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Comparative efficacy of feline leukemia virus inactivated whole virus vaccine and canarypox 1

virus-vectored vaccine during virulent FeLV challenge and immunosuppression 2

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M. Patela, K. Carritta, J. Lanea, H. Jayappaa, M. Stahlb, M. Bourgeoisb# 4

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Merck Animal Health, Elkhorn, NEa, Merck Animal Health, Madison, NJb 6

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Running Head: Comparative efficacy killed and vectored FeLV vaccine 8

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#Address correspondence to Melissa Bourgeois, [email protected] 10

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CVI Accepted Manuscript Posted Online 13 May 2015Clin. Vaccine Immunol. doi:10.1128/CVI.00034-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 22

Four vaccines for feline leukemia virus (FeLV) are available in the United States. This 23

study’s purpose was to compare efficacy of Nobivac® Feline 2-FeLV (inactivated, adjuvanted 24

whole virus vaccine) and PureVax® Recombinant FeLV (live canarypox virus-vectored vaccine) 25

following FeLV challenge. Cats were vaccinated at 9 and 12 weeks with Nobivac® Feline 2-FeLV 26

(Group A, n =11) or PureVax® Recombinant FeLV (Group B, n=10). Group C (n=11) were 27

unvaccinated controls. Three months post-vaccination, cats were immunosuppressed and 28

challenged with FeLV-A/61E. Outcome was monitored 12 weeks post-challenge (PC) for 29

persistent antigenemia and 3-9 weeks PC for proviral DNA and viral RNA. 30

Persistent antigenemia was observed in 0/11 Group A, 5/10 Group B, and 10/11 Group C 31

cats. Group A was significantly protected compared to B (P <0.013) and C (P <0.0001). No 32

difference was found between Group B and C (P >0.063). Preventable fraction was 100% for 33

Group A and 45% for B. Nine weeks PC, proviral DNA and viral RNA were detected 1/11 Group 34

A, 6/10 Group B, and 9/11 Group C cats. Nucleic acid loads were significantly lower in Group A 35

than C (P <0.01). Group A had significantly lower proviral DNA loads than B weeks 6-9 (P <0.02). 36

Viral RNA loads were significantly lower in Group A than B weeks 7-9 (P <0.01). 37

The results demonstrate Nobivac® Feline 2-FeLV vaccinated cats were fully protected 38

against persistent antigenemia and had significantly lower amounts of proviral DNA and plasma 39

viral RNA loads than PureVax® Recombinant FeLV vaccinated cats and unvaccinated controls. 40

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INTRODUCTION 42


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Feline leukemia virus (FeLV) is a retroviral infection of cats that is transmitted mainly 43

through saliva, although other body fluids can transmit the virus as well1,2. Infected cats 44

demonstrate a wide range of clinical signs, including cytoproliferative disorders (lymphoid or 45

myeloid tumors), cytosuppressive disorders (infectious diseases associated with 46

immunosuppression, anemia, myelosuppression), inflammatory disorders, neurological 47

disorders, abortions, enteritis, and more3,4 . 48

The outcome of FeLV exposure is dependent on a variety of factors, including host 49

immune status, host age, viral strain, viral load, and exposure route. Previous classifications, 50

including the classifications of persistent antigenemia, transient antigenemia, and elimination 51

of infection were mainly defined by tests for antigenemia (p27 ELISA), virus isolation, and 52

immunofluorescence assays3,5. Tests for antigenemia are highly useful in clinical applications, 53

and in determining whether the clinical disease is a result of actively circulating virus. 54

The use of polymerase chain reaction (PCR) testing for FeLV infection has altered the 55

understanding of the pathophysiology and clinical course of FeLV infection6,7,8,9. PCR can detect 56

low levels of viral RNA circulating in the blood stream, as well as low levels of proviral DNA 57

integrated into the cat’s genome, resulting in more sensitive assays for FeLV status. Because of 58

PCR testing, it is now generally accepted that there are three major outcomes for cats that are 59

exposed to FeLV: progressive, regressive, and abortive infection2,6,10. 60

Progressive infection is characterized by an inadequate immune response to FeLV 61

infection. In these cats, the virus will actively replicate and circulate in blood, bone marrow, 62

and tissues. FeLV is spread in the saliva of these cats and they typically succumb to FeLV 63


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infection within years. These cats will test positive for FeLV antigenemia (p27 antigen) by ELISA, 64

as well as viral RNA and proviral DNA by PCR2,6,10. 65

Regressive infection is characterized by an effective immune response, with viral 66

containment occurring shortly after infection. There may be transient antigenemia as tested by 67

p27 enzyme-linked immunosorbent assay (ELISA). These cats do test positive for proviral DNA, 68

and may test transiently or persistently positive for viral RNA. These cats are at little risk for 69

succumbing to FeLV associated disease and do not spread the virus2. In fact, some of these cats 70

that have low concentrations of proviral DNA with little to no circulating viral RNA may 71

completely clear the infection with time, resembling an abortive infection6. However, there 72

may be an association between persistently high proviral DNA and viral RNA concentrations 73

with reactivation of infection6,7,11,12. 74

Cats that undergo abortive infection are able to completely clear the virus, and will test 75

negative for p27 antigen, viral RNA, and proviral DNA. To-date, there are no large scale 76

prevalence studies in North America based on PCR results and the classification of cats as 77

undergoing regressive, progressive, abortive infection. All current prevalence studies are based 78

on the detection of persistent antigenemia by p27 ELISA. The most recent, large scale 79

seroprevalence study conducted in the United States demonstrated that 2.3% of 18,000 cats 80

tested seropositive for FeLV13. Based on this and previous studies, the rates of FeLV infection 81

appear to be decreasing, most likely due to effective vaccination protocols13,14. However, 82

different vaccines have been shown to have varying degrees of efficacy15. To demonstrate true 83

efficacy, vaccines should be tested by challenge model, and FeLV testing using at least p27 84


