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.     

Induction of virus specific and H-2 restricted cytotoxic T cells by UV inactivated murine cytomegalovirus

Summary Infection of C3H mice with live or UV-inactivated murine cytomegalovirus (MCMV) was able to generate population(s) of lymphocytes in the spleen (CTL) which could exert a lytic effect against L cells infected with MCMV but not against uninfected or those infected with HSV-1. The effector cells proved to be theta-bearing T cells and the lysis of target cells was H-2 restricted. Data presented show that early viral protein synthesis but not viral DNA synthesis was necessary for the appearance of relevant antigenic determinant(s) on target cells. The results of co-capping experiments suggest that H-2 molecules may have close association with MCMV induced product(s) as also with murine leukemia virus glycoprotein (gp 70) which is carried by normal L cells. Despite this observation, anti-H-2 serum effectively blocked the cytolysis whereas anti-gp 70 and anti-MCMV sera failed. Anti-MCMV serum was effective in blocking cytolysis, only if the L cells were infected for 24 hours and then used as targets. MCMV infected L cells which were coated externally with inactivated Sendai virus could be effectively recognised by MCMV as also by sendai specific CTL. That the cytotoxicity exerted on such targets was of specific nature was revealed by the results of competitive blocking experiments with unlabelled targets.     

Complement-mediated cytolysis of HSV-1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody.

Surface antigens of BHK-21 cells infected with HSV-1 or HSV-2 were radioiodinated (125I) with lactoperoxidase, immune precipitated and analysed by polyacrylamide gel electrophoresis (PAGE). Experiments using antiserum to HSV-1 or HSV-2, absorbed with appropriate hemotypic or heterotypic antigens, revealed that both type-specific (homotypic) and cross-reactive antibody combined with surface glycoproteins to form a single large radioactive peak. This peak, which constituted the major glycoprotein region (region a) observed in electropherograms, represented a range in mol. wt. from 115000 to 130000. Sensitization of cells to complement lysis, neutralization of infectious virus and immune precipitation of surface glycoproteins (region a) were found to be generally correlated properties of all the antibody preparations analysed, including antibody prepared specifically against region a antigens. These findings suggest a major immunological role for the surface glycoproteins migrating in PAGE region a.     

Rotavirus-specific cytotoxic T lymphocytes recognize overlapping epitopes in the amino-terminal region of the VP7 glycoprotein.

Rotavirus-specific cytotoxic T lymphocytes (CTL) play an important role in the resolution of rotavirus infection. The outer capsid glycoprotein, VP7, elicits a class I MHC-restricted CTL response. Vaccinia virus recombinants expressing the VP7 genes from simian rotavirus SA11 (serotype G3) and from the RF strain of bovine rotavirus (serotype G6) were used to analyze the CTL activity to this antigen in BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice neonatally infected with homologous and heterologous rotaviruses. A vaccinia virus recombinant expressing the first amino-terminal 88 amino acids of VP7 was constructed and used to search for cross-reactive CTL against this region of the protein. By using synthetic Kb, Db, and Kd motif-fitting peptides two overlapping CTL epitopes have been identified located in the first hydrophobic domain (H1) of VP7. Splenocytes obtained from rotavirus SA11-infected C57BL/6 mice induced the strongest CTL response against target cells sensitized with a peptide containing a Kb-restricted CTL epitope (amino acids 8-16). A second Kd-restricted epitope (residues 5-13) was recognized by splenocytes derived from rotavirus-infected BALB/c mice. These findings reveal the existence of CTL epitopes in the H1 signal sequence of the VP7 glycoprotein that coexist with a CTL epitope (residues 31-40) previously described within the H2 region.     

HLA-restricted T lymphocyte-mediated cytotoxicity against herpes simplex virus-infected cells in humans.

