Immunization with DNA vaccine expressing herpes simplex virus type 1 gD and IL-21 protects against mouse herpes keratitis

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 ?g/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-? and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-? and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.     

Clinical trials of prophylactic and therapeutic herpes simplex virus vaccines

Herpes simplex virus type 2 (HSV-2) is a suitable target for a vaccine, despite available antiviral therapies, because the virus causes lifelong infection and significant medical and psychosocial morbidity. A vaccine has the potential to reduce HSV acquisition, disease severity and the number of cases of neonatal herpes. It could also reduce transmission of HIV, which is epidemiologically linked to HSV. Prophylactic vaccines for HSV-2 must give broad and durable immunity across all mucosal surfaces to be effective. This is a significant challenge, as the major determinants of effective immunity have not yet been identified. Even if full protection cannot be achieved, vaccines would still be useful if they could increase the threshold of infection, or prevent clinical disease. However, it is possible that a vaccine could reduce symptomatic disease, but not eliminate asymptomatic shedding, which could inadvertently increase transmission from individuals who believe they are not infectious. Investigated prophylactic vaccines for HSV-2, including subunit vaccines encoding HSV glycoproteins packaged with adjuvants, have shown some benefits. The Chiron gD2gB2-MF59 vaccine gave transient protection of less than 6 months. The GlaxoSmithKline gD2-alum MPL vaccine conferred a 73-74% reduction in acquisition of symptomatic HSV-2 disease and a 38-42% reduction in the acquisition of HSV-2 infection in HSV-seronegative women, but gave no protection in men or HSV-1 seropositive women. Therapeutic vaccines aim to prevent HSV recurrences or minimise disease severity and duration, thereby reducing transmission. Research indicates that to be effective, therapeutic vaccines need to stimulate strong cell-mediated immune responses. Vaccines have induced HSV-specific antibody responses alone but have failed to protect recipients from recurrences. Further research is needed to define determinants of immunity to HSV-2, including identifying HSV-2 antigens, in order to design more effective vaccines.     

The importance of MHC-I and MHC-II responses in vaccine efficacy against lethal herpes simplex virus type 1 challenge.

To investigate the importance of major histocompatability complex (MHC) class I- and MHC class II-dependent immune responses in herpes simplex virus-1 (HSV-1) vaccine efficacy, groups of beta 2% (MHC I-) and Ab% (MHC II-) mice were inoculated with various vaccines, and then challenged intraperitoneally with HSV-1. Following vaccination with either live avirulent HSV-1, expressed HSV-1 glycoprotein D (gD), or a mixture of seven expressed HSV-1 glycoproteins (7gPs), Ab% (MHC-II-) mice developed no enzyme-linked immunosorbent assay (ELISA) or neutralizing antibody titres. In contrast, significant ELISA and neutralizing antibody titres were induced in beta 2m% (MHC-I-) mice by all three vaccines. The neutralizing antibody titres were similar for all three vaccines, but were only approximately 1/4 to 1/3 of that developed in C57BL/6 (parental) mice vaccinated with the same antigens. All three vaccines protected 100% of the wild-type C57BL/6 mice against lethal challenge with 2 x 10(7) plaque-forming units (PFU) of HSV-1. The live virus vaccine and the 7gPs vaccine also protected 80% of the beta 2m% mice against the same lethal HSV-1 challenge dose. In contrast, in Abo/o mice, none of the vaccines provided significant protection against the same lethal challenge dose of HSV-1. However, at a lower challenge dose of 2 x 10(6) PFU, all three vaccines protected 70-80% of the vaccinated Ab% mice (compared to only 10% survival in mock vaccinated controls). Thus, vaccination provided some protection against lethal HSV-1 challenge in both beta 2m% and Ab% mice; however, the protection was less than that seen in the parental C57BL/6 mice. In addition, Ab% mice were less well protected by vaccination than were beta 2m% mice. Our results suggest that (1) both MHC-I and MHC-II are involved in vaccine efficacy against HSV-1 challenge; (2) both types of responses must be present for maximum vaccine efficacy: and (3) the MHC-II-dependent immune response appeared to be more important than the MHC-I-dependent immune response for vaccine efficacy against HSV-I challenge.     