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ELISA, PCR for viral RNA, and PCR for proviral DNA conducted. PCR testing is of paramount 85

importance in determining the true FeLV status in challenged or vaccinated and challenged 86

cats. 87

Four vaccines for FeLV are available in the United States, including two whole virus, 88

adjuvanted killed vaccines; a dual adjuvanted, multiple antigen vaccine; and a nonadjuvanted, 89

canarypox virus-vectored vaccine. Previous work has demonstrated the efficacy of whole virus, 90

adjuvanted killed vaccines after challenge, including testing vaccinated and unvaccinated cats 91

for viral RNA, proviral DNA, FeLV antibodies, and p27 antigen16,17,18. There is limited data 92

evaluating the efficacy of the nonadjuvanted recombinant FeLV vaccine available for use19. 93

Therefore, the purpose of this study was to compare the efficacy of two commercially available 94

feline leukemia vaccines, Nobivac® Feline 2-FeLV (inactivated whole virus vaccine) and PureVax® 95

Recombinant FeLV (live canarypox virus-vectored vaccine) following challenge with virulent 96

feline leukemia virus. 97

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MATERIALS AND METHODS 99

Study Animals. Thirty-two 8 week old cats were enrolled in the study. All cats tested 100

negative for FeLV infection by p27 ELISA assay kit (optical density <0.200, IDEXX, Westbrook, 101

ME) prior to vaccination, booster, and challenge. Cats were housed separately during the 102

vaccination phase of the study. During the challenge phase, placebo cats were randomly 103

divided and housed with each of the vaccinated groups. All cats were housed to meet the 9CFR 104

USDA Animal Welfare Regulations (Chapter 1) and Institutional Animal Care and Use Committee 105


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(IACUC) guidelines. Cats were observed daily throughout the course of the study and were 106

euthanized if they became unwell or at the end of the study. All male cats were castrated at 21 107

weeks of age according to standard veterinary procedures. 108

Immunization. Vaccine was administered subcutaneously to cats at 9 and 12 weeks of 109

age with Nobivac® Feline 2-FeLV vaccine (Group A, n =11) or PureVax® Recombinant FeLV 110

vaccine (Group B, n=10), three weeks apart per manufacturer’s label. Group C (n=11) served as 111

age-matched, unvaccinated controls. Injection site and systemic reactions were noted (if 112

present) 4-8 hours after vaccination, daily for 2 days following vaccination, and 1-2 times per 113

week thereafter until challenge. Nobivac® Feline 2-FeLV vaccine is a whole virus, adjuvanted 114

killed vaccine that contains FeLV Subtype A/Rickard strain. PureVax® Recombinant FeLV is a a 115

nonadjuvanted, canarypox virus-vectored vaccine that contains the mutated env, gag, and part 116

of pol proteins of the FeLV Subtype A/Glasgow-1 strain. 117

Challenge. Viral challenge occurred 3 months after second vaccination. Blood was 118

collected for PCR testing prior to challenge. During the entire challenge phase of the study, the 119

placebo vaccinated cats were randomly divided and co-mingled with the commercially 120

vaccinated groups. Commercially vaccinated groups remained separated from each other. On 121

the day of challenge, all cats were administered 10mg/kg of methylprednisolone acetate (MPA) 122

intra-muscularly > 4 hours pre-challenge. Three mL of feline leukemia virus (105.5 PFU/mL), 123

strain 61E (subtype A) grown on NCE-F161 cells, was administered via oronasal route. Pre- and 124

post- challenge samples of the virus were back titrated to check concentration on Clone 81 125

cells. One week post challenge all cats were administered 10mg/kg of methylprednisolone 126


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acetate (MPA) intra-muscularly. From alignment and analysis of amino acid sequences of 127

vaccine and challenge viruses, this is considered a heterologous challenge. 128

Sample Collection and Monitoring. Cats were observed daily for clinical signs of illness. 129

Blood samples were collected weekly in ACD tubes for 3-10 weeks post challenge, in EDTA 130

tubes for 3-9 weeks post challenge, and in SST tubes on weeks 11 and 12 post challenge. Blood 131

from SST tubes was allowed to clot at room temperature and centrifuged to obtain serum. 132

Serum and ACD blood was tested for antigenemia by p27 ELISA (PetCheck™, IDEXX, Portland, 133

OR; performed by Merck Animal Health; Elkhorn, NE). Blood from EDTA tubes was tested by 134

qPCR for viral RNA and proviral DNA (Zoologix Chatsworth, CA). All personnel testing 135

laboratory samples and performing clinical observations were blinded as to the treatment 136

groups. 137

ELISA to Detect Antibodies to FeLV. FeLV antibody detection on serum samples was 138

performed by indirect ELISA. Briefly, plates were coated with capture antigen (1:1,000 dilution, 139

swine pox virus expressing FeLV gp70 glycoprotein). Plates were washed once with PBST and 140

incubated with blocking buffer (5% fish gelatin) for 90 minutes at 36oC. Plates were washed 141

once with PBST and incubated with the positive control, negative control, and test samples 142

(diluted 1:250) in triplicate at 36oC for 90 minutes. Plates were washed 4 times with PBST and 143

incubated with goat anti-cat IgG (H+L) peroxidase conjugate (Kirkegaard and Perry Laboratories 144

Inc) 1:10,000 dilution at 36oC for 90 minutes. Plates were washed 4 times with PBST and TMB 145

substrate (Kirkegaard and Perry Laboratories Inc) was added to each well. The plates were 146

incubated at 36oC for 20 minutes. 100 μL of 1.5M phosphoric acid was added to the wells to 147


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stop the reaction. The absorbance of each well was measured at 650 nm and the mean OD of 148

the blank wells subtracted from the controls and samples. The status of a sample was evaluated 149

by the sample to positive ratio (S/P ratio). Sample to positive ratio was calculates as follows: 150

S/P ratio = [(Sample O.D. – negative control O.D) / (positive control O.D. – negative control 151

