Immunogenicity of a trivalent subunit vaccine for …...Rationale for HSV-2 vaccine • Prevention...
Transcript of Immunogenicity of a trivalent subunit vaccine for …...Rationale for HSV-2 vaccine • Prevention...
Immunogenicity of a trivalent subunit
vaccine for genital herpes in Rhesus
macaques.
Sita Awasthi, PhD
World Congress on Virology, 2015
Atlanta, December 7-9
Global prevalence of HSV-2 Infection
Looker KJ. Plos One Jan. 2015
Rationale for HSV-2 vaccine
• Prevention of genital herpes
15% of adult population is infected with HSV-2 worldwide
20 million new infections are added each year globally
• Treatment of genital herpes
In US ~ 50 million people are HSV-2 positive
Economic burden of treatment is ~billion dollars annually in
US
• Curb acquisition and transmission of HIV
• There is no cure or FDA approved vaccine
Rationale for a trivalent genital herpes
vaccine
Entry protein
Inhibits
complement
IgG Fc Receptor
Glycoprotein C protects virus against human
complement
gC2 HSV-2 WT
gD2
C3b
C1q
HSV-2 gC null
gD2
C3bC5-9
C3b
C1q
Awasthi et al J Virol 2011
Glycoprotein E protects virus from IgG Fc
mediated ADCC and C’activation
WT virus: Antibody bipolar
bridging
HSV-2 WT
gD2
HSV-2 gE mutant: No antibody
bridging
HSV-2 gE del
gD2
gI
C1q
Awasthi et al J Virol 2014
gC2/gD2/gE2 as a prophylactic vaccine
Goal: Elimination of acute and recurrent
disease, and asymptomatic shedding of HSV-2
DNA in pre-clinical model.
0-acute
0-recurrent
0-shedding of HSV-2 DNA
High ELISA titers to all immunogens and neutralizing titers to HSV-2
ELISA titers
Mock gD2 Mock gC2 Mock gE20.0
0.5
1.0
1.5
2.0
2.5
gD2 Ag gE2 AggC2 Ag
OD
at
40
5 n
m
Mock gC2/gD2/gE2
20
40
80
160
320
640
1280
2560
<20
Neutralization titers
Se
rum
Dil
uti
on
Immunogenicity studies: ELISA responses and
neutralizing Ab titers
gC2/gD2/gE2 as a prophylactic vaccine
Table. Recurrent disease
Recurrent genital disease
Days post-challenge
Cu
mu
lati
ve
lesi
on
da
ys/
an
ima
l
* P<0.001
Results: Trivalent vaccine highly
protective against recurrent
disease.
Mock gD2 gC2/gD2/gE2
Incidence of
recurrent genital
disease
7/8
(88%)⌘⌘⌘⌘16/25
(64%)**
10/36
(28%)**
Days with
lesions/total
observation days
(15-60)
85/340
(25%)¶
77/1087
(7%)***
16/1509
(1%)***
0
2
4
6
8
10
12
14
Mock
gD2
Trivalent
No HSV DNA
151-1000
103- 105
>105 HSV-2 DNA
>105 DNA and
Infectious HSV-2
Day 28 Day 48
GP 1
GP 9
Day 28 Day 48
GP 1
GP 9
HSV-2 DNA and infectious virus from vaginal swabs
gD2
Trivalent
Summary of efficacy studies in
guinea pigs
Acute and recurrent disease: Trivalent vaccine is very
effective at preventing acute and recurrent genital disease
and comes close to meeting 0 acute – 0 recurrent disease
goals.
Asymptomatic DNA shedding: Trivalent vaccine reduced
vaginal HSV-2 DNA shedding, but did not eliminate it.
However, no infectious HSV-2 was recovered from vaginal
swab during recurrent phase in trivalent vaccinated guinea
pigs.
1ST 3rd2nd
Translational efforts: Immunogenicity in
rhesus macaques
0 1 2 8 12 month
Immunizations and assessments
Groups
Immunogenicity Endpoints
ELISA antibody titers for each antigens, mucosal antibody titers,
neutralization titers, cellular immune responses, C3b blocking ability,
Fc receptor blocking ability.