Coexpression of interleukin-2 enhances the immunization effect of a DNA vaccine expressing herpes simplex 1 glycoprotein D

In this study, DNA vaccines consisting of vector IRES-gD expressing Herpes simplex virus 1 (HSV-1) glycoprotein D (gD) and vector IRES-gD-IL-2 coexpressing HSV-1 gD and interleukin-2 (IL-2), respectively, were constructed. After intramuscular inoculation, both vaccines induced in BALB/c mice antibodies as assayed by ELISA and virus neutralization. However, IRES-gD-IL-2 elicited significantly higher levels of IgG (ELISA) and neutralizing antibodies than IRES-gD. Isotyping of sera from mice injected with IRES-gD-IL-2 revealed predominantly IgG2a antibodies. IRES-gD-IL-2 also elicited a higher delayed-type sensitivity (DTH) reaction. However, there was no difference in the protection against lethal challenge with HSV-1 between the two vaccines (P>0.05). The results suggest that the vaccination with IRES-gD-IL-2 can efficiently enhance the immune response of mice to HSV-1, particularly through increased cellular immunity.     

Immunization with Combined HSV-2 Glycoproteins B2:D2 Gene DNAs: Protection against Lethal Intravaginal Challenges in Mice

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2:D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOS>B2:D2 cocktail>D2>B2 gene vaccines. The vaginal external diseases in the B2:D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2:D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.     

HSV-2gD模拟抗原表位、HBsAg与IL-18融合蛋白DNA疫苗的免疫效果观察

目的:研究串联重组核酸疫苗pc(pcDNA3.1)-S(HBsAg)-P6-IL18和pc(pcDNA3.1)-S(HBsAg)-NP6-IL18对机体的免疫效果,P6是我们前期采用噬菌体展示技术筛选出的HSV-2gD的模拟抗原表位,NP6为与HSV-2gD模拟抗原表位P6最相似的天然抗原表位序列。方法:分别将空质粒pcDNA3.1(阴性对照组)和构建的真核表达质粒pc-S-P6-IL18和pc-S-NP6-IL18肌内注射免疫接种BALB/c小鼠3次,每次间隔2周。末次免疫后2周眼眶静脉采血,ELISA法检测小鼠血清特异性抗体滴度、IFN-γ及IL-18含量;末次免疫后一月,处死小鼠,无菌分离脾脏,用刀豆蛋白A刺激淋巴细胞,采用MTT法测定脾淋巴细胞增殖率。结果:重组核酸疫苗pc-S-P6-IL18和pc-S-NP6-IL18免疫小鼠后可刺激血清特异性抗体(抗HBsAg抗体和抗HSV-2gD模拟表位抗体)的产生,与阴性对照组相比可诱导分泌较高水平的IFN-γ和IL-18,可促进小鼠脾淋巴细胞的增殖。结论:重组核酸疫苗pc-S-P6-IL18和pc-S-NP6-IL18能诱导较强的细胞免疫和体液免疫,可用于今后预防HBV和HSV-2感染的研究。     

Glycoprotein D adjuvant herpes simplex virus vaccine

Herpes simplex virus (HSV) Type-1 and -2 are common infections that can cause primary and recurrent herpes labialis and genitalis, as well as gingivostomatitis, keratoconjunctivitis, encephalitis, disseminated infections in immunocompromised persons and neonatal infections. Despite several decades of HSV vaccine development, no effective vaccine has been developed until recently. The following review of the genital HSV-2 glycoprotein D (gD2t, t is for truncated) subunit vaccine formulated with a new adjuvant (AS04) containing alum and 3-O deacylated monophosphoryl lipid A (MPL) provides a background in which to evaluate the vaccine as well as a brief review of other approaches to herpes vaccines. The gD2t-AS04 vaccine has been demonstrated to be safe in several large clinical trials. In two trials, the vaccine reduced genital herpes disease by 73 and 74%, but only in females with no previous HSV infection. A large ongoing trial in HSV seronegative females will provide additional data on protection from HSV disease and infection.     

Estimation of the B lymphocyte precursor frequencies to herpes simplex type 1 glycoproteins by a limiting dilution assay

The precursor frequency of B lymphocytes from Balb/c mice producing HSV-1 glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) antibody was determined by limiting dilution analysis under conditions to detect antibody from the clonal progeny of a single B cell precursor. In spleens of naive mice the average gC frequency was 1/48,917 +/- 5,550, while gD was 1/73,330 +/- 15,898, and gB frequency was in excess of 1/100,000. Immunization with live HSV-1 (KOS) increased the B cell frequencies of all three glycoproteins to approximately 1:3,000; however, the serum gB antibody ELISA titer was fivefold higher than gC or gD.     

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.     

