Resistance to herpetic stromal keratitis in immunized B-cell-deficient mice

This study evaluates the role of antibody as an indicator of immunity to ocular challenge with herpes simplex virus (HSV). Two genotypes of mice, BALB/c or BALB/c with mu-chain knockout (muK/O; which lack functional B cells), were immunized systemically either with nonvirulent infectious virus or with a eukaryotic expression plasmid encoding glycoprotein B (gB). Whereas naive muK/O mice were 10- to 100-fold more susceptible to HSV infection than BALB/c mice, following immunization both groups showed similar levels of resistance to ocular challenge. Thus both HSV-immunized groups cleared virus within 3 days and showed no signs of ocular lesions. gB DNA-immunized mice cleared virus less rapidly (5 days), and the incidence of lesions was 10 and 25% in BALB/c and muK/O mice, respectively. Since muK/O mice failed to produce detectable anti-HSV antibody, the mechanism of rapid viral removal was assumed to have a T cell basis. However, T cells would likely not mediate any protection directly since such cells were absent in infected corneas during clearance. A likely mechanism of immunity could involve innate defenses, perhaps enhanced by the action of cytokines released from antigen-reactive CD4+ cells in vascularized tissue adjacent to the cornea. Thus an abrupt inflammatory response consisting principally of neutrophils occurred in the corneal stroma in immune mice, and this subsided when virus disappeared. These data reveal that even though the deficiency in generating antibody renders mice more susceptible to HSV infection, once primed, resistance to disease expression is mediated solely by the cellular components and their products.     

Mucosal application of plasmid-encoded IL-15 sustains a highly protective anti-Herpes simplex virus immunity

In a DNA immunization against Herpes simplex virus (HSV), we examined the ability of plasmid-encoded interleukin-15 (pIL-15) to induce and maintain the mucosal B and T cell immune response. pIL-15 generated memory CD8(+) T cell responses that were threefold higher and mainly maintained in the spleen, but high levels of immunoglobulin A antibodies were induced and maintained long-term in the vaginal mucosa. Both of these enhanced components of the immune responses were recalled rapidly upon challenge with a lethal dose of HSV McKrae, affording protection in mice immunized with codelivery of pIL-15. Our results show for the first time that intranasal administration of pIL-15 along with plasmid-encoded glycoprotein B of HSV leads to enhancement of primary and memory CD8(+) T cell responses as well as humoral immune response. Therefore, a mucosal immunization strategy that incorporates a potent cytokine such as IL-15 as an adjuvant might induce protective mucosal immune responses that constitute the initial barrier at mucosal portals of pathogen entry.     

Assessment of a cellular vaccination approach consisting of crawling dendritic cells (CDCs) transduced with HSV-1-†pac vectors

Crawling dendritic cells (CDCs) and herpes simplex virus-1 (HSV-1) amplicon vectors were utilized in this study: (1) to evaluate whether CDCs can be transduced by HSV-1 amplicon vectors; (2) to assess the effects of HSV-1 infections on structure and functions of CDCs; (3) to assess the capabilities of the transduced CDC to express, process, and present the transgene products; and (4) to induce in vitro and in vivo priming of T cells and B cells. CDC supported amplicon-mediated transgene expression while retaining the ability to perform mixed lymphocyte reaction (MLR) and priming of naive T cells. Then it was tested whether transduced CDC were able to initiate immunity ggainst either the amplicon particle and/or the product encoded by the delivered transgene by injecting groups of mice with transduced CDCs expressing GFP or LacZ. Spleen cells of these mice were stimulated by co-incubation with cells expressing: (1) either one of the transgenes (GFP or LacZ), (2) peptides of β-gal, or (3) peptides of HSV-1 glycoprotein B (gB). Interestingly, no significant cytotoxic T lymphocyte (CTL) activity against the transgenes or against gB was observed. In contrast, mice developed high levels of antibodies against gB and LacZ. Mainly, the findings that CDCs not only express amplicon-delivered transgene, but were able to induce MLR and priming of naïve T cells against the transduced antigen. open up unexpected possibilities and the likelihood to use CDCs as a vehicle for cellular immunization against any transduced antigens. However, these results indicate that HSV-1 amplicon-transduced CDCs induce effective priming and a humoral response, but no strong cell-mediated immune response.     

