B R I E F R E P O R T
Better Neutralization of Herpes
Simplex Virus Type 1 (HSV-1) Than
HSV-2 by Antibody From Recipients
of GlaxoSmithKline HSV-2
Glycoprotein D2 Subunit Vaccine
Sita Awasthi,1Robert B. Belshe,2and Harvey M. Friedman1
1Infectious Disease Division, Department of Medicine, Perelman School of
Medicine, University of Pennsylvania, Philadelphia and2Division of Infectious
Disease, Allergy, and Immunology, Saint Louis University, Missouri
The Herpevac Trial evaluated a herpes simplex virus type 2
(HSV-2) glycoprotein D (gD2) subunit vaccine to prevent
genital herpes. Unexpectedly, the vaccine protected against
genital HSV-1 infection but not genital HSV-2 infection.
We evaluated sera from 30 women seronegative for HSV-1
and HSV-2 who were immunized with gD2 in the Herpevac
Trial. Neutralizing antibody titers to HSV-1 were 3.5-fold
higher than those to HSV-2 (P< .001). HSV-2 gC2 and gE2
on the virus blocked neutralization by gD2 antibody, while
HSV-1 gC1 and gE1 did not block neutralization by gD2
antibody. The higher neutralizing antibody titers to HSV-1
offer an explanation for the Herpevac results, and shielding
neutralizing domains provides a potential mechanism.
protein C; glycoprotein E; neutralizing antibody; Herpevac Trial.
HSV-1; HSV-2; vaccine; glycoprotein D; glyco-
Two publications reporting use of a herpes simplex virus type 2
(HSV-2) glycoprotein D2 (gD2) subunit vaccine adjuvanted
with monophosphoryl lipid A (MPL) and aluminum hydroxide
(alum) demonstrated mixed efficacyagainstHSV genital disease
[1, 2]. The first report enrolled couples discordant for genital
HSV-2 infection. In a subset analysis, the gD2 vaccine provided
approximately 70% protection against HSV-2 genital disease in
women seronegative for both HSV-1 and HSV-2 but not in
women seropositive for HSV-1 or in men of any serostatus
.The results were sufficiently compelling to initiate the Her-
pevac Trial for Women, which enrolled >8000 women seroneg-
ative for both HSV-1 and HSV-2 . The Herpevac Trial
subjects were not restricted to discordant couples. Unexpected-
ly, the gD2 vaccine provided significant protection against
HSV-1 but not HSV-2 genital infection and disease . Sixty
percent of genital disease in control subjects was caused by
HSV-1, which has emerged asthe leading cause of primary gen-
ital disease [1, 3]. Few HSV-1 infections were detected in the
first report, owing to selection of HSV-2–discordant couples.
HSV-2 gD2 is required for virus entry into cells .In choos-
ing gD2 as an immunogen, a goal is to induce potent neutral-
izing antibodies to block entry. Enzyme-linked immunosorbent
assay (ELISA) antibody titers but not cellular immune responses
to gD2 were highly correlated with protection in the Herpevac
Trial . A possible explanation for protection against HSV-1
but not HSV-2 may relate to the relative ease of blocking entry,
as measured by neutralization of the 2 viruses. Therefore, we
evaluated the hypothesis that neutralizing antibody titers were
higher to HSV-1 than to HSV-2 in the Herpevac Trial.
We measured neutralizing antibodies by 2 methods. One ap-
proach evaluated the 50% end point neutralizing titer by incu-
bating serial 2-fold dilutions of heat-inactivated serum with 100
plaque-forming units (PFU) of virus for 1 hour at 37°C, inocu-
lating the virus-antibody mix onto Vero cells, overlaying with
methylcellulose, and counting plaques at 72 hours .The sec-
ond assay measured the log10titer reduction by using 106PFU
of virus mixed with a 1:40 dilution of heat-inactivated serum for
1 hour at 37°C and measuring plaques at 72 hours .The log10
neutralization was calculated as the difference in titer, using
serum from immunized individuals and nonimmune human
serum as a control. ELISA was performed at a 1:500 serum di-
lution, using baculovirus-expressed gD1 (bac-gD1[306t]) or
gD2 (bac-gD2[306t]) as antigen [6, 8].