O.D.)]. 152

ELISA to Detect FeLV p27 Antigen. FeLV p27 antigen testing on serum samples was 153

performed by ELISA (PetCheck™, IDEXX, Portland, OR). Briefly, 50 µL of serum or plasma was 154

added to anti-p27 antigen coated wells and then 50 µL horseradish peroxidase conjugate was 155

added to each well. Plates were incubated for 5 minutes at 15 - 30° C and then washed 5 times 156

with wash buffer. TMB substrate was then added, 100 µL per well, and then the plate 157

incubated 5 minutes at 15 - 30° C. Following incubation, 50 µL stop solution was then added to 158

each well and plates read visually and absorbance measured. Development of distinct blue 159

color was considered positive for FeLV p27 antigen for visual measurement. Absorbance of 160

each well was also measured at 650 nm and samples were considered positive if OD >0.200. 161

qRT-PCR to Detect FeLV Viral RNA and qPCR to Detect FeLV DNA. FeLV proviral DNA 162

and plasma viral RNA loads were quantified using real time PCR and reverse transcription, real 163

time PCR, respectively. For DNA extraction, 200 ul of EDTA whole blood was extracted using 164

the Qiagen Blood DNA mini kit (Qiagen, Valencia, CA). DNA was eluted in TE buffer. For RNA 165

extraction, approximately 600 ul of EDTA whole blood was spun down to separate out the 166

plasma from the cell layer. 140 ul of plasma was used for RNA extraction using the Qiagen Viral 167

RNA extraction kit (Qiagen, Valencia, CA). RNA was eluted in sterile water. 168


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Proviral DNA was quantitated by real-time PCR (Zoologix Inc., Chatsworth, CA). The limit of 169

detection was approximately 400 copies of DNA/ 1 mL. Plasma viral RNA was quantitated by 170

real-time RT-PCR in a one step reaction (Zoologix Inc., Chatsworth, CA). The limit of detection 171

was approximately 1000 copies of RNA/ 1 mL. . Primers and TaqMan probes were the same as 172

used in previous studies and methodology11,20. Primers and probes were directed against the 173

unique region (U3) of the long terminal repeat (LTR) of FeLV types A, B, and C. Endogenous 174

FeLV sequences are not detected based on this primer selection. 175

The viral loads of all samples were determined by comparing the samples’ threshold 176

cycle (CT) with the co-amplified standard curve. Both RNA and DNA standards were cloned U3 177

regions of FeLV subtype A/Glasgow-1, as per previous studies and methodology11,20. Ten-fold 178

dilutions from 109 to 10-2 copies/5μL were used to generate the standard curve. 179

Necropsy. Three cats were euthanized during the challenge phase of the study due to 180

adverse reactions to MPA administration (weight loss, dehydration, and lethargy). One cat did 181

not recover from anesthesia during the course of the study shortly after blood collection. 182

Necropsy was performed on all of these cats at the Cornell University Animal Health Diagnostic 183

Center. The remaining cats were euthanized at the conclusion of the study. 184

In Vivo Infectivity Analysis. Following challenge, blood was collected from persistently 185

viremic control cats. PBMCs were isolated and stimulated for 4 days with Con-A and then co-186

cultured with CRFK cells. The supernatant was harvested and stored in liquid nitrogen until use. 187

Five non-vaccinated control cats were then challenged oronasally with 105.7 PFU/cat for two 188

consecutive days. Prior to ≥4 hours pre-challenge and one week post challenge, cats were 189


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administered 10mg/kg of methylprednisolone acetate (MPA) intra-muscularly. Persistent 190

viremia was measured by ELISA to detect p27 antigen (see section 2.6). 191

Data Analysis. Persistent FeLV antigenemia was defined as the presence of FeLV p27 192

antigen in serum or plasma, detected by ELISA, for three consecutive weeks between the third 193

and twelfth week post-challenge or, five or more occasions, consecutive or not. The incidence 194

of FeLV persistent antigenemia was compared between the vaccinated groups and between the 195

controls and vaccinated groups using Fisher’s Exact Test in SAS 9.3, where P-values <0.05 means 196

significant difference. Further statistical analysis for each week was performed between the 197

vaccinated groups and between the controls and vaccinated groups using the Wilcoxon Exact 198

Rank Sum Test (Mann-Whitney U test). Statistical significance was set at p-value<0.05. 199

Statistical analysis using the Wilcoxon Exact Rank Sum Test was performed between the 200

vaccinated groups and the vaccinated groups and controls (p<0.05). 201

Plasma viral RNA (viremia) and proviral DNA were analyzed by qRT-PCR or qPCR, 202

respectively. The log10 DNA titer and the log10 RNA titer were analyzed separately by Wilcoxon 203

Exact Rank Sum Test (also called the Mann–Whitney U test ) between the vaccinated groups 204

and between the controls and vaccinated groups. Statistical significance was set at p-205

value<0.05. Additionally, the percentage of positive results (DNA or RNA titer>0) between 206

vaccinated groups and between the controls and vaccinated groups was analyzed by Fisher’s 207

Exact Test. Statistical significance was set at p-value<0.05. Prevented fraction to determine 208

vaccine efficacy was calculated as follows: 209


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1 – [(Incidence of persistent antigenemia in vaccinates)/(Incidence of persistent antigenemia in 210

controls)] 211

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RESULTS 213

Antibodies to FeLV Post Vaccination and Pre Challenge. Antibodies (IgG) to FeLV were 214

detected using indirect ELISA. All cats were negative for FeLV antibody on study day 0, prior to 215

vaccination. Ten of eleven cats in the Nobivac® Feline 2-FeLV group were positive for FeLV 216

antibody on study days 20 (S/P ratio =3.0544 +/-1.007) and 111 (S/P ratio= 1.002 +/-0.48). One 217

cat was not tested on study day 20 due to low serum sample volume. All cats in the PureVax® 218

Recombinant FeLV group as well as the control group remained negative for FeLV antibody 219

through the entire vaccination phase. Levels of IgG antibody in Nobivac® Feline 2-FeLV were 220

significantly lower at days 20 and 111 compared to Purevax® Recombinant FeLV (p=0.00) and 221

controls (p=0.00) (Figure 1). 222

FeLV p27 Antigen (Persistent Antigenemia) and Vaccine Efficacy. FeLV persistent 223

antigenemia was determined by p27 ELISA to detect circulating viral antigen in blood. 224

Development of distinct blue color combined with an OD reading >0.200 was considered 225

positive. Following challenge, ten of eleven (91%) control cats developed persistent 226

antigenemia (average challenge phase OD=0.726). No cats (0/11) in the Nobivac® Feline 2-FeLV 227

group became persistently viremic (average challenge phase OD=0.047). In comparison, 5 of 10 228

cats (50%) vaccinated with Purevax® Recombinant FeLV became persistently FeLV viremic 229

