

nc

hio

liti

s s

co

re

$

$

**

R S V -N F I (m g /k g )

B a s e lin e 0 1 4 1 4

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

Sy

nc

yti

al

ce

lls

sc

ore

$

$

$

R S V -N F I (m g /k g )

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

Ep

ith

eli

um

ne

cro

sis

sc

ore

$

$

**

R S V -N F I (m g /k g )

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Ep

ith

eli

um

hy

pe

rp

las

ia s

co

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$

$

$

R S V -N F I (m g /k g )

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

Ne

utr

op

hil

s s

co

re

$

*

**

R S V -N F I (m g /k g )

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Pe

rib

ro

nc

hio

lar l

ym

ph

oc

yti

c

infi

ltra

tes

sc

ore

n .s .

*

R S V -N F I (m g /k g )

n .s .

B a s e lin e 0 1 4 1 4

0 .0

0 .5

1 .0

1 .5

2 .0

Pe

riv

as

cu

lar l

ym

ph

oc

yti

c

in

filt

ra

tes

sc

ore

n .s .

R S V -N F I (m g /k g )

$

n .s .

a b

d e

c

f

g

Figure 4-8: Effect of RSV-NFI on the development of RSV associated histologic lesion.

Graphs show an integer-based score of Bronchiolitis (a), Syncytial cells (b), Epithelial cells necrosis (c),

epithelial cells hyperplasia (d), neutrophils (e), Bronchial nodules (f), and perivascular nodules (g) RSV-

NFI greatly reduce the RSV associated microscopic lesion in lung tissue. Animal infected with RSV and

treated with either vehicle only (0) or different concentration of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs

are not infected and treated with vehicle only. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. n.s.: not

significant. . $: on/off pattern indicating an obvious effect.

124

B a s e lin e 0 1 4 1 6

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

Re

lati

ve

IP

-10

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

**

*

B a s e lin e 0 1 4 1 6

0

5 0 0

1 0 0 0

1 5 0 0

Re

lati

ve

MC

P-1

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

**

*

B a s e lin e 0 1 4 1 6

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

Re

lati

ve

MIP

-1

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

**

*

B a s e lin e 0 1 4 1 6

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

Re

lati

ve

PD

L-1

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

**

*

B a s e lin e 0 1 4 1 6

0

1 0 0 0

2 0 0 0

3 0 0 0

21 0 0 6

41 0 0 6

61 0 0 6

Re

lati

ve

IF

N-

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

*

*

**

B a s e lin e 0 1 4 1 6

0

5 0

1 0 0

1 5 0

2 0 0

Re

lati

ve

IL

-13

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

n .s .

n .s .

B a s e lin e 0 1 4 1 6

0

5 0

1 0 0

1 5 0

Re

lati

ve

CC

10

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

n .s .

n .s .

B a s e lin e 0 1 4 1 6

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

Re

lati

ve

RA

NT

ES

ex

pre

ss

ion

(%

)

R S V -N F I (m g /k g )

n .s .

n .s .

**

a b c

d ef

g h

Figure 4-9: Effect of RSV-NFI inflammatory chemokine RNA expression in lung tissue.

Chemokine RNA expression IP-10 (a), MCP-1 (b), MIP-1α (c), PDL-1 (d), INF-λ (e), IL-13 (f), RANTES

(g). All are shown as average + SEM. Treatment with RSV-NFI greatly reduces IP-10, MCP-1, MIP-1α,

PDL-1, INF-λ. Animal infected with RSV and treated with either vehicle only (0) or different concentration

of RSV-NFI (1, 4, 16 mg/kg). Baseline lambs are not infected and treated with vehicle only. *p<0.05,

**p<0.01, ***p<0.001,****p<0.0001. n.s.: not significant.

125

CHAPTER 5. GENERAL CONCLUSION

Secondary Bacterial Co-Infection

The neonatal lamb model of RSV infection is well established, and many aspects of the

model were investigated in our laboratory such as different viral strains, different viral cultures,

different inoculation techniques, viral kinetics, immune response, and associated clinical signs [1–

