Abstract. Much attention is presently focused on the
vaccination with certain epitopes of an antigen. To fur-
ther study the ability of neutralizing epitopes mapped
in the first 1515 nucleotides of glycoprotein B of herpes
simplex virus type-1 (gB-1) to induce neutralizing anti-
bodies, a DNA immunization approach was employed.
Vaccination of mice with a plasmid expressing the neu-
tralizing epitopes induced humoral immune responses,
although the antibody titre was significantly lower
than that of antibodies induced by the full-length gB-1
gene. Furthermore, the plasmid DNA could not protect
the mice against HSV-1 lethal challenge, but could sig-
nificantly prolong the survival time compared to mock-
Herpes simplex virus type 1 (HSV-1) is responsible
for a wide range of human diseases from the localized
infection such as orolabial or corneal lesions to life-
threatening encephalitis, neonatal disease and pneumo-
nia in the immunocompromised individuals (Hwang et
al., 1999; Stanberry et al., 2000; Roizman et al., 2001).
Primary infection is every time followed by the estab-
lishment of a latent infection in the nervous system,
where it can be reactivated, resulting in the reinfection
and spreading of the virus among uninfected popula-
tions. To control the infection, the development of an
effective vaccine that prevents or reduces the primary
and recurrent infections would be of great importance.
It seems that DNA vaccination would be a promising
approach to achieve an effective immunity against HSV
infections (Stanbery et al., 1996; Bernstein et al., 1999).
Such vaccines can be tailored to express only those epi-
topes involved in protection and to direct a selective
immunity (Rodrigues et al., 1998; An et al., 2000).
It has been shown that CTL activity, antibody and
T-helper responses can be induced by appropriate epi-
topes (Yu et al., 1998). Moreover, DNA vaccination
technology, as a discovery tool for the identification of
more immunogenic epitopes of an antigen, has facilitat-
ed the development of new effective vaccines.
HSV-1 glycoprotein B is essential for infectiveness,
virus penetration and cell fusion. It is also one of the
main targets for neutralizing immunity (Glorioso et al.,
1984). There are many reports indicating that gB-based
DNA immunization has provided the protection of ani-
mals against the lethal, latent and recurrent HSV infec-
tions (Mester et al., 2000; Gaselli et al., 2001; Baghian
et al., 2002). Beside the strong CD4+ and CD8+-related
immunity (Manickan et al., 1995; Wallace et al., 1999),
gB-1 elicits a considerable amount of neutralizing anti-
bodies against HSV-1 (Sanchez-Pescador et al., 1992).
Epitopes for neutralizing antibodies cluster in the three
major domains: D1, D2 and D5a (Navarro et al., 1992).
In the present study, using a DNA vaccination approach
the strength of the epitopes located in the D1 and D2
domains to induce neutralizing antibodies and protect
mice against HSV-1 lethal challenge has been investi-
gated. Mammalian expression vectors carrying the full-
length or a truncated derivate of the gB-1 gene encoding
D1 and D2 domains were constructed. The antibody
titre induced by the gB-1 derivate was measured and
compared to that of antibodies induced by the vaccine
candidate encoding full-length gB-1 or the wild-type
virus. Furthermore, the protection of vaccinated
BALB/c mice against the lethal challenge of HSV-1
was evaluated. The results showed that the full-length
Immunogenicity and Protective Effect of a DNA Construct
Encoding Certain Neutralizing Epitopes of Herpes Simplex
Virus Type-1 Glycoprotein B
( HSV-1 / glycoprotein B gene / neutralizing epitopes / DNA immunization )
T. BAMDAD1, M. H. ROOSTAEE1, M. SADEGHIZADEH2, F. MAHBOUDI3, A.
KAZEMNEJAD4, H. SOLEIMANJAHI1
1Department of Virology, School of Medical Sciences, 2Department of Genetics, School of Basic Sciences,
Tarbiat Modarres University, Tehran, Iran
3Department of Biotechnology, Pasteur Institute, Tehran, Iran
4Department of Biostatistics, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
Received April 15, 2005. Accepted June 15, 2005.
Corresponding author: Taravat Bamdad, Department of
Virology, School of Medical Sciences, Tarbiat Modarres
University, P.O.Box 14115-111, Tehran, Iran. Tel.: +98 21
8011001 ext 3880;
Fax: +98 21 8013030;
Abbreviations: CTL ñ cytotoxic T lymphocyte(s), gB ñ glyco-
protein B, HSV-1 ñ herpes simplex virus type 1, LD50 ñ lethal
dose 50%, TCID50 ñ tissue culture infectious dose 50%.
Folia Biologica (Praha) 51, 109-113 (2005)
gB-1 gene could protect all the inoculated mice against
the wild-type virus, while the truncated form could pro-
duce neutralizing antibody and delay the death follow-
ing the HSV-1 challenge.