(average OD=0.445) (Figures 2&3). 230


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Nobivac® Feline 2-FeLV demonstrated vaccine efficacy of 100% (prevented fraction), 231

compared to the control group. The prevented fraction for the PureVax® Recombinant FeLV 232

group was calculated at 45% compared to the control group. When compared to both the 233

control group and the PureVax® Recombinant FeLV group, the protection conferred by Nobivac® 234

Feline 2-FeLV was significantly higher (p <0.0001 and p = 0.0124, respectively). There was no 235

significant difference between the PureVax® Recombinant FeLV group and the control group (p 236

= 0.0635) (Table 1). 237

Persistent antigenemia data was statistically analyzed using the Wilcoxon Exact Rank 238

Sum Test. Groups were compared on day -1 (pre-challenge), then weekly post challenge from 239

weeks 3-12. Higher levels of p27 antigen were seen in the Purevax® Recombinant FeLV 240

vaccinates and controls compared to the Nobivac® Feline 2-FeLV vaccinates at all time points. 241

Statistically significant differences were seen between the Nobivac® Feline 2-FeLV and PureVax® 242

Recombinant FeLV groups at weeks 3, 4, 6 to 9 and 11 post challenge (p<0.01). Statistically 243

significant differences were seen between the Nobivac® Feline 2-FeLV and control groups at all 244

time points (p<0.01). No statistically significant differences were seen between PureVax® 245

Recombinant FeLV groups and control groups at any time point (p<0.01) (Figure 4). 246

Plasma Viral RNA. Circulating plasma viral RNA load was determined by real-time RT-247

PCR assay from 3 to 9 weeks post challenge to determine whether FeLV vaccination would 248

prevent circulating viral nucleic acid persistence (active replication). At the end of the 249

challenge, plasma viral RNA was detected in 1 of 11 (9%) of the Nobivac® Feline 2-FeLV 250

vaccinated cats, 6 of 10 (60%) of the PureVax® Recombinant FeLV vaccinated cats and 9 of 11 251


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(82%) of the unvaccinated control cats. Viral RNA concentrations were low for the Nobivac® 252

Feline 2-FeLV vaccinated group at < 1x10^6 copies/mL at all time points except for week 3 253

(median 1.5x10^5 copies RNA/mL, range 3.3x10^3 to 3.7x10^9 copies RNA/mL). In contrast, 5 254

of the 9 PureVax® Recombinant FeLV vaccinated cats had plasma viral RNA concentrations 255

exceeding 1x10^6 copies/mL for the majority of the time points that they were positive 256

(median 1.0x10^8 copies RNA/mL, range 5.3x10^3 to 1.7x10^11 copies RNA/mL). Ten of 11 257

control cats tested positive for plasma viral RNA at concentrations exceeding 1x10^6 copies/mL 258

for the majority of testing (median 6.5x10^8 copies RNA/mL, range5.9x10^7 copies RNA/mL to 259

4.7x10^11 copies RNA/mL). Concentrations of viral RNA between the groups were compared 260

using the Wilcoxon Exact Rank Sum Test. Plasma viral RNA loads were significantly lower in the 261

Nobivac® Feline 2-FeLV group than the control group at all time points (p < 0.01). Plasma viral 262

RNA loads were significantly lower in the Nobivac® Feline 2-FeLV group than the PureVax® 263

Recombinant FeLV group from week 7 to 9 (p < 0.01). There was a strong association seen 264

between plasma viral RNA loads and persistent antigenemia (Figures 2&5). 265

Plasma Proviral DNA. Circulating proviral DNA loads were determined by quantitative 266

PCR assay from 3 to 9 weeks post challenge to determine whether FeLV vaccination would 267

prevent nucleic acid persistence. At the end of the challenge, proviral DNA was detected in 1 of 268

11 (9%) of the Nobivac® Feline 2-FeLV vaccinated cats, 6 of 10 (60%) of the PureVax® 269

Recombinant FeLV vaccinated cats and 9 of 11 (82%) of the unvaccinated control cats. All cats 270

in the Nobivac® Feline 2-FeLV vaccinated group had proviral DNA concentrations below 1x10^6 271

copies/mL except one (median 2.2x10^5 copies proviral DNA/mL, range 1.5x10^3 to 1x10^9 272

copies proviral DNA/mL). In contrast, 5 of the 10 proviral DNA positive PureVax® Recombinant 273


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FeLV vaccinated cats had proviral concentrations >1x10^6 for the majority of the study (median 274

2.1x10^9 copies proviral DNA/mL, range 8.6x10^4 to 4.1x10^11 copies proviral DNA/mL). Ten 275

of 11 control cats tested positive for proviral DNA at concentrations exceeding 1x10^6 276

copies/mL for the majority of testing (median 1.7x10^10 copies proviral DNA/mL, range 277

8.3x10^5 to 1.8x10^12 copies proviral DNA/mL). Concentrations of proviral DNA between the 278

groups were compared using the Wilcoxon Exact Rank Sum Test. Proviral DNA loads were 279

significantly lower in the Nobivac® Feline 2-FeLV group than the control group (p < 0.01). In 280

addition, the Nobivac® Feline 2-FeLV group had significantly lower proviral DNA loads than the 281

PureVax® Recombinant FeLV group from weeks 6 to 9 ( p < 0.02). Proviral DNA loads were 282

strongly associated with the persistently viremic cats (Figures 2&5). 283

Adverse Events and Clinical Disease. One cat (Group B PureVax® Recombinant FeLV) 284

did not recover from anesthesia following the week 7 blood collection. Additionally, three cats 285

(two Group B PureVax® Recombinant FeLV, one Group C Control group) were euthanized during 286

the course of the study due to weight loss, dehydration and lethargy, secondary to MPA 287

administration. No fever or clinical signs were observed in any of the vaccinated or control 288

groups due to FeLV infection. 289

In Vivo infectivity Analysis. Four out of the 5 naïve control cats (80%) demonstrated 290

persistent antigenemia as measured by p27 ELISA for the infectivity analysis. 291

