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