6]. Investigating mechanisms of bacterial infections secondary to RSV infection is the next step to

understand the pathogenesis of bacterial coinfection and utilize the model to test the efficacy of

therapeutic agents in complex infections which are associated with enhanced disease severity and

death. This study demonstrated the susceptibility of lamb to Streptococcus pneumoniae (Spn)

infection, and the enhanced disease severity when lambs challenged with Spn 3 days after primary

RSV nebulization in comparison to lambs receiving either RSV or Spn alone. The titer of RSV

were higher in the RSV-Spn infected group than RSV alone, while there were no differences

between Spn infected lambs in Spn lung titer. There was an increase in severity of lung lesions in

the RSV-Spn group, which was characterized by a higher number of infiltrating neutrophils, and

increased epithelial necrosis and degeneration. These findings were in agreement other studies of

viral-bacterial co-infection, and demonstrated the enhanced infection by both pathogens.

Efficacy of Anti-RSV Fusion Inhibitor

The neonatal lamb model of RSV infection which mimics RSV infection in infants provides

an excellent tool to evaluate the efficacy of antiviral compound on the development of RSV-

associated acute lower respiratory tract infection. This study demonstrated the efficacy of JNJ-

49214698, which is an RSV small molecule fusion inhibitor that binds to the RSV prefusion

conformation preventing critical F protein transformation to postfusion conformation and

126

subsequently preventing multicyclic RSV infection. In three treatment regimen,JNJ-49214698

had a very protective effect when given prophylactically 1 day before RSV nebulization. These

lambs had no infectious RSV or mRNA in the lung, and had minimal clinical signs, and almost no

RSV associated lung lesion. Although prophylactic administration of JNJ-49214698 is not its

intended use, the finding demonstrates the ability of fusion inhibitor to prevent RSV infection.

When JNJ-49214698 given 1 day and 3 days post-RSV nebulization, it had significant decrease in

infectious RSV in the BALF with a less pronounced reduction in the mRNA. There were

reductions in RSV associated clinical signs, gross and microscopic lung lesion, and RSV protein

and mRNA expression in lung tissue. The effect of JNJ-49214698 was more evident when given

1 day after RSV nebulization. These data suggest a protective effect of JNJ-49214698 in reducing

RSV replication and subsequently RSV associated signs and lesions. Therefore, the compound is

a promising antiviral candidate against RSV infection in infants.

Efficacy of Anti-RSV Non-Fusion Inhibitor

Another mechanistic type of anti-RSV compounds are the non-fusion inhibitors, which bind

and inhibit viral structural protein other than F protein. In this study, we evaluated the efficacy of

the non-fusion inhibitor JNJ-64417184 in the neonatal lamb model of RSV infection. In this study,

3 different doses were used to treat lambs 1 day after RSV nebulization (16, 4, or 1 mg/kg body

weight). The study demonstrated that JNJ-64417184 markedly reduced all RSV associated lesions

in treated lambs in comparison to RSV infected vehicle-treated lambs. Additionally, the compound

significantly reduced infectious RSV titer and mRNA expression in BALF and lung tissue

homogenate and markedly reduced RSV antigen and mRNA expression in lung tissue sections.

These results were accompanied by a reduction in immunomodulatory cytokines IP-10, MCP-1,

127

MIP-1α, PDL-1, and IFN-λ, and correlated with the reduction of inflammatory cells in the lung

tissue observed in histological evaluation. These results indicate the ability of JNJ-64417184 to

reduce RSV replication and disease progress.

Future Directions

The neonatal lamb model of RSV infection in infants is a well-characterized model and

currently used to evaluate anti-RSV therapeutics[7,8]. We demonstrated that neonatal lambs

infected with RSV are also susceptible for infection with Spn and lead to enhance disease severity

in lambs infected with both RSV-Spn. Several aspects of this model can be modified and further

developed in future studies of viral-bacterial co-infection such as: expanding the length of time of

infection, assessing different inoculation time points of Spn following RSV, assessing different

doses of Spn, and different types of Spn delivery (e.g., intranasal, intrabronchial, aerosolization).