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DISCUSSION 293


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This study reinforced previous work demonstrating the efficacy of killed, whole virus 294

adjuvanted vaccines in protecting against feline leukemia virus challenge16,17,18. In this study, 295

FeLV p27 antigen could not be detected in any of the cats vaccinated with Nobivac® Feline 2-296

FeLV, a killed whole virus adjuvanted FeLV vaccine at any point in the 12 weeks post challenge. 297

The prevented fraction calculated for the Nobivac® Feline 2-FeLV vaccine was 100%. This is 298

consistent with prevented fractions in previous studies using this vaccine ranging from 90-299

100%16,17,18. In contrast, 5 out of 10 (50%) of cats that were vaccinated with the nonadjuvanted 300

live canary-pox vectored vaccine (PureVax® Recombinant FeLV) tested positive for FeLV p27 301

antigen. The prevented fraction calculated for the PureVax® Recombinant FeLV vaccine was 302

45%. This prevented fraction was higher than that seen in a previous report with a 303

recombinant, canary-pox virus vectored vaccine at 20%19. Ten of 11 (91%) control cats were 304

positive for FeLV p27 antigen, demonstrating a strong viral challenge. Significantly higher levels 305

of p27 antigen were measured at weeks 3, 4, 6 to 9 and 11 post challenge (p<0.01) in the 306

PureVax® Recombinant FeLV vaccinated cats compared to the Nobivac® Feline 2-FeLV 307

vaccinated cats. Furthermore, it was found that vaccination with the Nobivac® Feline 2-FeLV 308

vaccine produced detectable levels of IgG directed against FeLV pre-challenge. Ten of eleven 309

cats in this group tested positive for FeLV-specific antibodies at day 20 and day 111. No 310

antibodies could be detected in either the PureVax® Recombinant FeLV vaccinated or control 311

groups, and this was statistically significant (p=0.00). 312

These results support the finding that vaccination with killed, whole virus adjuvanted 313

vaccines can help to prevent persistent antigenemia. Clinically, this is highly relevant based on 314

the availability of in-clinic diagnostic tests relying on the presence of viral antigen. Vaccination 315


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with the nonadjuvanted live canary-pox vectored vaccine did not confer the same level of 316

protection, based on persistent antigenemia and immunoglobulin testing. In this study it 317

appears that vaccination with killed, whole virus adjuvanted Nobivac® Feline 2-FeLV vaccine can 318

help to prevent active viral replication after severe challenge. 319

Both the vaccines and the challenge virus contained FeLV subtype A. Only FeLV subtype 320

A is considered infectious (other subtypes arise from mutation within the cat after infection)21. 321

Nobivac® Feline 2-FeLV vaccine is a whole virus, adjuvanted killed vaccine that contains FeLV 322

Subtype A/Rickard strain. PureVax® Recombinant FeLV is a nonadjuvanted, canarypox virus-323

vectored vaccine that contains the pol and env proteins of the FeLV Subtype A/Glasgow-1 324

strain. The envelope proteins of Nobivac® Feline 2-FeLV and Purevax® Recombinant FeLV 325

contain amino acids that differ significantly from the envelope protein of the challenge strain 326

FeLV Subtype A/61E (data not shown). Thus the challenge for both vaccines would be 327

considered a heterologous challenge. 328

The requirement for virus neutralizing antibodies for protection from FeLV is 329

controversial. The passive transfer of virus specific antibodies has been shown to provide 330

protection against disease after FeLV exposure22. Other studies have shown the development 331

of virus neutralizing antibodies after FeLV exposure may be important in long-term FeLV 332

challenge outcomes. In one study, high titers of virus neutralizing antibodies developed in 333

challenged cats that were able to overcome infection, but not in challenged cats that became 334

persistently viremic23. In this same study, the development of cytotoxic T lymphocytes 335

occurred before virus neutralizing antibodies, and the adoptive transfer of these CTLs was able 336


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to lower the FeLV viremic load23. This lends support to the fact that other immune mechanisms 337

may be involved in protection from FeLV. This is highlighted in a previous study in which 338

vaccination with a DNA construct vaccine containing env/gag/pol and a gene adjuvant 339

containing IL-12 and IL-18 conferred protection against FeLV challenge without the 340

development of virus neutralizing antibodies until after challenge24. Thus the development of 341

detectable levels of IgG seen in this study with Nobivac® Feline 2-FeLV pre-challenge may or 342

may not have significance when compared to the PureVax® Recombinant FeLV vaccinated or 343

control groups which did not develop detectable levels of IgG. The IgG ELISA data should be 344

evaluated in combination with the PCR data, persistent antigenemia data, and challenge 345

outcome to compare efficacies of the vaccines. 346

The limit of detection for both viral RNA and proviral DNA was similar at 1000 copies/mL 347

and 400 copies/mL in this study to other studies. This includes studies detecting the proviral 348

DNA load from 5 copies/DNA qPCR reaction and 5 proviral copies/10^6 cells to 1150 RNA 349

copies/mL and 2250 viral RNA copies/ mL plasma8,18,28. Caution should always be taken when 350

comparing results across different studies, as different PCR techniques may be employed at 351

different facilities. However, based on the limits of detection of the assays used in this study, it 352

can be concluded that the sensitivity of this assay was high, consistent with other PCR assays 353

for FeLV virus detection, and even very low limits of viral integration and replication were 354

detected. 355

Previous studies have shown that vaccination does not confer sterilizing immunity, and 356

no vaccine to date has been shown to completely prevent proviral DNA integration and plasma 357


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viral RNA circulation after challenge. However, the concentration of viral RNA and proviral 358

DNA, the duration of active infection as detected by PCR, and the degree and length of 359

persistent antigenemia are all factors that may be used in predicting the outcome of viral 360

infection and likelihood of viral reactivation. In a study examining both naturally and 361

experimentally infected cats, cats that were p27 antigen positive and progressively infected had 362

significantly higher proviral DNA loads than cats that were p27 antigen negative and 363

regressively infected. In the naturally infected cats, the mean proviral load in the regressively 364

infected cats was 300 times lower than the progressively infected cats11. This is reflected in 365

another study, in which cats that recovered from FeLV infection had lower proviral DNA 366

burdens compared to cats that were persistently infected23. 367

These results are mirrored when plasma viral RNA loads are examined. In multiple 368

studies looking at infection outcome (recrudescence) and viral RNA loads, higher viral RNA 369

concentrations were significantly associated with viral reactivation, even more so if the cats 370

were persistently viremic7,12. One study demonstrated differences in viral RNA concentration 371

and infection outcome and classified the cats as regressively infected without antigenemia 372