Subjects were immunized at 0, 1, and 6 months [1, 2]. Paired
sera were obtained before the first immunization and at
month 7 from 35 Herpevac Trials subjects; 30 had received
20 µg of gD2 with MPL/alum, whereas 5 received hepatitis A
vaccine as a control . An additional 13 sera were obtained
at month 7 from subjects seronegative for both HSV-1 and
HSV-2 who were enrolled in GSK gD2 vaccine trials between
Received 11 February 2014; accepted 14 March 2014; electronically published 20 March
Correspondence: Harvey M. Friedman, MD, 522E Johnson Pavilion, Infectious Disease Divi-
sion, University of Pennsylvania, Philadelphia, PA 19104-6073 (firstname.lastname@example.org).
The Journal of Infectious Diseases2014;210:571–5
© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases
Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@
BRIEF REPORT • JID 2014:210 (15 August) • 571
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1995 and 1997. Ten had received gD2 with MPL/alum vaccine,
and 3 were MPL/alum controls. These sera were stored at
−80°C. Seven of 13 sera were from subjects enrolled at the Uni-
versity of Pennsylvania in HSV-007 (3 controls and 4 subjects
immunized with 20 µg gD2 with MPL/alum) . Five of 13 sera
were from subjects enrolled in a dose-ranging study that used
20, 40, or 80 µg of gD2 with MPL/alum (HSV-014), and 1 sub-
ject was immunized with 20 µg gD2 with MPL/alum to evaluate
diluent volumes (HSV-015).
Four low-passage HSV-1 and 3 low-passage HSV-2 clinical iso-
lates were used for neutralization assays. The gC and gE deletion
strains were derived from low-passage HSV-1 and HSV-2 wild-
type (WT) viruses NS and 2.12, respectively [9–11]. The Uni-
versity of Pennsylvania Institutional Review Board approved
Methods for calculating statistical significance are described in
the legends of Figures 1 and 2.
Sera from subjects enrolled in the Herpevac Trial were selected
by biostatisticians to include 10 each with high, intermediate,
0 and 7 from hepatitis A (HepA; n=5) and glycoprotein D2 (gD2; n=30) immunized subjects. B, Neutralization at each serum dilution of gD2-immunized
subjects (n =30). The dotted line represents 50% neutralization. The solid lines represent the mean percentage neutralization at each dilution. The curves
were compared by 2-way analysis of variance and the Bonferroni test for repeated measures. The values at each point are significantly different, except for
1:640. C, Correlation of gD2 enzyme-linked immunosorbent assay (ELISA) titers with HSV-1 (P<.001) or HSV-2 (P<.001) neutralizing antibody titers of
subjects immunized with gD2 (n=30). P values were calculated using the Pearson correlation for nonparametric data and a2-tailed analysis. D, Fifty percent
end point neutralizing antibody titers to HSV-1 or HSV-2 by sera obtained between 1995 and 1997 at month 7 from placebo recipient (n =3) or gD2-
immunized subjects (n =10; 4 from HSV-007 and 6 from HSV-014 and 015). E, The same 6 HSV-014 and 015 sera were tested against 3 low-passage
HSV-1 strains and 2 low-passage HSV-2 strains (each strain represented by a different color). Note that the y-axis scale differs in panel E. P values for
panels A and D were calculated by 1-way analysis of variance, using the Dunn multiple comparisons test, whereas the P value in panel E was calculated by
the Mann–Whitney test. In panels A, D, and E, data are geometric mean titers with 95% confidence intervals.