Relatedness of glycoproteins expressed on the surface of simian herpesvirus virions and infected cells to specific HSV glycoproteins

Summary The antigenic relatedness of the surface glycoprotein antigens of six herpesviruses indigenous to human and nonhuman primates was examined. Binding of anti-viral sera to viral antigens expressed on the surface of infected cells demonstrated that the surface antigens of herpes simplex virus type 1 (HSV 1), HSV 2, simian agent 8 (SA 8), andHerpesvirus simiae (B virus) exhibit extensive cross-reactivity. Surface antigens of two viruses isolated from South American primates,H. saimiri 1 (HVS 1) andH. ateles 1 (HVA 1), were comparatively more virus-specific in their antigenic reactivity. Endpoint neutralization tests performed in the presence and absence of complement confirmed these results. Immunoprecipitation of viral proteins was used to identify those representing cross-reactive surface antigens. A glycoprotein of approximately 110,000–125,000 Daltons (110–125 k) was immunoprecipitated from cells infected with each of the six primate herpesvirus by antisera to each of the viruses. Using monospecific antisera, these glycoproteins were shown to be antigenically related to the gB glycoproteins of HSV. Although these glycoproteins were antigenically conserved among all six viruses, antibodies to the gB glycoproteins did not cross-neutralize heterologous viruses. A glycoprotein of approximately 60–70 k was precipitated from HSV 1, HSV 2, SA 8, and B virus infected cells by antisera to each of these four viruses. These SA 8 and B virus glycoproteins were shown to be antigenically related to the gD glycoproteins of HSV 1 and HSV 2 and to be involved in cross-neutralization among these viruses. Antisera to HVS 1 and HVA 1 did not recognize these gD glycoproteins nor was a glycoprotein of similar molecular weight precipitable from HVS 1 or HVA 1 infected cells by antisera to the other four viruses. Southern blot hybridizations using probes for HSV glycoprotein genes confirmed the conservation of the gB glycoproteins among all the simian viruses and of the gD gene in SA 8 and B virus. A glycoprotein of approximately 75–80 k was, however, precipitated from HVS 1 and HVA 1 infected cells by antisera to either of these two viruses. In addition, at least one glycoprotein which appeared to be predominantly virus-specific in its reactivity was identified for five of the viruses.     

Protection of mice against lethal challenge with herpes simplex virus by vaccination with an adenovirus vector expressing HSV glycoprotein B

Increasing attention has been focused on the use of recombinant mammalian viruses as potential vaccines. Recombinant human adenoviruses are one of the more promising vaccine vectors because they can be easily constructed and because live adenovirus vaccines have been administered orally to large numbers of military recruits without adverse reactions. In order to examine the efficacy of human adenoviruses as vaccines we have studied the immunity induced by a recombinant adenovirus vector, AdgB2, which induces high level expression of herpes simplex virus (HSV) glycoprotein B (gB) in human and murine cells. Mice inoculated with AdgB2 produced antibodies specific for gB which neutralized HSV in the presence of complement. Although mice inoculated with AdgB2 showed no ill-effects after AdgB2 inoculation and we were unable to detect replication of human adenoviruses in mice, the mice were protected from a lethal challenge with HSV after a single inoculation with AdgB2.     

Rescue of memory CD8+ T cell reactivity in peptide/TLR9 ligand immunization by codelivery of cytokines or CD40 ligation

The capability of cellular immune components to rapidly recall upon challenge in most situations decides the efficacy of a vaccine. Here, we show that immunization of mice with SSIEFARL peptide (immunodominant epitope in glycoprotein B of herpes simplex virus type 1, aa498-505) combined with TLR9 ligand in the absence of helper CD4(+) T cell activation generates a functionally impaired CD8(+) T cell memory response. Codelivery of IL-12, IL-15, or anti-CD40 together with MHC class-I-restricted peptide combined with TLR9 ligand at inception of immunization resulted in generation of memory CD8(+) T cells that were several fold less compromised than immunization with peptide alone. Furthermore, administration of either plasmid DNA encoding IL-15 or anti-CD40 mAb but not rIL-12 during the memory phase restored the reactivity of memory CD8(+) T cells. Moreover, the rescued CD8(+) T cells preserved their cytotoxic capability and were able to clear a recombinant vaccinia virus encoding glycoprotein B of HSV. Our results indicate that good memory CD8(+) T cell response to peptide immunization can be achieved by using costimulatory procedures at the time of priming or recall immunization.     