(median 1.8×10^3 copies/ml plasma), regressively infected with antigenemia (median 8.4×10^4 373

copies/ml plasma), or progressively infected (median 4.7×10^7 copies/ml plasma), and 374

correlated these outcomes to long-term (12 year) survival (worse survival rates were seen in 375

cats with higher concentrations of virus and antigenemia)12. Similarly, cats that test antigen 376

negative but remain plasma viral RNA positive may be at higher risk of FeLV reactivation than 377

cats that become viral RNA negative8. This positive to negative viral RNA status may be a result 378

of the virus replicating initially in peripheral immune cells (such as monocytes and lymphocytes) 379


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before being contained by an effective, vaccine induced immune response. Thus it can be 380

supposed that while vaccination may not confer sterilizing immunity, viral RNA and proviral 381

DNA concentrations as well as persistent antigenemia status should be examined when trying 382

to determine the degree of protection that a vaccine offers from infection. 383

Throughout this study, Nobivac® Feline 2-FeLV vaccinated cats had significantly lower 384

proviral DNA and plasma viral RNA loads, and were positive for much shorter periods of time, 385

than the PureVax® Recombinant FeLV vaccinated cats. In addition, no Nobivac® Feline 2-FeLV 386

vaccinated cat ever tested positive for persistent antigenemia, while half of the PureVax® 387

Recombinant FeLV vaccinated cats were persistently viremic. A median concentration of 388

1.5x10^5 copies RNA/mL and 2.2x10^5 copies proviral DNA/mL was seen in the Nobivac® Feline 389

2-FeLV vaccinated cats (with all time points < 1x10^6 copies/mL except for week 3). A median 390

concentration of 1.0x10^8 copies RNA/mL and 1.7x10^10 copies proviral DNA/mL was seen in 391

the PureVax® Recombinant FeLV vaccinated cats (with all cats >1x10^6 copies/mL for the 392

majority of the time points). In the Nobivac® Feline 2-FeLV group, while 7 of 11 cats tested 393

positive for circulating plasma viral RNA and/or proviral DNA at least once in the 3 to 9 weeks 394

post challenge, only 1 of these cats remained positive for both plasma viral RNA and proviral 395

DNA at the conclusion of testing (9 weeks). In contrast, 9 of 10 PureVax® Recombinant FeLV 396

vaccinated cats tested positive for circulating plasma viral RNA and/or proviral DNA at least 397

once in the 3 to 9 weeks post challenge, with 6 of these cats remaining positive for both viral 398

RNA and proviral DNA at the conclusion of the study. Ten of 11 control cats tested positive for 399

both proviral DNA and viral RNA, at concentrations exceeding 1x10^6 copies/mL for the 400


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majority of testing (median 6.5x10^8 copies RNA/mL, median 1.7x10^10 copies proviral 401

DNA/mL). 402

From this data, it appears that Nobivac® Feline 2-FeLV vaccinated cats either 403

experienced abortive infection (in 4 cats that never tested PCR positive), or regressive infection 404

that was contained by an effective, vaccine-induced immune response (7 cats that had low viral 405

nucleic acid concentrations but were never viremic). Although long-term outcomes were not 406

examined, these cats with low PCR loads and no persistent antigenemia might be less likely to 407

reactivate infection, and may even eventually clear infection12. In contrast, PureVax® 408

Recombinant FeLV vaccinated cats that were PCR positive with high PCR loads (9 cats total) and 409

were persistently (4 cats) or transiently (1 cat) viremic would be much more likely to remain 410

progressively infected or become regressively infected with a higher likelihood of viral 411

reactivation12. However, because bone marrow samples were not obtained and analyzed, the 412

ability of the two vaccines to prevent latency cannot be established.Nobivac® Feline-2 FeLV 413

vaccine provided excellent protection in the face of severe viral challenge accompanied by 414

immunosuppression. Nobivac® Feline 2-FeLV vaccinated cats were fully protected against 415

persistent antigenemia and had significantly lower amounts of proviral DNA and plasma viral 416

RNA loads compared to PureVax® Recombinant FeLV vaccinated cats and unvaccinated control 417

cats. In this study, it appears that Nobivac® Feline 2-FeLV (a whole virus, adjuvanted killed 418

vaccine) provided superior protection against FeLV infection when compared to PureVax® 419

Recombinant FeLV (a nonadjuvanted, canarypox virus-vectored vaccine). Effective vaccination 420

is an important means of controlling FeLV, and the efficacy of the vaccine chosen should be 421

examined closely to obtain the best protection possible. 422


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423

CONFLICT OF INTEREST 424

The authors are employees of Merck Animal Health, the company marketing Nobivac 425

Feline 2-FeLV. Merck Animal Health provided the funding for the study. All animal research was 426

run at a contracted, independent research facility (Liberty Research) by blinded investigators. 427

All PCR analysis was run at a contracted, independent research facility (Zoologix) by blinded 428

investigators. Merck employees performed the ELISA analysis and were blinded. The 429

manuscripts has been read and approved by all named authors. 430

431

ACKNOWLEDGEMENTS 432

The study was supported by Merck Animal Health. Dr. M. Patel and Dr. M. Stahl 433

designed the study. Dr. M. Patel, K. Carritt, and J. Lane supported the in life phase, data 434

acquisition, laboratory work and vaccine acquisition. Dr. M. Patel and Dr. H. Jayappa 435

interpreted the data. Dr. M. Bourgeois wrote the article and contributed to the design of the 436

study. The authors would like to acknowledge L. Deng for data analysis and Dr. K. Heaney for 437

providing valuable inputs during article writing. The authors thank Dr. A. Torres for evaluating 438

the study protocol. 439

440

REFERENCES 441


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1. Pacitti AM, Jarrett O, Hay D. 1986. Transmission of feline leukaemia virus in the milk of a 442

non-viraemic cat. Vet Rec 118:381-384. 443

2. Levy J, Crawford C, Hartmann K, Hofmann-Lehmann R, Little S, Sundahl E, Thayer V. 2008. 444

American Association of Feline Practitioners’ feline retrovirus management guidelines. 445