A, Fifty percent end point neutralizing antibody titers of herpes simplex virus type 1 (HSV-1) and HSV-2 Herpevac Trial sera obtained at months
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and low gD2 ELISA titers to reflect the distribution of ELISA
responses in the entire study population . Selected subjects
were not infected with HSV before month 7. The geometric
mean 50% end point neutralization titer of sera taken at
month 7 was 1:101 for HSV-1, compared with 1:29 for HSV-
2 (3.5-fold difference; Figure 1A). The mean percentage neutral-
ization at each serum dilution was higher against HSV-1 than
against HSV-2 (Figure 1B). A significant correlation was detect-
ed between gD2 ELISA titers (provided by the Herpevac inves-
tigators) and neutralizing antibody titers to HSV-1 and HSV-2
for the 30 subjects immunized with gD2 (Figure 1C) . We
evaluated neutralizing antibody titers of 13 additional sera
from subjects enrolled in GSK gD2 vaccine trials between
1995 and 1997, including 3 sera from placebo-immunized sub-
jects and 10 from subjects immunized with gD2 with MPL/
alum. The geometric mean neutralizing antibody titer was
1:422 against HSV-1 and 1:97 against HSV-2 (4.4-fold
difference; Figure 1D). Six of the 10 sera (those from HSV-
014 and 015) were tested for neutralizing antibody titers against
3 additional low-passage HSV-1 strains and 2 additional low-
passage HSV-2 strains. The geometric mean neutralizing anti-
body titer was 1:242 against HSV-1 and 1:106 against HSV-2
(2.3-fold difference; Figure 1E).
As a potential explanation for the greater neutralization of
HSV-1 than HSV-2, we hypothesized that HSV-2 gC2 and
gE2 may shield neutralizing domains on HSV-2 gD2 more ef-
fectively than gC1 and gE1 shield domains on HSV-1 gD1. This
hypothesis is based on our prior studies demonstrating that gC1
and gE1 block access of neutralizing antibodies to HSV-1 glyco-
proteins involved in virus entry . We compared neutraliza-
tion of WT HSV-1 and HSV-2 with gC or gE mutant strains
derived from the WT viruses. Six sera from gD2-immunized
subjects (from HSV-014 and 015; Figure 1E) were evaluated.
Neutralization was significantly greater against WT HSV-1
resent standard errors of the mean. B, Enzyme-linked immunosorbent assay (ELISA) titers at a 1:500 dilution of vaccine sera (n=6). The dotted line shows the
ELISA value of serum specimens seronegative for both herpes simplex virus type 1 (HSV-1) and HSV-2. Results represent geometric mean titers with 95%
confidence intervals. P values in panels A and B were calculated by 1-way analysis of variance with Tukey posttest analysis for pair-wise comparison. C,
A, Log10neutralization of wild-type (WT), gCnull, and gEnull mutant viruses by sera from gD2 HSV-014 and 015 vaccinees (n=6). Error bars rep-
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(2.2 log10) than against WT HSV-2 (1.0 log10; Figure 2A), con-
firming results using the 50% end point neutralizing titer (Fig-
ure 1). The gD2 vaccine sera reduced the titer of HSV-1 gCnull
(NS-gCnull) virus by 2.2 log10and HSV-1 gEnull (NS-gEnull)
virus by 1.8 log10, which are not statistically different from find-
ings for WT HSV-1 strain NS, indicating that gC1 and gE1 do
not block neutralization by gD2 antibodies (Figure 2A). Strik-
ingly, gC2 and gE2 had a significantly greater effect on blocking
HSV-2 strain 2.12 neutralization, since the gD2 vaccine sera re-
duced the titer of HSV-2 gCnull (2.12-gC2null) virus by 3.3
Importantly, ELISA revealed that antibody produced by immu-
nization with the gD2 vaccine reacted equally well to soluble,
purified gD1 and gD2 proteins (Figure 2B), suggesting that an-
tibody bound comparably to both glycoproteins.
The gD2 amino acid sequence included in the vaccine has 84%
identity with gD1 ; therefore, cross-protection against HSV-1
is not surprising. However, it is surprising that neutralizing ti-
ters were significantly higher to HSV-1 than to HSV-2. The im-
pressive difference in neutralizing antibody titers provides a
plausible explanation for the protection against HSV-1 infec-
tion and disease reported in the Herpevac Trial. The low
HSV-2 mean neutralizing titer of 1:29 at month 7 may explain
the lack of protection observed against HSV-2.