Mixed vaginal infections of Balb/c mice with low virulent herpes simplex type 1 strains result in restoration of virulence properties: vaginitis/vulvitis and neuroinvasiveness

Vaginal infections of BALB/c Ann mice with herpes simplex virus type 1 (HSV-1) were studied. Mice were inoculated with virulent strains ANG path and 17 syn⁺ or low-virulent recombinant strains 27/III and 17-syn3 that differ from parental strains in their glycoprotein B (gB) gene sequences. When low-virulent strains were inoculated separately, no vaginitis/vulvitis was produced despite replication in the vagina. In contrast, after coinfection of mice with the two low-virulent strains, vaginitis/vulvitis was produced and virus could be recovered from the central nervous system (CNS). Two of the CNS isolates produced vaginitis/vulvitis, neuroinvasiveness and death of mice after vaginal infection. Restriction fragment analysis and sequencing were used to assess recombination events in the gB gene sequence of the CNS isolates. After mixed vaginal infection recombination between non-virulent HSV strains occurs, resulting in vaginitis/vulvitis and neuroinvasiveness. No correlation was detected between the syncytial phenotype and local vaginal virulence. Virulence of HSV is not solely dependent on gB function; it seems to be more probable that several genes act in concert to induce virulence and neuroivasiveness. Received: 28 May 1996     

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL.     

Specific and nonspecific immune responses to Marek's disease virus

Marek's disease (MD) virus (MDV) has provided an important model to study immune responses against a lymphoma-inducing herpesvirus in its natural host. Infection in chickens starts with a lytic infection in B cells, followed by a latent infection in T cells and, in susceptible birds, T cell lymphomas develop. Non-specific and specific immune responses are important for the control of virus infection and subsequent tumor development. Interferon-gamma and nitric oxide are important for the control of virus replication during the lytic phase of infection and are also important to prevent reactivation of MDV replication in latently infected and transformed cells. Cytotoxic T cells (CTLs) are the most important of the specific immune responses in MDV. In addition to antigen-specific CTL against MDV proteins pp38, glycoprotein B (gB), Meq, and ICP4, ICP27-specific CTL can also be detected as early as 6 to 7 days post infection. The epitope for gB recognized by CTLs from P2a (MHC: B(19)B(19)) chickens has been localized to the Eco47III-BamHI (nucleotides 1515-1800) fragment. A proposed model for the interactions of cytokines and immune responses as part of the pathogenesis of MD is discussed.     

Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.     

Enveloped Virus-Like Particle Expression of Human Cytomegalovirus Glycoprotein B Antigen Induces Antibodies with Potent and Broad Neutralizing Activity

A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection.     

Deletion of the carboxy-terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B does not affect oligomerization,heparin-binding activity,or its ability to protect against HSV challenge

Summary A recombinant vaccinia virus designated VgBt which expresses a truncated secreted herpes simplex virus gB (gBt) was constructed and compared to V11gB, a vaccinia recombinant previously studied which expresses gB exclusively on the surface of infected cells. Indirect immunofluorescence assay (IFA) revealed that gBt was strongly associated with the surface of infected cells despite being released slowly into the cell culture medium. Both gB and gBt existed as oligomers, and both membrane bound and secreted forms of gBt exhibited heparin-binding activity. In protection studies VgBt and V11gB conferred equivalent protection against both homologous (HSV-1) and heterologous (HSV-2) challenge with HSV.     

Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties.

A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.     

Interferon-γ- and interleukin-4-producing T cells can be primed on dendritic cells in vivo and do not require the presence of B cells

The antigen-presenting cell (APC) requirements for the in vivo induction of Th1-and Th2-type responses were investigated using a severe combined immunodeficiency (SCID)mouse chimera model. SCID mice adoptively transferred with either T cells [SCID(T)] or T + B cells [SCID(T + B)] and immunized with antigen in adjuvant were able to generate antigen-specific T cells which could produce both interferon (IFN)-γ and interleukin (IL)-4 upon in vitro restimulation. This suggests that B cell APC are not necessary for the priming of either IFN-γ- or IL-4-producing T cells in vivo. The ability of different APC to activate Th2-dependent effector mechanisms was also investigated. SCID(T) and SCID(T + B) mice were infected with the nematode parasite Nippostrongylus brasiliensis and analyzed for the development of IL-5-dependent peripheral blood eosinophilia. Following infection both SCID(T) and SCID(T + B) mice generated similar numbers of peripheral blood eosilnophils, suggesting that similar amounts of IL-5 had been produced. Therefore, B cell APC are also not required for the in vivo activation of Th2 cells to lymphokine production. To establish more precisely which APC prime T cells to produce IFN-γ and IL-4, normal mice were immunized by injection of syngeneic splenic dendritic cells which had been pulsed with antigen in vitro. T cells from these immunized mice were able to produce good IFN-γ and IL-4 responses upon in vitro restimulation with specific antigen; therefore, dendritic cells appear to be sufficient APC for the in vivo priming of both IFN-γ- and IL-4-producing T cells.     