Cytotoxic T lymphocytes (CTL) against herpes simplex virus (HSV) were induced in vitro from human peripheral blood lymphocytes by stimulation with HSV antigen. CTL generated by HSV type 1 (HSV-1) antigen stimulation killed not only HSV-1-infected target cells but also HSV type 2 (HSV-2)-infected target cells, though at a lower level. This evidence suggests that CTL against HSV recognize the HSV type-specific and type-common determinants on HSV-infected target cells. These CTL were generated from high responders against HSV-1 antigen as measured by antigen-specific T lymphocyte proliferation in vitro, but not to such an efficient degree from low responders. The cytotoxic activities of CTL against the allogeneic HSV-infected target cells were high when at least one of the HLA-A or -B antigens was shared. However, the HLA-A and -B nonidentical target cells were not killed effectively. The data presented here suggest the possibility of HLA restriction of HSV-specific CTL in humans.     

Cell-mediated immunity against herpes simplex induction of cytotoxic T lymphocytes.

The conditions required for the induction of both primary cytotoxic T lymphocytes (CTL) in vivo and secondary CTL in vitro against herpes simplex virus type 1 (HSV-1)-infected cells were defined. Primary CTL responses occurred only in mice exposed to infectious HSV-1. These responses, which were shown to be mediated by T lymphocytes, peaked at 1 week and had disappeared by 2 weeks after infection. The level of primary cytotoxicity was enhanced by treatment of mice with cyclophosphamide before infection. Secondary in vitro CTL responses were more pronounced and were induced by some forms of inactivated virus as well as by infectious HSV-1. Thus, both ultraviolet light- and glutaraldehyde-inactivated preparations of HSV-1 induced CTL, but heat-inactivated and detergent-extracted antigens failed to do so. The reasons for the differing efficiency of infectious and noninfectious HSV-1 for induction of CTL are discussed.     

Immune responses of cattle to Theileria parva (East Coast fever): specificity of cytotoxic cells generated in vivo and in vitro.

Examination of the specificity of cytotoxicity generated in vitro and in vivo against infected bovine lymphoblasts revealed that cytotoxic T lymphocytes (CTL) obtained from cattle immune to Theileria parva recognized parasite-induced alterations associated with major histocompatibility complex (MHC) antigens on the membrane of infected autologous cells. By comparison, cytotoxicity generated in vitro in an autologous Theilerial-lymphocyte culture (AuTLC) contained both CTL and activity akin to that of natural-killer (NK) cells. The addition to the AuTLC of 2 inhibitors of glycosylation, tunicamycin (Tun) and 2-desoxy-D-glucose (2-DOG) abolished both the proliferative response and the generation of cytotoxicity. While the addition of Tun or 2-DOG in conventional cell-mediated lympholysis (CML) assays did not modify the effector function of cytotoxic cells, pretreatment of target cells with either compound prevented lysis by CTL, but not by NK cells. Although parasite-induced antigens have not been purified from infected bovine lymphoblasts, the present study indicated that these are likely to be glycoprotein or carbohydrate in character, and that their recognition on autologous cells is a consistent feature of CTL from immune cattle.     

Protective immunization of mice with specific HSV-1 glycoproteins.

Two herpes simplex virus type 1 (HSV-1) antigens of apparent molecular weight 123,000 and 63,000 which are associated with the viral glycoprotein complex have been identified and purified using virus-specific monoclonal antibodies. Mice immunized with either glycoprotein showed marked resistance to challenge with virulent HSV-1. Therefore purified glycoproteins of HSV-1 free of nucleic acid can be used in protective immunization of mice against human herpes simplex type 1.     

Specifically immune mouse T-cells can destroy H-2 compatible murine target cells infected with herpes simplex virus types 1 or 2.

Lymphocytes from the spleen (SL) or peritoneal cavities (PL) of HSV-1- or HSV-2-immunized C 3 H mice expressed pronounced cytotoxicity, as measured by 51Cr release assay, against L cells experimentally infected with HSF-types 1 or 2 but not against uninfected L cells. HSV-1 or HSV-2 immune lymphocytes induced substantially more 51Cr release when L-cell targets were infected with homotypic virus compared to those infected with heterotypic virus. Inasmuch as the cytotoxicity of specifically immune C 3 H SL for HSV-infected L cells was selectively eliminated by treatment with AKR anti-theta C 3 H serum plus complement, the effector cells in present system were theta-bearing T cells. Evidence has been provided which indicates that specifically immune T cells express cytotoxicity exclusively directed against different HSV-infected target cells (mouse embryo cells or mouse peritoneal macrophages) which share H-2 antigens with the effector cells.     

