local and systemic antibody responses to hsv
TRANSCRIPT
Betsy C. Herold, M.D.
Albert Einstein College of Medicine
The Key Role of FcR Activating
Antibodies in Generating
"Sterilizing" Immunity against
Herpes Simplex Virus
Science at the heart of medicine
Scope of the Problem HSV-1 and HSV-2 cause lifelong recurrent infectious mucocutaneous
ulcers
HSV-1 predominant cause of oral lesions (gingivostomatitis)
HSV-2 more common cause of genital lesions worldwide
However, in US and Europe, HSV-1 has emerged as a more common cause of genital herpes
HSV-1 also leading cause of sporadic fatal encephalitis and corneal blindness
HIV-HSV-2 syndemic:
HSV-2 associated with increased risk of HIV transmission and acquisition
Both serotypes transmitted perinatally with potentially devastating outcomes to infants even with treatment
Worldwide prevalence:
HSV-1 ~3.7 billion
HSV-2 ~417 million
History of HSV prophylactic vaccines
• Subunit vaccines targeting major envelope glycoproteins gD and gB that elicit high titer neutralizing antibodies dominated the field
• Neutralizing antibodies presumed to be surrogates of protection
• Two vaccines completed Phase 3 clinical trials with negative outcomes:
• gD-2/gB-2 + MF59 Phase III (Chiron)
• Elicited high titer neutralizing antibodies
• Overall vaccine efficacy was 9% (95% CI, -29% to 36%) (JAMA, 1999)
• gD-2+ Alum + MPL Phase III (GSK-Herpevac)
• Elicited high titer gD specific neutralizing Abs (higher than natural infection) and CD4 T cell responses
• Serodiscordant partners: vaccine protective in ♀ who were doubly seronegative but not HSV-1+ ♀ and not in ♂ (NEJM, 2002)
• Field study HSV-1/HSV-2 seronegative ♀ 18-30 yrs, not protective against genital HSV-2 disease or infection
• Efficacy against genital HSV-1 disease was 57% (CI 12 to 80).
• Both vaccines were protective in “standard” murine & guinea pig models
What would happen if instead of focusing
on gD as immune target, we vaccinated
with ΔgD virus?
Would we generate immune response against other antigenic
targets?
What would be the functionality of the immune responses
elicited?
Hypotheses:
Vaccine would be safe
gD required for entry and cell-to-cell spread
Vaccine would elicit polyantigenic response unmasked by removal
of the immunodominant gD
Vaccine might elicit functionally different immune responses
reflecting loss of immunomodulatory gD protein
Engineering ΔgD virus on HSV-1 gD
complementing cells
Allelic exchange
substrate
ori
gfpRL
L RUS6 (gD)US5 (gJ) US7 (gI)
parental HSV-2 (G) DNA
L RUS5 (gJ) US7 (gI)
gfp
recombinant HSV-2 (∆gD::gfp) DNA
HSV-2 ΔgD-/+gD-1
Complementing
HSV-1 gD cell line(VD60)
HSV-2 DNA ∆gD::gfp
Engineering ΔgD virus on HSV-1 gD
complementing cells
Complementing
HSV-1 gD cell line (VD60)
ΔgD-/+
Non- complementing
Cell line (vero)
ΔgD-/-
gB
GFP
DAPI
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