J Feline Med Surg 10:300–316. 446

3. Hoover EA, Mullins JI. 1991. Feline leukemia virus infection and diseases. J Am Vet Med 447

Assoc 199: 1287–1297. 448

4. Levy JK, Crawford PC. 2005. Feline leukemia virus. In: Ettinger SJ, Feldman EC (eds), 449

Textbook of Veterinary Internal Medicine (6th edn). Philadelphia, WB Saunders. 450

5. Rojko JL, Kociba GJ. 1991. Pathogenesis of infection by the feline leukemia virus. J Am Vet 451

Med Assoc 199: 1305–10. 452

6. Torres AN, Mathiason CK, Hoover EA. 2005. Re-examination of feline leukemia virus: host 453

relationships using real-time PCR. Virology 332: 272–283. 454

7. Hofmann-Lehmann R, Tandon R, Boretti FS, Meli ML, Willi B, Cattori V, Gomes-Keller MA, 455

Ossent P, Golder MC, Flynn JN, Lutz H. 2006. Reassessment of feline leukaemia virus (FeLV) 456

vaccines with novel sensitive molecular assays. Vaccine 24: 1087-1094. 457

8. Hofmann-Lehmann R, Cattori V, Tandon R, Boretti FS, Meli ML, Riond B, Lutz H. 2008. How 458

molecular methods change our views of FeLV infection and vaccination. Vet Immunol 459

Immunopathol 123: 119–123. 460

9. Torres AN, O’Halloran KP, Larson LJ, Schultz RD, Hoover EA. 2008. Development and 461

application of a quantitative real-time PCR assay to detect feline leukemia virus RNA. Vet 462

Immunol Immunopathol 123: 81–89. 463


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10. Major A, Cattori V, Boenzli E, Riond B, Ossent P, Meli ML, Hofmann-Lehmann R, Lutz H. 464

2010. Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection. 465

Vet Res 41:17. 466

11. Hofmann-Lehmann R, Huder JB, Gruber S, Boretti F, Sigrist B, Lutz H. 2001. Feline 467

leukaemia provirus load during the course of experimental infection and in naturally infected 468

cats. J Gen Virol 82: 1589-1596. 469

12. Hofmann-Lehmann R, Cattori V, Tandon R, Boretti FS, Meli ML, Riond B, Pepin AC, Willi B, 470

Ossent P, Lutz H. 2007. Vaccination against the feline leukaemia virus: outcome and response 471

categories and long-term follow-up. Vaccine 25:5531-5539. 472

13. Levy JK, Scott HM, Lachtara JL, Crawford PC. 2006. Seroprevalence of feline leukemia virus 473

and feline immunodeficiency virus infection among cats in North america and risk factors for 474

seropositivity. J Am Vet Med Assoc 228: 371-376. 475

14. Lee IT, Levy JK, Gorman SP, Crawford PC, Slater MR. 2002. Prevalence of feline leukemia 476

virus Infection and serum antibodies against feline immunodeficiency virus in unowned free-477

roaming cats. J Am Vet Med Assoc 220(5):620-2. 478

15. Sparkes AH. 1997. Feline leukaemia virus: a review of immunity and vaccination. 479

J Small Anim Pract 38(5):187-94. 480

16. Hines DL, Cutting JA, Dietrich DL, Walsh JA. 1991. Evaluation of efficacy and safety of an 481

inactivated virus vaccine against feline leukemia virus infection. J Am Vet Med Assoc 199: 1428–482

1430. 483

17. Pedersen NC. 1993. Immunogenicity and efficacy of a commercial feline leukemia virus 484

vaccine. J Vet Intern Med 7: 34–39. 485


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18. Torres AN, O'Halloran KP, Larson LJ, Schultz RD, Hoover EA. 2010. Feline leukemia virus 486

immunity induced by whole inactivated virus vaccination. Vet Immunol Immunopathol 134(1-487

2):122-31. 488

19. Stuke K, King V, Southwick K, Stoeva MI, Thomas A, Winkler MT. 2014. Efficacy of an 489

inactivated FeLV vaccine compared to a recombinant FeLV vaccine in minimum age cats 490

following virulent FeLV challenge. Vaccine 32(22):2599-603. 491

20. Cattori V, Hofmann-Lehmann R. Absolute Quantitation of Feline Leukemia Virus Proviral 492

DNA and Viral RNA Loads by TaqMan® Real-time PCR and RT-PCR. In: Methods in Molecular 493

Biology, Vol. 429: Molecular Beacons: Signalling Nucleic Acid Probes, Totowa, NJ: Humana Press 494

Inc. 495

21. Greggs WM, Clouser CL, Patterson SE, Mansky LM. 2011. Broadening the use of 496

antiretroviral therapy: the case for feline leukemia virus. Ther Clin Risk Manag 7: 115–122. 497

22. Haley PJ, Hoover EA, Quackenbush SL, Gasper PW, Macy DW. 1985. Influence 498

of antibody infusion on pathogenesis of experimental feline leukemia virus infection. J 499

Natl Cancer Inst 74(4):821-7. 500

23. Flynn JN, Dunham SP, Watson V, Jarrett O. 2002. Longitudinal analysis of feline leukemia 501

virus-specific cytotoxic T lymphocytes: correlation with recovery from infection. J Virol 502

76(5):2306-15. 503

24. Hanlon L, Argyle D, Bain D, Nicolson L, Dunham S, Golder MC, McDonald M, McGillivray 504

C, Jarrett O, Neil JC, Onions DE. 2001. Feline leukemia virus DNA vaccine efficacy is enhanced 505


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by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors. J Virol 506

75(18):8424-33. 507

25. Tartaglia J, Jarrett O, Neil JC, Desmettre P, Paoletti E. 1993. Protection of Cats against 508

Feline Leukemia Virus by Vaccination with a Canarypox Virus Recombinant, ALVAC-FL. J Virol 509

