Comparison of the Adjuvant Activity for the Glucosaminyl-Muramyl Dipeptide and the Granulocyte-Macrophage Colony-Stimulating Factor Gene in Gene Immunization against the Herpes Simplex Virus

The adjuvant activity in DNA immunization against herpes simplex virus type 1 (HSV1) was studied for the granulocyte-macrophage colony-stimulating factor (GM-CSF) synthesized from an eukaryotic expression plasmid (pDNAGM-CSF) and for the synthetic glucosaminyl-muramyl dipeptide (GMDP). A plasmid containing the HSV gD gene (pDNAgD) was used as an immunogen. GMDP and pDNAGM-CSF each enhanced the T-cell immune response to DNA immunization. The protective effect of DNA immunization increased from 63 to 100% when the two plasmids were injected simultaneously and to 96% when pDNAgD was injected one day after injecting GMDP. The results showed that DNA vaccines combined with genetic or peptide adjuvants are promising for DNA immunization against HSV.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 504–512.Original Russian Text Copyright © 2005 by Kozlov, Klimova, Shingarova, Boldyreva, Nekrasova, Guryanova, Andronova, Novikov, Kushch.     

单纯疱疹病毒I型糖蛋白D胞外区的真核表达及生物学活性分析

单纯疱疹病毒 (Herpes simplex virus,HSV) 包膜糖蛋白D (glycoprotein D,gD) 是HSV的结构蛋白之一,具有重要抗原表位,是目前疫苗研究的热点。为了分离纯化HSV gD1糖蛋白胞外区片段并对其生物学活性进行分析,本研究将化学合成的gD1胞外区基因片段克隆至真核表达载体pCEP4,重组质粒转染HEK293细胞进行瞬时表达,产物经Western blotting检测后用亲和层析法进行分离纯化,ELISA检测其抗原性。以纯化的重组蛋白作为抗原免疫小鼠,ELISA测血清特异性抗体效价以评价其免疫原性。构建的重组质粒经测序显示基因序列完全正确。表达产物的Western blotting分析发现,在相对分子量约46 kDa处有外源蛋白表达,与预期蛋白带一致。用Ni柱得到了纯化的重组gD1蛋白,ELISA检测显示其具有良好的抗原性,免疫小鼠7周后血清中抗体效价达到5×103。重组gD1蛋白的抗原性及免疫原性分析为HSV检测试剂和基因重组亚单位疫苗的研制提供了实验依据。     

真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析

为在真核细胞中表达并纯化I型单纯疱疹病毒（HSV I）包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。     

Effect of tumor necrosis factor DNA on immune response to DNA immunization against herpes simplex virus

A study was made of the adjuvant effect of the mouse tumor necrosis factor alpha (mTNF alpha) on DNA immunization against the herpes simplex virus type 1 (HSV1). The HSV1 gD gene (pDNAgD) served as an immunogen; mTNF alpha or its gene cloned in an eukaryotic expression vector (pDNAmTNF) were used to modulate the immune response. Double immunization with pDNAgD led to a sixfold increase in the in vitro T-cell response, a high (1:2000) titer of anti-HSV1 antibodies (including virus-neutralizing antibodies), an increase in IgG2a/IgG1 (suggesting a shift of the immune response to the Th1 type), and no change in CD4/CD8 T-cell ratio. A single injection of mTNF alpha along with inactivated HSV1 allowed a twice higher antibody titer and a fourfold higher T-cell response as compared with immunization with HSV1 alone. Double immunization with both pDNAgD and pDNAmTNF increased the titer of anti-HSV1 antibodies and the T-cell response by factors of 8 and 1.5, respectively, as compared with immunization with pDNAgD alone. However, the protective effect was significantly lower with the two plasmids than with pDNAgD (73 vs. 100%). Thus, DNA immunization with pDNAgD induced both B- and T-cell responses and completely protected mice from a lethal doze of HSV1. The adjuvant properties of mTNF alpha and pDNAmTNF need further investigation.     

Functional interaction between herpes simplex virus type 2 gD and HVEM transiently dampens local chemokine production after murine mucosal infection