D a y s p o s t - i n f e c t i o n
Pe
rc
en
t s
ur
viv
al
H S V - 2 W T / i v a g 1 04
P F U
H S V - 2 W T / i v a g 1 05
P F U
H S V - 2 g D- / +
/ i v a g 1 06
P F U
H S V - 2 g D- / +
/ i v a g 1 07
P F U
ΔgD causes no disease in SCID Mice
SCID
Wt or gD-/+
exposure end of study
Sacrifice morbid-bound animals
26 days
15
20
25
30
35
40
45
qP
CR
Ct
(UL30)
Genital tract
cu
t o
ff
Neural tissue
*** *** *** ***
WT ivag 104 PFU
DgD ivag 107 PFU
WT sc 105 PFU
DgD sc 107 PFU
WT ivag 104 PFU
DgD ivag 107 PFU
WT sc 105 PFU
DgD sc 107 PFU
Day 5
Petro and Gonzalez, et al., 2015, eLife
ΔgD Protects C57BL/6 and Balb/C Mice
against vaginal HSV-2(4674)
C57Bl/6
Balb/C
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
2 0
4 0
6 0
8 0
1 0 0
D a y s p o s t-c h a lle n g e
Pe
rc
en
t s
urv
iva
l * * *
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
2 0
4 0
6 0
8 0
1 0 0
D a y s p o s t - c h a l l e n g e
Pe
rc
en
t s
ur
viv
al
C o n t r o l L D 9 0
* * *
g D 1 0 x L D 9 0
C o n t r o l 1 0 x L D 9 0
g D L D 9 0
Petro and Gonzalez, et al., 2015, eLife
ΔgD immunized mice have no detectable HSV-2 in
vaginal at 5 days or neural tissue up to 28 days
post-challenge
NO viral titer in vaginal washes
NO viral reactivation from explanted co-
cultured neural tissue
NO viral DNA in excised tissue
Lo
g1
0H
SV
-2
p
fu
/m
l
D a y 2 D a y 4
0
1
2
3
4 * * * * * *
V a g i n a l T r a c t N e u r a l t i s s u e
0
1
2
3
4
5
Lo
g1
0H
SV
-2
p
fu
/g
r
* * * * * *
5 7 9 1 1 1 3 1 5 1 7 1 9 2 1
0
2 5
5 0
7 5
1 0 0
D a y s e x v i v o c e l l c o - c u l t u r e
Pe
rc
en
t o
f
re
ac
tiv
ate
d D
RG
s
g D
C o n t r o l
* * *
Adoptive transfer scheme
21 days 21 days
15 days
sc sc
Prime+ Boost
Challenge
HSV-2 WT
IV
Pan T cell
OR
Serum
IP ivag
Passive transfer of ∆gD immune serum
protects naïve mice from HSV-2 challenge
D a y s p o s t-c h a lle n g e
Pe
rc
en
t s
urv
iva
l
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
2 0
4 0
6 0
8 0
1 0 0
gD- / +
P a n T c e lls
gD- / +
s e ru m
C o n tro l P a n T c e lls
C o n tro l s e ru m
gD- / +
Ig -d e p le te d
s e ru m
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
5 0
1 0 0
D a y s p o s t - c h a l l e n g e
Pe
rc
en
t s
ur
viv
al
C o n t r o l s e r u m
g D- / +
s e r u m
Intravaginal challenge Skin challenge
Petro and Gonzalez, et al., 2015, eLife
2 3 4 5 6 7 8
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
L o g 1 0 re c ip ro c a l d ilu t io n o f s e ru m
To
tal
HS
V-2
Ig
(OD
45
0n
m)
g D - /+
C o n tro l
1 :8 0 0 ,0 0 0
Total serum anti-HSV-2
antibodies
0.0
0.2
0.4
0.6
0.8
1.0
an
ti-H
SV
-2 Ig
(O
D450 n
m)
D7 p-boost D4 p-challenge
***
21 days 21 days 15 days
sc sc
Prime
HSV-2 ΔgD-/+
Boost
HSV-2 ΔgD-/+
Challenge
HSV-2 WT
∆gD elicits a robust systemic and mucosal
HSV-2 Ab response
Total mucosal anti-HSV-2
antibodies
HSV529 (single cycle replication impaired virus)