Pathogenic potentials of glycoprotein C-negative syncytial mutants from rabbit T cells infected persistently with herpes simplex virus type 1

Human T cell lymphotropic virus type I (HTLV-I)-transformed T cells of rabbits were infected persistently with Herpes simplex virus type 1 (HSV-1) strain KOS. These infected cells yielded syncytial mutants, either glycoprotein C (gC)-negative or -positive, which predominated over and replaced the wild-type virus in a long-term culture for 2 years. An alignment of nucleotide sequences showed multiple mutations in glycoprotein B (gB) and gC genes of these mutants, which are or may be responsible for the mutant phenotypes. One of four mutants analyzed produced extensively large syncytia and possessed point mutations within the cytoplasmic domain of gB. All four mutants possessed multiple point mutations in gC and two possessed single insertions which resulted in a frame shift, leading to the premature termination of the gC polypeptide chain. The supernatant of the 2-year culture of cells infected persistently, containing only gC-negative syncytial mutants, induced encephalitic symptoms in B/Jas inbred rabbits, when injected intravenously. One gC-negative syncytial isolate from an encephalitic lesion, together with those from the culture supernatant, were examined for pathogenic potential in vitro and in vivo. All these mutants were more cytotoxic and more susceptible to complement inactivation than the parental virus, and could infect and replicate in adrenal glands when injected intravenously into rabbits. Invasion into the central nervous system appeared to be blocked at the portal of entry, the adrenal gland, i.e., none exhibited neuroinvasive potential by itself. Syncytial gC-negative mutants could thus be pathogenic in rabbits.     

Immunogenicity of purified glycoprotein gB of herpes simplex virus

Summary The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB.With 6 Figures     

A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo

CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to beta2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered.     

Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major virion component, essential for various steps of virus replication in cells, such as entry and maturation, and cell fusion. In addition, gB is a strong inducer of the immune response in humans and has been involved in neuropathogenesis. To analyze gB during infection, a recombinant HSV-1 was generated containing gB fused to the green fluorescent protein (GFP). The GFP-gB fusion protein was incorporated into fully infectious viral particles. In cells infected with the recombinant KGFP-gB, the spontaneous fluorescence emitted by the fusion protein was observed as early as 5 h post infection, and its transport through cell compartments was followed during an entire viral replication cycle. The results show that GFP can be inserted into an essential viral envelope component of HSV-1 such as gB while preserving the infectivity of the resulting recombinant. This virus allows the investigation of several events of the viral life cycle involving gB, and provides the basis for the development of new diagnostic assays.     

Identification of an Epstein-Barr virus glycoprotein which is antigenically homologous to the varicella-zoster virus glycoprotein II and the herpes simplex virus glycoprotein B

The Epstein-Barr virus (EBV) antigenic homologue of the varicella-zoster virus glycoprotein II and the herpes simplex virus (HSV) glycoprotein B (gB) was identified through cross-reactivity with anti-glycoprotein II and anti-glycoprotein B peptide sera. The homologue is the previously characterized EBV glycoprotein, with an apparent molecular weight of 125,000 Da, which is synthesized late during productive EBV infection and appears to be encoded by the BamHI A EBV fragment. This glycoprotein, but not other EBV proteins, reacted with the antisera in immunoprecipitation experiments and by ELISA. In addition, absorption of the sera with the purified EBV 125-kDa glycoprotein removed the cross-reacting antibody. Whether the EBV gB homologue has the same biological functions associated with HSV gB has yet to be determined.     

Characterization of B virus glycoprotein antibodies induced by DNA immunization

Genes encoding glycoproteins gB, gC, gD, gE, and gG of herpes B virus (species Cercopithecine herpesvirus 1) were cloned into mammalian expression vector pcDNA3.1/V5-His. Abilities of the plasmid constructs to express recombinant glycoproteins were confirmed by Western blot analysis of transfected CHO-K1 and COS-7 cells. Antibody production was induced in rabbits by intramuscular injections with the expression constructs at four-weekly intervals. Antibodies to gB were detected after the second DNA inoculation, while it took an additional plasmid injection to induce responses to gC, gD and gE. The gG plasmid failed to stimulate antibody production. Antisera ELISA titers varied greatly depending on the gene, with gB inducing highest (21,000) and gE inducing lowest (60) antibody titer. The induced antibodies were predominantly conformation-dependent. The gB, gC, and gD antisera contained HSV cross-neutralizing antibodies, but only gB antisera contained B virus neutralizing antibodies. The gB antisera cross-reacted with HSV antigens in Western blot, ELISA, dot-blot, plaque immunostaining and immunoprecipitation assays, whereas gD and gC antisera were mostly B virus-specific. Thus, polyclonal antibodies to B virus glycoproteins can be generated by DNA immunization and used as diagnostic and research reagents.