Synthesis of the gB and gC glycoproteins of herpes simplex virus type 1 in the absence of viral DNA synthesis

Using an inhibitor of DNA synthesis as well as a temperature-sensitive (ts), DNA negative mutant of herpes simplex virus type 1 (HSV-1), we have examined the role of viral DNA synthesis on the expression of the virus-specific glycoproteins. Analysis by immunoblotting employing monospecific antisera revealed that only gC glycoprotein synthesis was completely inhibited in the absence of viral DNA synthesis. In contrast, the gB glycoprotein was detectable in significant, albeit somewhat reduced, amounts. These data suggest that gC is a ‘true-late’ protein of HSV-1 while gB is not. In addition, temperature shift-up experiments with the ts mutant (tsA1) suggest that some viral gene product(s) is needed continuously to achieve wild-type levels of gC synthesis.     

The immune response to herpes simplex virus: comparison of the specificity and relative titers of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections

Employing an immunoblotting technique, the polypeptide specificity and relative titers of anti-HSV IgG reactive with denaturation-resistant epitopes on HSV proteins were determined in patients experiencing primary HSV-1 infections at various anatomical sites. Early sera from previously seronegative patients with primary HSV-1 infections were found to have comparatively low levels of antibody directed against the major viral glycoprotein antigens (gB, gC, and gD) relative to titers present in sera of individuals with long-standing, latent orofacial HSV-1 infections. Patients with primary infections did however have high titers of antibody directed against a series of low molecular weight HSV polypeptide antigens. These antigens were found to be antigenically related to a structural component of virion nucleocapsids. At later times postinfection, titers of antibodies directed against other viral polypeptides including the major glycoproteins increased to levels more closely approximating those observed in latently infected individuals. These results indicate that the anti-HSV IgG detected by immunoblot analysis which appears earliest following primary infection is not directed against the known major infected cell or virion glycoprotein surface antigens but rather against an internal capsid protein of HSV.     

Studies on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai virus).

A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F₀, NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F₀, and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.     

Herpes simplex virus protein synthesis in the presence of 2-deoxy-D-glucose.

The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [¹⁴C]amino acids or [¹⁴C]glucosamine in the presence or absence of 2-deoxy-d-glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 mM), the major envelope glycoprotein (VP123, MW 123,000) labeled with [¹⁴C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [¹⁴C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [¹⁴C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose.     

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1.

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2 infected LL-1 cells demonstrated a block in HSV-2-specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSV-specific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.     

Type-specific reovirus antiserum blocks the cytotoxic T-cell-target cell interaction: evidence for the association of the viral hemagglutinin of a nonenveloped virus with the cell surface.

It has previously been demonstrated that spleen cells from mice immunized with reovirus type 1 or 3 generate virus-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation. Such cytotoxic T cells lyse H-2 identical targets that are infected with the appropriate reovirus type. Viral recombinants were used to demonstrate that the S1 gene is the predominant viral gene determining the specificity of the CTL. Reoviruses are nonenveloped, non-membrane-maturing viruses; therefore, it was important to determine whether viral products were being recognized by CTL on the surface of target cells. Antiserum blocking was utilized to investigate this issue. Using viral recombinants and antisera to reoviruses types 1 and 3, we were able to demonstrate that the major viral antigen recognized by the CTL on the target cell surface is the sigma 1 polypeptide encoded by the S1 genome segment. Thus, viral antigens on the target cell membrane seem to be important in the CTL response to a nonenveloped, non-membrane-maturing virus.     