showed reduced efficacy against African HSV isolate
in murine model
Dudek, et al., 2011, JID
HSV-2ΔgD-2 provides complete protection following
intravaginal or skin challenge with US clinical isolate
with vaccine doses as low as 5x104 PFU
Clinical Isolates are Genetically Diverse
B3x HSV-1 and HSV-2 isolates provided from MMC Clinical Lab
sd90 (African clinical isolate) gift from D. Knipe
But Variable Virulence in Mice
HSV-1 isolates5x10^5pfu/mouse
HSV-2 isolates1x10^5pfu/mouse
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
D a y s p o s t c h a lle n g e
Pe
rc
en
t s
urv
iva
l
B3
x 1 .1
B3
x 1 .2
B3
x 1 .4
B3
x 1 .5
H S V -1 1 7
H S V -1 F
B3
x 1 .3
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
D a y s p o s t c h a lle n g e
Pe
rc
en
t s
urv
iva
l
B3
x 2 .1
B3
x 2 .2
B3
x 2 .3
B3
x 2 .4
B3
x 2 .5
3 3 3 -Z A G
S D 9 0
H S V -2 G
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
0
1
2
3
4
5
D a y s P o s t C h a lle n g e
Sk
in D
ise
as
e S
co
re
B3
x 1 .1
B3
x 1 .2
B3
x 1 .4
B3
x 1 .5H S V -1 1 7
H S V -1 F
B3
x 1 .3
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
0
1
2
3
4
5
D a y s p o s t c h a lle n g e
Sk
in D
ise
as
e S
co
re
B3
x 2 .1
B3
x 2 .2
B3
x 2 .3
B3
x 2 .4
B3
x 2 .5
3 3 3 -Z A G
S D 9 0
H S V -2 G
Immunization with HSV-2-ΔgD protects
against most virulent clinical isolates
21 days 21 days 15 days
sc sc
Prime
HSV-2 ΔgD
Boost
HSV-2 ΔgD
Challenge
HSV-1 or 2
SD90 B3x2.3 B3x1.1
Immunization with HSV-2-ΔgD confers protection
against virulent HSV clinical isolates
21 days 21 days 15 days
sc sc
Prime
HSV-2 ΔgD
Boost
HSV-2 ΔgD
Challenge
HSV-1 or 2
SD90 B3x2.3 B3x1.1
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
D a y s P o s t C h a lle n g e
Pe
rc
en
t s
urv
iva
l
C tr l B3
x 1 .1
g D -2 B3
x 1 .1
C tr l S D 9 0
g D -2 S D 9 0
***
C tr l B3
x 2 .3
g D -2 B3
x 2 .3
ΔgD immunized mice are protected against
10-100x LD90 of Bx1.1 and SD90
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
D a y s P o s t C h a lle n g e
Pe
rc
en
t s
urv
iva
l
***
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
1
2
3
4
5
D a y s P o s t C h a lle n g e
Sk
in D
ise
as
e S
co
re
Disease Survival
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
1
2
3
4
5
D a y s P o s t C h a lle n g e
Sk
in D
ise
as
e S
co
re C tr l B
3x 1 .1
g D -2 1 x B3
x 1 .1
g D -2 1 0 x B3
x 1 .1
C tr l S D 9 0
g D -2 1 x S D 9 0
g D -2 1 0 x S D 9 0
g D -2 1 0 0 x S D 9 0
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5
0
1
2
3
4
5
D a y s P o s t C h a lle n g e
Sk
in D
ise
as
e S
co
re C tr l B
3x 1 .1
g D -2 1 x B3
x 1 .1
g D -2 1 0 x B3
x 1 .1
C tr l S D 9 0
g D -2 1 x S D 9 0
g D -2 1 0 x S D 9 0
g D -2 1 0 0 x S D 9 0
ΔgD elicits high titer IgG2 response in skin
following challenge with clinical isolates
Anti HSV-2 IgG (ELISA)
Antiviral antibody responses
Trkola, Nature, 2014.