Herpes virus entry mediator (HVEM) is one of two principal receptors mediating herpes simplex virus (HSV) entry into murine and human cells. It functions naturally as an immune signaling co-receptor, and may participate in enhancing or repressing immune responses depending on the natural ligand used. To investigate whether engagement of HVEM by HSV affects the in vivo response to HSV infection, we generated recombinants of HSV-2(333) that expressed wild-type gD (HSV-2/gD) or mutant gD able to bind to nectin-1 (the other principal entry receptor) but not HVEM. Replication kinetics and yields of the recombinant strains on Vero cells were indistinguishable from those of wild-type HSV-2(333). After intravaginal inoculation with mutant or wild-type virus, adult female C57BL/6 mice developed vaginal lesions and mortality in similar proportions, and mucosal viral titers were similar or lower for mutant strains at different times. Relative to HSV-2/gD, percentages of HSV-specific CD8(+) T-cells were similar or only slightly reduced after infection with the mutant strain HSV-2/gD-Δ7-15, in all tissues up to 9 days after infection. Levels of HSV-specific CD4(+) T-cells five days after infection also did not differ after infection with either strain. Levels of the cytokine IL-6 and of the chemokines CXCL9, CXCL10, and CCL4 were significantly lower in vaginal washes one day after infection with HSV-2/gD compared with HSV-2/gD-Δ7-15. We conclude that the interaction of HSV gD with HVEM may alter early innate events in the murine immune response to infection, without significantly affecting acute mortality, morbidity, or initial T-cell responses after lethal challenge.     

The Effect of the Tumor Necrosis Factor DNA on the Immune Response in DNA Immunization against the Herpes Simplex Virus

A study was made of the adjuvant effect of the mouse tumor necrosis factor (mTNF) on DNA immunization against the herpes simplex virus type 1 (HSV1). The HSV1 gD gene (pDNAgD) served as an immunogen; mTNF or its gene cloned in an eukaryotic expression vector (pDNAmTNF) were used to modulate the immune response. Double immunization with pDNAgD led to a sixfold increase in the in vitro T-cell response, a high (1:2000) titer of anti-HSV1 antibodies (including virus-neutralizing antibodies), an increase in IgG2a/IgG1 (suggesting a shift of the immune response to the Th1 type), and no change in CD4/CD8 T-cell ratio. A single injection of mTNF along with inactivated HSV1 allowed a twice higher antibody titer and a fourfold higher T-cell response as compared with immunization with HSV1 alone. Double immunization with both pDNAgD and pDNAmTNF increased the titer of anti-HSV1 antibodies and the T-cell response by factors of 8 and 1.5, respectively, as compared with immunization with pDNAgD alone. However, the protective effect was significantly lower with the two plasmids than with pDNAgD (73 vs. 100%). Thus, DNA immunization with pDNAgD induced both B- and T-cell responses and completely protected mice from a lethal doze of HSV1. The adjuvant properties of mTNF and pDNAmTNF need further investigation.     

Transplantation of syngeneic transfected cells to probe the in vivo immune response to viral proteins.

BALB/3T3 cells were transfected with the glycoprotein D (gD) gene of herpes simplex virus (HSV) and a cell line expressing gD on the cell surface was isolated. In vitro, 51Cr release tests showed that the transfected cells were destroyed by anti-HSV antibody in the presence of complement. To investigate in vivo immune response, the gD-transfected cells were transplanted into the footpads of syngeneic HSV-immunized or unimmunized BALB/c mice. In unimmunized mice, transfected cells remained intact for 7 days or longer, and the site of injection showed only slight lymphocyte infiltration. In contrast, in immunized mice, transfected cells elicited massive lymphocyte infiltration and were mostly destroyed by day 4. Analysis of infiltrating cells revealed that they were mainly Thy1+ and CD8+ lymphocytes along with small numbers of CD5+, CD4+, and B lymphocytes. These studies show that transfected murine cells expressing gD can be used to study the in vivo immune response to single viral proteins and they argue that the immune response contributes to the pathogenesis of HSV infection.     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.     

A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms

We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.     

Efficacy of recombinant herpes simplex virus 1 glycoprotein D candidate vaccines in mice

To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.     

Modulation of the immune response mediated by oral transgene administration of IL-10

In the current study, we analyze the immunomodulatory effect of oral transgene administration of IL-10 using a mice model of viral inflammation. Salmonella harboring a plasmid encoding the IL-10 gene (SLIL10) was administered by the oral route together with Salmonella carrying a plasmid encoding the glycoprotein D or B (SLgD, SLgB) of Herpes simplex virus type 2 (HSV-2). This resulted in a high inhibition of the cellular and human immune response against the viral proteins. When mice immunized against the HSV proteins were challenged with 10 lethal doses of HSV-2 by the intravaginal route, only those that had also received SLIL10 showed severe lesions and died. When Salmonella harboring pIL10 was administered orally to mice immunized by the intramuscular route with a plasmid encoding gD, inhibition of cellular and humoral immune responses were also observed but to a lesser extent than with oral immunization. By means of adoptive transfer experiments and in vitro experiments, we have subsequently determined that the mechanism possibly involved in the inhibition of the immune response could be a reduced antigenic presentation when mice receive SLIL10 that induced a state of anergy on specific T lymphocytes.     