H-2 restriction and serotype crossreactivity of anti-reovirus cytotoxic T lymphocytes (CTL)

Murine anti-reovirus cytotoxic T lymphocytes (CTLs) were analyzed for H-2 restricted recognition of virus infected target cells and for potential cross-reactivity with cells infected by reovirus serotype 1 (T1; Lang strain) or by serotype 3 (T3; Dearing strain). Anti-reovirus CTL specifically lysed virus infected cells and lysis was shown to be H-2 restricted by the H-2Dd, H-2Ld, H-2Kd, H-2Kb, and H-2Kk antigens. No H-2 antigens were identified which failed to restrict virus recognition by anti-reovirus CTL. Anti-T1 and anti-T3 CTLs were also shown to crossreact completely with cells infected with the opposite virus serotype. Thus, anti-reovirus CTLs are restricted by a broad spectrum of H-2 antigens and they detect common rather than unique structural components of these two viral serotypes.     

Deoxyribonucleoprotein complexes and DNA synthesis of herpes simplex virus type 1

Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleoprotein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.     

Expression of cellular proteins Bcl-X(L), XIAP and Bax involved in apoptosis in cells infected with herpes simplex virus 1 and effect of pavine alkaloid (-)-thalimonine on virus-induced suppression of apoptosis

The expression of three cellular proteins involved in the modulation of apoptosis, namely antiapoptotic Bcl-X(L) and XIAP and proapoptotic Bax, was investigated in cells infected with Herpes simplex virus 1 (HSV-1). To assess whether the regulation of apoptosis in virus-infected cells depends on strain specificity the wild-type (wt) strain Victoria and the mutant R-100 resistant to acyclovir (ACV) were used. In addition, the expression of Bcl-X(L), XIAP and Bax was studied in cells infected with HSV-1 and treated with pavine alkaloid (-)-thalimonine. Our previous work has demonstrated that (-)-thalimonine irreversibly inhibits the replication of wt HSV-1 in cultured cells. Our data showed that (-)-thalimonine down-regulates the expression of viral proteins U(L)17, VP11-12, VP22, VP24 and gamma1 34.5, and affects negatively the posttranslational processing of glycoproteins D (gD) and G (gG). As both gamma1 34.5 and glycoprotein D possess antiapoptotic activity, we investigated whether the antiviral effect of the alkaloid could also be due to its ability to suppress the antiapoptotic activity of the virus. Our results demonstrated that: (i) the virus induced overexpression of antiapoptotic proteins Bcl-X(L) and XIAP; (ii) (-)-thalimonine reduced their overexpression, and (iii) this effect was stronger with the acyclovir resistant mutant R-100 than with the wt virus. Taken together, these data suggest that: (i) the virus abolishes apoptosis by means of virus-induced up-regulation of cell-specific prosurvival proteins Bcl-X(L) and XIAP, and (ii) (-)-thalimonine, apart from affecting essential viral targets, inhibits the infectious progeny production via restoration of apoptosis during viral replication.     

Induction of receptors for complement and immunoglobulins by herpesviruses of various species

Cells infected with herpes simplex virus type 1 (HSV-1) express a viral glycoprotein on the cell surface, which can function as a receptor for a cleavage product of complement factor 3 (C3b), and it has been suggested that this has biological relevance in the infected host (Smiley et al., 1985, J. Virol. 19, 217). As herpesviruses of different species share common determinants on their glycoproteins, a possible conservation of biological function was investigated for bovine herpesviruses type 1 and 2 (BHV-1 and -2), equine herpesvirus type 1 (EHV-1) and HSV-1 and -2, respectively. Only HSV-1 and EHV-1 induced C3b-receptors on infected cells. Nevertheless, BHV-1 infected cells could be killed by complement-dependent neutrophil mediated cytotoxicity (CDNC) as could EHV-1-infected cells. HSV-1-infected cells were not killed by this mechanism, but were highly susceptible to direct C-lysis. Four different scenarios for interaction between herpesvirus-infected cells and the nonspecific host defense system are presented.