Material and Methods
Virus and cell line
An HSV-1 isolate was obtained from a patient with
herpetic encephalitis signs. The isolated virus was con-
firmed to be HSV-1 by specific fluorescent mAb. The
virus was propagated in HeLa cell monolayer and
stored at -80∞C until further use.
Construction of plasmids
Glycoprotein B gene of the KOS strain of HSV-1
was cut out from the clone pAcgB-1 (a gift from Dr.
Ghiasi, UCLA, Los Angeles, CA) (Ghiasi et al., 1999)
using BamHI restriction enzyme. The pcgB plasmid
expressing gB-1 was constructed by the insertion of the
gB-1 gene into the BamHI site of the pcDNA3 vector
under the control of CMV promoter. The clones were
screened and the orientation of the inserted DNA was
determined using PvuI restriction enzyme.
To construct a vector expressing neutralizing epi-
topes of gB-1, a 1515 nt fragment of the gene was
obtained by cutting the pAcgB-1 with BamHI and PstI
restriction enzymes. This fragment encodes 475 amino-
terminal residues of the mature protein carrying the
major continuous and discontinuous neutralizing epi-
topes. The PstI adaptor was engineered to encode a stop
codon in all three reading frames. The adaptor was
synthesized by GENSET SA (Paris, France) in two sep-
arate strands (5íG TAGATAGATAGT3í and 3íACGT-
CATCTATCTATCAGATC5í). Formation of ds-oligo-
mers created PstI and XbaI overhangs at the 5í and 3í
ends, respectively. The synthetic fragments were not
phosphorylated at the 5íends to eliminate the possibili-
ty of tandem formation. Thus, ligation of the fragments
and the adaptors with the pcDNA3 vectors digested
with BamHI and XbaI took place in a one-step reaction.
The presence of the fragment in the constructed vector
(pc1500) was then determined with suitable restriction
enzyme digestion, Southern blot and DNA sequence
Transfection and expression analysis
The subconfluent COS-7 cells grown on coverslips
were transfected with each construct using DOTAP
liposome (Roche Inc., Mannheim, Germany) according
to the manufacturerís instruction. Regarding the pcgB
vector 72 h after transfection, the cells were washed 3
times with PBS and fixed with absolute methanol at
ñ20∞C for 10 min. The expression of gB-1 was analysed
by indirect immunofluorescence. The cells were incu-
bated for 45 min at 37∞C with hyperimmune human
serum diluted 1/100 in PBS. The cells were then
washed and incubated at 37∞C for 45 min with fluores-
cein isothiocyanate-conjugated antihuman IgG and
counterstained with 0.01% Evans blue. The coverslips
were mounted with 80% glycerol in phosphate buffer
and examined by fluorescent microscopy.
To analyse the expression of the truncated form of
gB-1 the same protocol was used except that for cyto-
plasmic detection of the secretory protein, a time course
analysis was applied.
Immunization of mice
Groups of 3ñ4 weeks old female BALB/c mice
(6/group) were each given three injections of pcgB or
pc1500 plasmids, at 21 day intervals, at a dose of 90 µg
per mouse intramuscularly. The control groups were
injected with a sublethal dose of HSV-1 as positive con-
trol and pcDNA3 and PBS as two separate negative
Fourteen days after the last immunization, the sera
were collected from the vaccinated mice to check for
specific antibodies. The sera were heat-inactivated at
56∞C for 30 min. One hundred µl of two-fold dilutions
of each serum were added to a 96-well plate and 100 µl
of the virus suspension containing 100 TCID50of HSV-1
were then added to each well. The plate was incubated
at 37∞C for 1 h. The reaction mixtures were then added
to the 3 × 103HeLa cells grown on a 96-well plate.
After 1 h adsorption, 100 µl of DMEM supplied with
4% FBS were added to each well. The highest dilution
of each serum that neutralized the virus in 24 h was
taken as the serum titre.
Three weeks after the last inoculation, all the mice
were challenged with 10 LD50of the virus intraperi-
toneally. The challenged mice were monitored for a
month. To confirm that the death of the challenged mice
was due to the HSV-1 infection, samples of brains and
lungs of the sacrified mice were subjected to HSV-1
For comparison of antibody titres, analysis of vari-
ance (ANOVA) and a multiple comparison test of LSD
(Least Significant Differences) were used. Mice sur-
vival data were analysed for time using Kaplan-Meier
Cloning and in vitro expression
We generated two DNA constructs; pcgB and
pc1500 using the pcDNA3 expression vector. The plas-
mid pcgB was constructed by the insertion of the full-
length gB-1 into the BamHI site of the pcDNA3 vector.