The neutralizing titers of Herpevac Trial sera cannot be com-
pared with sera from the 1995–1997 studies since Herpevac
samples, not the 1995–1997 samples, were intentionallyselected
to include subjects with high, medium, and low ELISA titers.
In addition, higher antigen doses were used in some of the
1995–1997 studies. Nevertheless, the sera can be compared
for neutralizing titers to HSV-1 and HSV-2, since the same
serum sample was tested against both viruses. Neutralizing
titers were significantly higher to HSV-1 than to HSV-2 in
sera from subjects immunized with the gD2 vaccine prepared
years earlier than the Herpevac trial, which suggests that varia-
tions in vaccine lots cannot explain the higher HSV-1 titers.
Higher neutralizing titers to HSV-1 than to HSV-2 were
noted using a total of 4 HSV-1 and 3 HSV-2 low-passage iso-
lates, which supports the generalizability of the observation.
We previously reported that HSV-1 gC1 and gE1 did not
block neutralization by gD antibodies; however, gC1 and gE1
blocked neutralization when gD antibodies were combined
with neutralizing antibodies to other glycoproteins involved in
entry, including gB and gH/gL. We interpreted these results as
indicating that gC1 and gE1 block domains of interaction be-
tween gD1 and other entry molecules . Here we hypothe-
sized that gC2 and gE2 shield gD2 from neutralizing
antibodies, as a mechanism to explain the neutralizing antibody
results. The ratio of gC1 to gD1 molecules on HSV-1 strain NS
is 1:14, which may help explain the lackof blocking by gC1 .
Similar evaluations have not been performed with HSV-2;
therefore, comparing the stoichiometry of glycoproteins on
HSV-1 and HSV-2 is not yet possible.
Glycosylation of envelope proteins prevents antibody access
to neutralizing domains on HIV-1 gp120 and influenza hemag-
glutinin .Our results suggest that neighboring glycoproteins
also may block antibody access. A hypothetical model depicting
differences between gC and gE of HSV-1 and HSV-2 in block-
ing antibody access is shown in Figure 2C and 2D. Other poten-
tial mechanismsto explain greater neutralization of HSV-1 than
HSV-2 include the possibility that epitopes recognized by anti-
body on gD1 may be more essential for virus entry than anal-
ogous epitopes on gD2 or that fewer gD molecules are expressed
on HSV-1, making it easier to neutralize.
In the Herpevac Trial, ELISA titers correlated with protection
against HSV-1, while CD4+or CD8+T-cell responses did not
[1, 5]. We demonstrated here that ELISA titers correlated with
neutralizing antibody titers; however, it is possible that other
antibody responses are also important, including antibody-
dependent cellular cytotoxicity and mucosal antibodies. T-cell
responses that were not measured in the trial may also be im-
portant for protection.
Our results suggest that avaccine containing gD2 antigen will
likely induce higher neutralizing antibody titers to HSV-1 than
to HSV-2, which is encouraging in terms of potential benefits of
a vaccine that includes gD2. However, approaches are required
to improve the neutralizing titersto HSV-2. One possibility isto
induce antibodies to gD2 domains that are not shielded, while
other possibilities include using higher concentrations of anti-
gen, using more-potent adjuvants, or preventing antibody and
complement immune evasion by the virus to enhance the effi-
cacy of the antibodies produced .
providing gD1 and gD2 antigens.
of Allergy and Infectious Diseases (subcontract of grant N01-AI-45250, to
Saint Louis University) and GlaxoSmithKline.
Potential conflicts of interest.
R. B. served on a data safety monitoring
boardfor GSKonan unrelated vaccine.Allotherauthorsreportnopotential
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
We thank Gary Cohen and Roselyn Eisenberg for
This work was supported by the National Institute
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