人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

目的 研发高效广谱的人高致病性禽流感病毒H5N1实验疫苗.方法 首先构建了含H5N1(安徽株)结构基因[血凝素(HA)、神经氨酸酶(NA)、基质蛋白M1与M2]的两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒(rTTV天坛株)疫苗,采用不同剂量(104 PFU或107PFU)或组合(疫苗单独或联合)方式于0、4周二次免疫BALB/c小鼠,初步比较分析抗原特异的体液(HA血凝抑制抗体、NA特异性抗体、中和抗体)与细胞免疫应答(IFN-γ ELISPOT)特点.结果 重组痘苗病毒疫苗可有效表达H5N1靶抗原;高剂量组的重组痘苗病毒疫苗可快速激发较强的针对各个抗原的抗体与针对血凝素与神经氨酸酶蛋白的细胞免疫应答,含血凝素蛋白的重组痘苗病毒疫苗亦可诱导明显的中和抗体;但各组重组痘苗病毒疫苗所激发的针对基质蛋白(M1,M2)的细胞免疫应答均较弱;两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒疫苗联合应用所激发的针对基质蛋白2(M2)的体液免疫应答明显强于单双顺反子(HAop/M2)疫苗单独应用.结论 本研究中制备的各组重组痘苗病毒疫苗可诱导多个抗原特异的体液与细胞免疫应答,该研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础.     

Protective immune correlates can segregate by vaccine type in a murine herpes model system.

A central tenet of vaccine development is to identify immune correlates of protection. Both plasmid-encoded gD as well as recombinant protein gD can protect mice from lethal herpes simplex virus (HSV) challenge. It is known that different vaccine modalities should induce different immune phenotypes. Yet, paradoxically, it is also thought that the basis for protection should rely on exploitation of vulnerabilities of the pathogen and therefore that the overlapping properties of these different vaccines would reveal insight into common immune mechanisms responsible for protection. We sought to investigate this question by comparing two different vaccine modalities in the HSV-2 mouse model. We observed that gD protein was a strong inducer of T(h)2-type immune responses, and overall antibody titers of IgG, IgE and IgA were significantly higher than those induced by plasmid gD vaccines. In contrast, the plasmid gD vaccine induced a strong T(h)1 bias. Following high-dose challenge the gD protein was most effective at providing protection. However, at lower lethal dose challenge, while both vaccines were protective with regards to survival, only the plasmid-vaccinated animals were protected from HSV-2 infection-induced morbidity. These studies suggest that these different vaccine modalities induce protection through unique non-overlapping mechanisms, supporting that vaccine correlates are associated with the types of immunogen rather than solely the pathogen.     

Protective immunity against lethal HSV-1 challenge in mice by nucleic acid-based immunisation with herpes simplex virus type-1 genes specifying glycoproteins gB and gD

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radio-immunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996     

Immunogenicity evaluation of DNA vaccines that target guinea pig cytomegalovirus proteins glycoprotein B and UL83

Vaccines are needed for control of congenital human cytomegalovirus (HCMV) infection. Although the species-specificity of cytomegaloviruses precludes preclinical evaluation of HCMV vaccines in animal models, the guinea pig cytomegalovirus (GPCMV), which causes disease in utero, is a relevant model for the study of vaccines against congenital infection. We investigated whether DNA vaccines that target two GPCMV proteins, glycoprotein B (gB) and UL83 (pp65), are capable of eliciting immune responses in vivo. After cloning each gene into an expression vector, DNA was delivered by intramuscular inoculation and by pneumatic epidermal delivery. In Swiss-Webster mice, anti-gB titers were significantly higher after epidermal delivery. After epidermal inoculation in guinea pigs, all gB-immunized animals (n = 6) had antibody responses comparable to those induced by natural infection. Viral neutralization titers ranged from 1:64 to greater than 1:128. A GPCMV UL83 DNA vaccine also elicited an antibody response in all immunized guinea pigs (n = 6) after epidermal administration. Immunoprecipitation and Western blot assays confirmed that immune sera were immunoreactive with virion-associated UL83 and gB proteins. We conclude that DNA vaccines against GPCMV structural proteins are immunogenic, and warrant further investigation in the guinea pig model of congenital CMV infection.     

DNA priming and killed virus boosting vaccination strategy can augment the immune response to pseudorabies virus

The immune efficacy of DNA vaccines containing three plasmids encoding gB, gC, and gD glycoproteins (Mix DNA) of Pseudorabies virus (PRV) or the plasmid for gC only (gC DNA), killed virus (KV) vaccine or combination of gC DNA, Mix DNA and KV vaccines was evaluated in mice using primeboost strategy. The mice vaccinated twice with Mix DNA, and once with KV generated higher levels of gCspecific and virus neutralization (VN) antibodies and a stronger cellular immune response than the mice vaccinated three times with the Mix DNA vaccine only. The highest level of VN antibodies were detected in mice vaccinated twice with KV vaccines alone or with combination of DNA and KV vaccines. The challenge of vaccinated mice with the lethal dose of PRV showed that the complete protection against PRV was achieved in the group of mice immunized with the DNA and KV vaccines combined. The results suggested that DNA priming followed by KV vaccine boosting could enhance the antibody response and cellular immunity against PRV infection in mice.     