T. Bamdad et al.110 Vol. 50
The pc1500 contained the gB-1 BamHI-PstI fragment
encoding the 475 amino-terminal residues of the gB-1.
The plasmids were characterized by restriction analy-
sis, Southern blot detection and sequence analysis of
the inserted genes. The sequenced regions were com-
pletely identical with those of the KOS strain of HSV-1.
The expression of full-length gB-1 was confirmed in the
transfected COS-7 cells by immunofluorescence reac-
tion using HSV-1 polyclonal antibody 72 h after the
transfection. For cytoplasmic detection of the secretory
protein expressed by pc1500, the cells were tested
20ñ60 h after transfection. The cytoplasmic detection of
gB in transfected cells showed an increased expression
To evaluate the humoral immune responses induced
by the neutralizing epitopes, the antibody titres induced
by pcgB, pc1500 and the live HSV-1 were compared in
the vaccinated mice. The sera were assayed for specific
antibody by the virus neutralization test. Table 1 shows
the average of log10 titres of neutralizing antibody in
each group. All the test and positive control groups
showed antibody responses; however, the mice in the
HSV-1 group had a higher antibody level than the other
groups. Compared to the live HSV-1 immunized group,
the average log10of the antibody level of the mice inoc-
ulated with pcgB and pc1500 were about 1.3 and 3.8
fold lower, respectively. However, the mean antibody
titre of the pc1500 group was higher than those of the
mock-infected animals. All differences among the anti-
body titres of the test groups and also among the test
groups and the control groups were statistically signifi-
cant (P < 0.05).
Protection against HSV-1 lethal challenge
A challenge dose of 10LD50was used to assess the
protection induced by the gB-1 gene or its derivate. All
the groups of mice were challenged by i.p. injection of
HSV-1 and monitored daily for clinical signs and mor-
tality (Fig. 1). Similar to those of HSV-1-vaccinated
animals, the survival rate of the mice receiving pcgB
was 100%, although one of them showed the sign of
transient paralysis in its legs. The protection rate in the
pc1500-injected animals against mortality was restrict-
ed to 33% of the cases, but the median survival time
was prolonged in comparison with the pcDNA3- and
PBS-injected groups. While 83% of the mock-vaccinat-
ed mice died within 8 days, in the pc1500 group the
median survival time was 15 days after the challenge
DNA immunization is a promising approach to
developing effective vaccines. In addition, compared to
other recombinant vaccination methods, the advantages
of DNA immunization as a discovery tool provide a
rapid way to identify more immunogenic epitopes of an
Immunogenicity and Protective Effect of a DNA ConstructVol. 50
Table 1. Antibody titres in the mice after immunization
The mice were immunized with various immunogens 3
times, at 21 day intervals. On day 14 after the last immu-
nization, serum samples were collected and titered for
specific antibody by the neutralization test. The data pre-
sented in the Table are the mean of log10antibody titres in
each group ± SEM. The LSD test showed significant dif-
ferences among the titres (P < 0.05).
Vaccine group No. of
1.76 ± 0.22
0.6 ± 0.33
2.3 ± 0.15
* mean ± SEM (log 10)
Table 2. Resistance to HSV-1 i.p. challenge in the immu-
Mean time of
± SEM (days)
14.17 ± 1.48
9 ± 1.49
9.17 ± 1.5
The mice were immunized with various immunogens 3
times, at 21 day intervals. On day 28 after the last immuni-
zation, the mice were challenged with 10 LD50of HSV-1.
The mice were monitored daily for survival.
Median time of survival shows that 50% of animals had
survived at the indicated time.
ND ñ no death was detected.
Fig. 1. The effect of immunization with pcgB and
pc1500 vectors on the survival of the mice infected with
10 LD50of HSV-1. Groups of mice injected with
pcDNA3, PBS and sublethal dose of HSV-1 were used as
control groups. The mice (6 mice /group) were immunized
3 times, at 21 day intervals intramuscularly. On day 21
post-immunization, mice were challenged with HSV-1
and monitored daily for mortality.
antigen and their capacity to elicit effective immunity.
Definition of these epitopes will provide a rationale for
tailoring recombinant vaccines.
Some of the viral envelope glycoproteins have been
used as immunogens in recombinant vaccines. Due to
the role of gB and gD in HSV infectiveness, they have
received the highest attention. We chose gB-1 because
it contains well-characterized epitopes for induction of
humoral and cellular immunity. Although there are
some reports of identification of the neutralizing epi-
topes of gB-1, little is known about the quantitative
antibody responses induced by these epitopes.