2370-2375. 510

26. Poulet H, Brunet S, Boularand C, Guiot AL, Leroy V, Tartaglia J, Minke J, Audonnet JC, 511

Desmettre P. 2003. Efficacy of a canarypox virus-vectored vaccine against feline leukaemia. 512

Vet Rec 153(5):141-5. 513

27. Jirjis FF, Davis T, Lane J, Carritt K, Sweeney D, Williams J, Wasmoen T. 2010. Protection 514

against feline leukemia virus challenge for at least 2 years after vaccination with an inactivated 515

feline leukemia virus vaccine. Vet Ther 11(2):E1-6. 516

28. Tandon R, Cattori V, Gomes-Keller MA, Meli ML, Golder MC, Lutz H, Hofmann-Lehmann R. 517

2005. Quantitation of feline leukaemia virus viral and proviral loads by TaqMan real-time 518

polymerase chain reaction. J Virol Methods 130: 124–132. 519

520

TABLE AND FIGURE LEGENDS 521

Table 1: Prevented Fraction based on Persistent Antigenemia 522

Group Treatment Persistent

Antigenemia (%)

Prevented Fraction

(%)

A Nobivac® Feline 2-FeLV 0 100

B PureVax® Recombinant FeLV 50 45


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C Placebo Control 91

523

Prevented fraction was calculated for all groups using the equation: 1 – [(Incidence of 524

persistent antigenemia in vaccinates)/(Incidence of persistent antigenemia in controls)]. 525

Nobivac® Feline 2-FeLV had a preventative fraction of 100%, PureVax® Recombinant FeLV of 526

45%. Statistically significant differences in protection was seen in the Nobivac® Feline 2-FeLV 527

group when compared to both the control group and the PureVax® Recombinant FeLV group (p 528

<0.0001 and p = 0.0124, respectively). There was no significant difference between the 529

PureVax® Recombinant FeLV group and the control group (p = 0.0635). 530

531

FIG 1. Serum IgG Antibodies to FeLV Vaccination Phase. All cats were negative for FeLV 532

antibody prior to vaccination on study day 0. Ten of eleven cats in the Nobivac® Feline 2-FeLV 533

group were positive for FeLV antibody on study days 20 (average S/P ratio =3.0544 +/-1.007) 534

and 111 (average S/P ratio= 1.002 +/-0.48). The PureVax® Recombinant FeLV cats and control 535

cats remained antibody negative during the vaccination phase of the study. Statistically 536

significant differences were seen between the Nobivac® Feline 2-FeLV group and Purevax® 537

Recombinant FeLV (*) and controls (**) (p=0.00). 538

539

FIG 2. p27 Antigen, Proviral DNA, and Viral RNA Results Weeks Post Challenge. All cats were 540

tested for p27 antigen, FeLV plasma viral RNA, and FeLV proviral DNA. Cats in the Nobivac® 541

Feline 2-FeLV group were negative for p27 antigen throughout the entire post challenge period. 542


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50% of the PureVax vaccinated cats and 91% of the Control cats became persistently 543

antigenemic post challenge. At week 9 post challenge, plasma viral RNA was detected in 1 of 11 544

(9%) of the Nobivac® Feline 2-FeLV vaccinated cats, 6 of 10 (60%) of the PureVax® Recombinant 545

FeLV vaccinated cats and 9 of 11 (82%) of the unvaccinated control cats. From weeks 7 to 9, 546

viral RNA loads were significantly lower in the Nobivac group than the PureVax® Recombinant 547

FeLV group (p < 0.01). Proviral DNA was detected in 1 of 11 (9%) of the Nobivac® Feline 2-FeLV 548


(82%) of the unvaccinated control cats. From weeks 6 to 9, the Nobivac® Feline 2-FeLV group 550

had significantly lower proviral DNA loads than the PureVax® Recombinant FeLV group ( p < 551

0.02). 552

553

FIG 3. FeLV Persistent Antigenemia. FeLV p27 antigen ELISA to detect persistent antigenemia 554

in the three groups (Group A- Nobivac® Feline 2-FeLV, Group B- PureVax® Recombinant FeLV, 555

Group C- Controls). OD = Optical Densities > 0.200 were considered positive for FeLV p27 556

antigen. Testing was performed during both the vaccination and challenge phases of the study. 557

Percentages of cats persistently antigenemic were calculated at 0% for the Nobivac® Feline 2-558

FeLV group, 50% for the PureVax® Recombinant FeLV group, and 91% for the controls. 559

560

FIG 4. FeLV p27 Antigenemia ELISA Data. FeLV p27 antigen ELISA to detect persistent 561

antigenemia in the three groups (Group A- Nobivac® Feline 2-FeLV, Group B- PureVax® 562

Recombinant FeLV, Group C- Controls). OD = Optical Densities > 0.200 were considered 563

positive for FeLV p27 antigen. Statistically significant differences were seen between the 564


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Nobivac® Feline 2-FeLV and control groups at all time points (p<0.01). Statistically significant 565

differences were seen between the Nobivac® Feline 2-FeLV and PureVax® Recombinant FeLV 566

groups at weeks 3, 4, 6 to 9 and 11 post challenge (p<0.01, Indicated by *). No statistically 567

significant differences were seen between PureVax® Recombinant FeLV groups and control 568

groups at any time points (p<0.01). 569

570

FIG 5. FeLV Plasma Viral RNA and Proviral DNA Results by PCR. Quantitative detection of FeLV 571

virus in proviral DNA by real time PCR, and plasma viral RNA by reverse transcription, real time 572

PCR. The limit of detection for DNA was approximately 400 copies of DNA/ 1 mL. The limit of 573

detection for RNA was approximately 1000 copies of RNA/ 1 mL. At the end of the challenge, 574

proviral DNA and plasma viral RNA were detected in 1 of 11 (9%) of the Nobivac® Feline 2-FeLV 575


(82%) of the unvaccinated control cats. Statistically significant differences were seen between 577

the Nobivac® Feline 2-FeLV and control groups at all time points for both plasma viral RNA and 578

proviral DNA (p<0.01). Statistically significant differences were seen between the Nobivac® 579

Feline 2-FeLV and PureVax® Recombinant FeLV groups at between weeks 7 and 9 for plasma 580

viral RNA (p<0.01) and between weeks 6 and 9 for proviral DNA (p<0.02). 581

582


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