Response impacted by antigenic target, isotype of Ab, FcR interactions and
glycans
Antibody responses
Retro-orbital or
cardiac bleed
HSV specific ELISA
for titer, isotype
Neutralization assay
Antigenic targets
Fc receptor activationPassive transfer
Serum neutralizing Ab
Immune serum from vaccinated animals
showed little neutralization of HSV in vitro
1:5 dilution of serum
HSV-2 HSV-1
∆gD elicits predominantly anti-HSV IgG2
antibodiesH
SV
-2 A
b (
OD
45
0n
m)
C o n tr o l g D- /+ g D- 1
C o n tr o l g D- /+ g D- 1
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
7 d a y s p o s t b o o s t
*** ***
***
***
******
8 d a y s P C
***
*
HS
V-2
Ab
(O
D4
50
nm
)
C o n tr o l g D- /+ g D- 1
C o n tr o l g D- /+ g D- 1
0 .0
0 .2
0 .4
0 .6
0 .8
4 d a y s P C 8 d a y s P C
**
***
***
***
- + - + - + - +
0
1 0
2 0
3 0
4 0
5 0
% A
DC
C
- F c R I I I / I I
* *
p - b o o s t p - c h a l l e n g e
* * * *
HSV-infected cell
SplenocyteKilling via
FcγR
engagement
Petro and Gonzalez, et al., 2015, eLife
Immune Serum Elicits Antibody-Dependent Cell
Mediated Cytotoxicity
Serum from ∆gD-vaccinated mice mediate Antibody-
Dependent-Cellular phagocytosis (ADCP) in vitroMonocyte/Macrophage
Phagocytosis
via FcγR
engagement
HSV-coated beads
Dotted- Control serum+HSV
Solid-∆gD-/+ serum+HSV
Solid-∆gD-/+ serum+HSV
Red-∆gD-/+ serum+Cell
V C V C
0
2
4
6
8
1 0
Ph
ag
oc
yti
c S
co
re
((%
po
sit
ive
XM
FI)
/10
6)
L y s a te c o a te d b e a d s :
***
***
V C V C
0
5
1 0
1 5
2 0
2 5
IFN
y (
pg
/ml)
*
c u t o f f
IFNγ
Decrease in inflammatory response in the skin by day 5 post
challenge in ΔgD versus Control-vaccinated mice
Decrease in inflammatory response in the skin by day 5 post
challenge in ΔgD versus Control-vaccinated mice
What are the antigenic targets of
immune serum?
Western blots of cellular lysates infected with HSV-2(4674) and probed with dilutions
of sera from HSV-2 ΔgD vaccinated mice 7 days post-boost
Conclusions
Subunit vaccines elicit neutralizing Abs (gD/gB) similar to natural infection
NOT sufficient to prevent infection in clinical trials
Did not prevent latency in “standard” murine models
High neutralization titers are observed in pts with frequent recurrences
ΔgD vaccine:
Safe and immunogenic
Elicits high titer FcR activating Abs and CD4 and CD8 T cell responses
Optimal protection observed with live, not inactivated, virus
Proves protection against array of clinical isolates in vaginal & skin murine models (male and female)
Prevents establishment of latency (98/100 mice)
Protects guinea pigs from lethal challenge and prevents virus reaching DRG (challenged with clinical isolate)
Preclinical studies should include evaluation of vaccine responses against multiple clinical isolates of HSV-1 and HSV-2
Speculations
Rapid recruitment of FcR activating Abs to sites of HSV
exposure required for optimal prevention
FcR activation may provide better correlate of vaccine
efficacy
The presence of gD may skew the immune response
towards generation of neutralizing Abs or…
Absence of gD promotes an IgG2 dominant ADCC
antibody response-
? WHY
Acknowledgements
Herold Lab
• Clare Burn
• Chris Petro
• Natalia Cheshenko
• Natalie Ramsey
• Naz Khajoueinejad
• Carol Kao
William Jacobs
• Brian Weinrick
• Kayla Weiss
• Joseph Dardick
• Bing Chen
• Pablo Gonzalez
43
David Knipe
Harvard Medical School
Kelsoe Lab
Duke University
Garnett Kelsoe
Masayuki Kuraoka
Akiko Watanabe
GlaxoSmithKline
Promega
Mei Cong
Vanessa Ott
Aileen Paguio