Regarding the earlier studies, the truncated form of gB-1
(707 aa), which lacks the transmembrane and cytoplas-
mic domains, could be an effective vaccine in the induc-
tion of humoral immune responses (McClements et al.,
1996). The aim of the present study is to further char-
acterize the gB-1 neutralizing epitopes in a DNA vacci-
nation approach and also to evaluate the strength of the
epitopes located on the first 475 amino acids of gB-1
(D1 and D2 domains) to induce antibody responses.
Furthermore, we wished to determine the ability of
these epitopes to protect against the lethal challenge
with HSV-1. Based on the previous works done on the
epitope map of gB-1(Navarro et al., 1992), we chose the
first 1515 nt of the gB-1 gene as the major neutralizing
epitopes. It has been shown that the gB-1 neutralizing
activity depends on the three major domains, D1, D2
and D5a. These epitopes are recognized by the comple-
ment-dependent and -independent neutralizing antibod-
ies (Navarro et al., 1992). D1 and D2 domains contain
continuous and discontinuous epitopes. Continuous
neutralizing epitopes are mapped between amino acids
32 and 47 and discontinuous residues are located
between amino acids 273 to 298, but additional residues
are required to assemble these discontinuous epitopes
(Qadri et al., 1999). At least 457 amino-terminal
residues are required to react antibodies with the dis-
continuous epitopes (Pereira et al., 1989). Other dis-
continuous epitopes have also been mapped in these
domains (Highlander et al., 1991). It has been shown
that similarly to the native glycoprotein, the 1-475
amino acid derivate of gB-1 translocates to the cell sur-
face (Navarro et al., 1991). The amino acids 1-475 were
chosen in this study to provide both the major neutral-
izing epitopes and the proper translocation of the pro-
tein. Although D5a contains a cluster of important
discontinuous epitopes, they are only active in the
dimer form of gB (Navarro et al., 1992), so it was not
possible to study the D5a domain as a separate region.
We constructed the vectors containing the full-length or
a truncated form of the gB-1 gene encoding intact gB or
epitopes of D1 and D2 domains. Beside measuring the
neutralizing antibodies induced by the vectors, a lethal
challenge model was used to compare the protective
effect of each vector in protection of the vaccinated
mice. Our data showed that immunization with both
vectors induced immune responses in the mice.
Although the antibody titre in the pcgB-injected group
was significantly lower than that of the HSV-1-vacci-
nated group, pcgB was as effective as HSV-1 in protec-
tion of mice. In comparison with pcgB, pc1500
generated lower antibody responses and provided only
minimal protection. However, it is of note that the
pc1500 significantly prolonged the survival time as
compared with negative controls.
It is difficult to interpret the effect of pc1500 in
immunization of mice. It is clear that full-length gB-1
bears additional epitopes that induce neutralizing anti-
bodies (600-690 amino acids). Besides, it is likely that
the folding of the truncated form of gB-1 used in this
experiment has been altered in comparison with the
native protein (Del Val et al., 1991; Higgins et al., 2000)
or, more probably, the processing of this secretory pro-
tein has been altered from that of the wild-type gB and
the protein was released from the cells more slowly
(Qadri et al., 1999). The presence of CTL and T-helper
inducing epitopes in D1 and D2 domains are not com-
pletely determined. Several studies have demonstrated
H-2d-restricted CTL (Hanke et al., 1991) and T-helper
epitopes in the N-terminus of gB, but it seems that they
are not the dominant epitopes for induction of cellular
immunity (Wallace et al., 1999). Our findings showed
that pc1500 could induce CD4+T-cell response, but the
stimulation index has been decreased in the pc1500-
immunized mice compared to those receiving pcgB
(data not shown). Beside the existence of neutralizing
epitopes there are some antibody-dependent cellular
cytotoxicity (ADCC)-related epitopes in these domains,
but lack of effective cell-mediated immunity in the
pc1500 vaccinee may be the main reason for the lower
protection induced by pc1500. In the context of protec-
tion, regarding the neutralizing activity of pc1500 and
with respect to the minimal cell-mediated immunity
induced by the vector, it is not surprising that pc1500
could not protect the animals against lethal challenge.
Neutralizing antibodies do not inhibit HSV replication
especially in the neurons, although they are among the
immune factors that determine the virus load and sever-
ity of the infection (Mikloska et al., 1999). ADCC and
neutralizing antibodies have been shown to affect the
outcome of HSV infection (Sanchez-Pescador et al.,
1992). These findings are in accordance with our data in
which the antibodies induced by pc1500 postponed the
death following challenge with the wild-type virus.
In conclusion, the results of this study might improve
our knowledge in the field of modern vaccinology using
molecular tailoring of antigens toward a desired direc-
tion of immunity and development of more effective
We thank Dr. Ghiasi for providing us with the
T. Bamdad et al. 112Vol. 50
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