Also regarding Spn, a colonization model similar to human and mice could be tested to evaluate

whether such mechanism of infection is a possibility in the lamb model. A third consideration is

to try assess other bacterial pathogens or combinations of:Staphylococcus aureus, Streptococcus

pyogenes, and Haemophilus influenza. These three bacterial pathogens are the most common

bacteria identified secondarily to viral infections in human.

Several characteristics make the neonatal lamb model an interest and compelling option for

pharmaceutical companies to assess their compounds. Although our laboratory has inoculated

lambs by various routes including intra-tracheal, bronchoscopy guided intra-bronchial inoculation,

nebulization of the virus has several advantages [2,5,6]. RSV nebulization provides more

homogenous distribution of the virus to different lung lobes and subsequently homogenized lung

pathology[5,9]. RSV nebulization is one feature of the neonatal lamb model that provides

consistency for RSV associated lower respiratory tract infection and does overwhelm areas of RSV

128

infection as can occur with intrabronchial deposition. We demonstrated that two antiviral

compounds which target fusion protein (entry of the virus) and non-fusion protein (replication of

the virus) had a strong effect in reducing RSV replication and associated pathology. In this same

fashion, other viral targets could be tested in the lamb model such as RSV N, SH, and L proteins.

Other potential studies could include a combination of compound that target 2 or more RSV protein

and different time points for treatment administration. However, treating within 2 days post viral

inoculation may be ideal because this likely is the time an infant may seek medical

intervention[10].

References

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Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV. PLOS ONE. 2013;

8(12):e81472.

2. Larios Mora A, Detalle L, Van Geelen A, et al. Kinetics of Respiratory Syncytial Virus

(RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs

as an RSV Infection Model for Human Infants. PLoS ONE [Internet]. 2015 [cited 2016

Jan 7]; 10(12). Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671688/

3. Meyerholz DK, Grubor B, Fach SJ, et al. Reduced clearance of respiratory syncytial virus

infection in a preterm lamb model. Microbes Infect. 2004; 6(14):1312–1319.

4. Sow FB, Gallup JM, Olivier A, et al. Respiratory syncytial virus is associated with an

inflammatory response in lungs and architectural remodeling of lung-draining lymph

nodes of newborn lambs. Am J Physiol-Lung Cell Mol Physiol. 2010; 300(1):L12–L24.

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5. Derscheid RJ, Geelen A van, McGill JL, et al. Human Respiratory Syncytial Virus

Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus

Grown in Vero Cells. Viruses. 2013; 5(11):2881–2897.

6. Olivier A, Gallup J, De Macedo MMMA, Varga SM, Ackermann M. Human respiratory

syncytial virus A2 strain replicates and induces innate immune responses by respiratory

epithelia of neonatal lambs. Int J Exp Pathol. 2009; 90(4):431–438.

7. Detalle L, Larios A, Gallup L, Van Geelen A, Duprez L, Stohr T. Delivery of ALX-0171

by inhalation greatly reduces disease burden in a neonatal lamb RSV infection model. 9th

Int Respir Syncytial Virus Symp Cape Town South Afr Abstr OP 72. 2014.

8. Olivier AK, Gallup JM, Geelen A van, Ackermann MR. Exogenous administration of

vascular endothelial growth factor prior to human respiratory syncytial virus a2 infection

reduces pulmonary pathology in neonatal lambs and alters epithelial innate immune

responses. Exp Lung Res. 2011; 37(3):131–143.

9. Grosz DD, Geelen A van, Gallup JM, Hostetter SJ, Derscheid RJ, Ackermann MR.

Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and

experimental infection. BMC Res Notes. 2014; 7:158.

10. Saleeby E, M C, Bush AJ, Harrison LM, Aitken JA, DeVincenzo JP. Respiratory

Syncytial Virus Load, Viral Dynamics, and Disease Severity in Previously Healthy

Naturally Infected Children. J Infect Dis. 2011; 204(7):996–1002.