Mock (CpG and alum) n=2
gC2 (CpG and alum) n=2
gD2/gC2/gE2 (CpG and alum) n=2
Vaccinations and bleeds
Collaboration with Tulane National Primate Center
4th Terminal
bleed
Plasma antibody and neutralization titers in
vaccinated rhesus macaques
Rhesus
#
Treatment ELISA titers Neutralization
titers
gD gC gE 3rd imm 4th Imm
1 CpG/alum <1:50 <1:50 <1:50 <1:20 <1:20
2 CpG/alum <1:50 <1:50 <1:50 <1:20 <1:20
3 gC2-CpG/alum NA 1:32000 NA 1:80 NA
4 gC2-CpG/alum NA 1:32000 NA 1:80 NA
5 Trivalent vaccine 1:32000 1:32000 1:32000 1:80 1:640
6 Trivalent vaccine 1:16000 1:32000 1:32000 1:320 >1:640
ELISA and neutralization titers in vaginal
mucosa
Rhesus
#
Treatment Mucosal IgG
(ELISA Ab titers)
Mucosal
Neutralization
titers
HSV-2gD gC gE
1 CpG/alum <1:25 <1:25 <1:25 <1:10
2 CpG/alum <1:25 <1:25 <1:25 <1:10
3 gC2-CpG/alum NA ND NA ND
4 gC2-CpG/alum NA ND NA ND
5 Trivalent vaccine 1:400 1:200 1:100 1:10
6 Trivalent vaccine 1:6400 1:1600 1:400 1:80
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Pre immune gC/gD/gE
Re
lati
ve
gE
bin
din
g
No IgG
IgG
Blocking of gE binding to the Fc end of human IgG by
vaccinated rhesus IgG
IgG
gE2
IgG
gE2
Immune IgGPre immune
• Plate was coated with human
IgG from an HSV-1/2 negative
donor
• gE2 was incubated with
rhesus IgG (0 or 200ng/µµµµl) at
37⁰C for 1h then added to IgG-
coated wells
• Bound gE was detected with
polyclonal rabbit anti-gE2 Ab
• * P<0.001*
Blocking of gE2 binding to IgG
Fc will enhance neutralizing
ability of gD2 antibody
Blocking of gC2 binding to C3b by vaccinated
rhesus IgG
C3b
gC2
C3b
gC2
Immune IgG Mock IgG
OD
at
40
5 n
m
gC2 binding to C3b
Antibodies that block gC2 binding to C3b will enhance neutralizing
ability of gD2 antibody
•Plate was coated
with C3b
•gC2 was incubated
with rhesus IgG (3rd
bleed) at 37⁰C for 1 h
then added to C3b-
coated wells
•Bound gC was
detected with
polyclonal rabbit
anti-gC2 Ab
No IgG
IgG
*
* p<.001
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Mock-Immune gC2-imm gC2/gD2/gE2
**
0
0.5
1
1.5
2
2.5
3
3.5
% I
FN
γp
osi
tive
CD
4+
ce
lls
0
2
4
6
8
10
12
14
% I
FN
γp
osi
tive
CD
8+
ce
lls
Cellular immune response in gC2-immunized
Rhesus macaques
CD4+ T cells CD8+ T cells
gC2 immunized rhesus PBMC had increased IFNγγγγ positive CD4 and CD8 T cells
when stimulated with gC2 peptide pool.
• PBMCs were isolated
following pre and post-
immunizations.
• Cells were stimulated
with a pool of
overlapping peptides
(gC2).
• IFNγ γ γ γ positive CD4 and
CD8 cell were measured
by surface and
intracellular staining,
followed by FACS
analysis
Vaccine specific CD4+ T cell responses in rhesus%
CD
4+
PB
MC
Glycoprotein C
0
0.05
0.1
0.15
0.2
Pre-immune Vaccinated
IL-2
TNF-a
IFNg
Glycoprotein E
% C
D4
+ P
BM
C
0
0.1
0.2
0.3
0.4
Pre-immune Vaccinated
IL-2
TNF-a
IFNg
0
0.03
0.06
0.09
0.12
0.15
Pre-immune Vaccinated
IL-2
TNF-a
IFNg
% C
D4
+ P
BM
C
Glycoprotein D• PBMCs were isolated following pre and post-
immunizations.
• Cells were stimulated with purified gC2, gD2
or gE2.
• IL-2, TNF-αααα, and IFNγ, γ, γ, γ, positive CD4+ cell
were measured by surface and intracellular
staining, followed by FACS analysis
Immunogen specific CD4+ T cell
responses are induced in vaccinated
rhesus
SummarySummary
• Trivalent vaccine is highly effective in eliminating primary and
recurrent disease in guinea pigs.
• High level ELISA titers to gD, gC and gE are detected
• High level of neutralizing antibody titers are noted in plasma
followed by trivalent vaccine immunization
• Vaccine specific mucosal IgG are detected in rhesus vagina
• C3b blocking and FcR blocking antibodies are produced in vaccinated
rhesus
• CD4 and CD8 T cell responses are induced in vaccinated rhesus
A trivalent vaccine that blocks evasion of host immunity is
a promising candidate from future human trials.
ConclusionsConclusions
Acknowledgments
Reagents: Gary Cohen, Roselyn Eisenberg,
Stuart Isaacs
GSK
Funding: NIH, Merck (2007-09), CFAR
Harvey FriedmanCarolyn Shaw
Lauren Hook
University of Pennsylvania
National Primate Center TulaneRon Veazey
Megan Gardner
Bapi Pahar
CD4+ T cell responses
Media Control gC2 SEBgE2
Animal IH34
Sample date
4/22/2014