人tPA信号肽和Yersinia Pestis保护性抗原F1-V融合基因的构建及其免疫效果观察

为获得含有鼠疫F1和V抗原编码基因以及人tPA信号肽基因的重组质粒tPA-pVAX1/F1-V,并测定其诱导特异性免疫应答的能力, 用PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序,构建pVAX1/F1-V融合重组质粒.PCR扩增tPA信号肽片段并将其插入到F1-V的上游,构建tPA-pVAX1/F1-V融合重组质粒;转染COS-7细胞,Western blot法鉴定目的蛋白的表达.重组质粒tPA-pVAX1/F1-V加GM-CSF佐剂免疫BALB/c小鼠,观察免疫效果.400个LD50强毒鼠疫菌皮下攻毒观察保护率.结果表明,tPA-pVAX1/F1-V在COS-7细胞中表达;免疫鼠体内产生特异性抗体;抗体亚型分析、细胞因子等指标的测定表明,所构建DNA疫苗以诱发Th1型免疫为主;攻毒保护率达90%.结果提示,已成功构建F1-V融合蛋白真核表达载体tPA-pVAX1/F1-V,且具有诱导特异性细胞免疫和体液免疫应答的能力, 对强毒鼠疫菌皮下攻毒有一定的保护效力,为鼠疫菌新型疫苗研制奠定了基础.     

DNA immunization with Plasmodium falciparum serine repeat antigen: regulation of humoral immune response by coinoculation of cytokine expression plasmid

We immunized mice with plasmid expressing the 47-kDa amino-terminal domain of the Plasmodium falciparum serine repeat antigen (SERA) using gene gun and investigated humoral immune response to SERA antigen. Significant SERA-specific IgG was observed in BALB/c mice after immunization three times with SERA expression plasmid. Furthermore, these levels were increased by the coinoculation of cytokine (IFN-gamma, IL-4, GM-CSF, or IL-12) expression plasmid. In respect to the SERA-specific Ig subclasses, coinoculation of IFN-gamma, GM-CSF, or IL-12 expression plasmid increased the levels of SERA-specific IgG2a, and these were much higher than that in mice immunized with SERA expression plasmid alone. In contrast to the SERA-specific IgG2a, coinoculation of any cytokine expression plasmid did not change the levels of SERA-specific IgG1. These results indicate that cytokine expression plasmid enhances and regulates humoral immune response elicited by SERA DNA immunization.     

Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-gamma) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-gamma and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-gamma and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.     

Expression of human immunodeficiency virus type 1 gp120 from herpes simplex virus type 1-derived amplicons results in potent, specific, and durable cellular and humoral immune responses

Herpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 10(6) infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8(+)-T-cell-mediated response to an H-2D(d)-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 10(6)-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 10(4) i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.     

Viral determinants of the variable sensitivity of herpes simplex virus strains to gD-mediated interference.

Cells that express glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) resist infection by HSV-1 and HSV-2 because of interference with viral penetration. The results presented here show that both HSV-1 and HSV-2 gD can mediate interference and that various HSV-1 and HSV-2 strains differ in sensitivity to this interference. The relative degree of sensitivity was not necessarily dependent on whether the cell expressed the heterologous or homologous form of gD but rather on the properties of the virus. Marker transfer experiments revealed that the allele of gD expressed by the virus was a major determinant of sensitivity to interference. Amino acid substitutions in the most distal part of the gD ectodomain had a major effect, but substitutions solely in the cytoplasmic domain also influenced sensitivity to interference. In addition, evidence was obtained that another viral gene(s) in addition to the one encoding gD can influence sensitivity to interference. The results indicate that HSV-1 and HSV-2 gD share determinants required to mediate interference with infection by HSV of either serotype and that the pathway of HSV entry that is blocked by expression of cell-associated gD can be cleared or bypassed through subtle alterations in virion-associated proteins, particularly gD.     

A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.     

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.     

Antibody-inducing properties of a prototype bivalent herpes simplex virus/enterotoxigenic Escherichia coli DNA vaccine

The antibody-inducing properties of a bacterial/viral bivalent DNA vaccine (pRECFA), expressing a peptide composed of N- and C-terminal amino acid sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) fused with an inner segment encoding the major structural subunit of enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae (CFA/I), was evaluated in BALB/c mice following intramuscular immunization. The bivalent pRECFA vaccine elicited serum antibody responses, belonging mainly to the IgG2a subclass, against both CFA/I and HSV gD proteins. pRECFA-elicited antibody responses cross-reacted with homologous and heterologous ETEC fimbrial antigens as well as with type 1 and type 2 HSV gD proteins, which could bind and inactivate intact HSV-2 particles. On the other hand, CFA/I-specific antibodies could bind but did not neutralize the adhesive functions of the bacterial CFA/I fimbriae. In spite of the functional restriction of the antibodies targeting the bacterial antigen, the present evidence suggests that fusion of heterologous peptides to the HSV gD protein represents an alternative for the design of bivalent DNA vaccines able to elicit serum antibody responses.