Comparative studies of HSV-1 antigens solubilised from infected cells by using non-ionic or zwitterionic detergents

HSV-1 antigen preparations solubilised from Vero cells by using either the non-ionic detergent Nonidet P40 or the zwitterionic detergent Empigen BB, and purified on sucrose density gradients or over a sucrose cushion, were tested by ELISA with anti-HSV-1 glycoprotein monoclonal antibodies and by radioimmunoprecipitation (RIP) with polyclonal HSV-1 antiserum. Amongst several proteins detected in these preparations, the four major HSV-1 glycoproteins, gB, gC, gD, and gE, were found to be present. Differences between NP40 or Empigen-solubilised HSV-1 antigen preparations with respect to two of these glycoproteins, gB and gE, were detected by using a small panel of monoclonal antibodies. Comparative studies in mice showed the Empigen-solubilised HSV-1 antigen preparations elicited greater antibody responses and greater protection against lethal HSV-1 challenge infection than the NP40-solubilised preparation.     

Zwitterionic detergent solubilisation of HSV-1 surface antigens

Summary A preparation was obtained from herpes simplex virus type 1 (HSV-1)-infected cells using a zwitterionic detergent, Empigen BB. The preparation was partially-purified either by ultracentrifugation over a cushion of 20% sucrose or on a sucrose density gradient. Partial characterisation of these materials by ELISA, using both polyclonal and monoclonal antibodies showed them to contain at least four major HSV glycoproteins, gB, gC, gD and gE. Comparison of Empigen-extracted HSV-1 antigen preparations with preparations obtained using the non-ionic detergents Nonidet P40 or Triton-X-100 indicate that, using conventional procedures, separation of glycoproteins, B, C, D, and E from unwanted proteins may be facilitated using the former detergent.Immunization of mice with Empigen-extracted, partially-purified or gradient-purified antigen preparations elicited good levels of antibody detectable by ELISA and a high degree of protection against both HSV-1 and HSV-2 challenge infection. Such protection could be achieved using aqueous antigen preparations, but was augmented using aluminium hydroxide gel as an adjuvant. In general, Empigen-extracted HSV-1 antigen preparations elicited higher ELISA antibody levels and more complete protection against HSV challenge infection than NP40 or Triton-X-100-extracted preparations.The value and usefulness of the detergent Empigen for obtaining HSV surface antigen preparations and the role of these as potential vaccines against HSV infections, is discussed.     

Immune response to vaccinia virus recombinants expressing glycoproteins gE, gB, gH, and gL of Varicella-zoster virus

Immunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs.     

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.     

The role of neutralizing antibody and T-helper subtypes in protection and pathogenesis of vaccinated mice following ocular HSV-1 challenge.

In order to determine the possible correlation of specific immune responses with protection against mortality and ocular disease following ocular herpes simplex virus type 1 (HSV-1) challenge, BALB/c mice were vaccinated with different doses and regimens of baculovirus-expressed gD. Neutralizing antibody, virus titres in the eyes, corneal scarring, and survival were measured. In addition, infiltration into the cornea of CD4+ T cells and cells containing the lymphokines interleukin-2 (IL-2), IL-4, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were monitored on days 3, 7, 10, 14 and 21 post-challenge by immunocytochemistry. The vaccination regimens used induced varying degrees of immune responses and protection upon ocular challenge with HSV-1. Our results suggest that neutralizing antibody was the most important immune response in protecting mice against lethal ocular challenge and corneal scarring. TNF-alpha and IL-2 were not crucial in terms of survival and corneal scarring, since gD1 (one vaccination with 1 microg of gD) and gD0.1 (one vaccination with 0.1 microg of gD), both of which provided high levels of protection, showed no TNF-alpha or IL-2 expression. However, TNF-alpha and IL-2 were crucial in terms of virus clearance from the eyes, since gD3 (three vaccinations with 1 microg of gD), which had less virus in their eyes, had high numbers of TNF-alpha and IL-2 infiltrates. Finally, mock-vaccinated mice were not protected from death and corneal disease following HSV-1 challenge. Eyes of mock-vaccinated mice had little or no TNF-alpha or IL-2 responses and the strongest IL-4 and IL-6 responses.