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.     

A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosylated processive intermediates bound to lentil lectin indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosylated, no gD and only a portion of pgD bound to lentil lectin when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1 and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.     

Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins

The specificity and neutralizing activity of antibodies against the major herpes simplex virus type 1 (HSV-1) glycoproteins were tested in serum samples of patients with a history of HSV-1 infection. By preabsorption of sera to preparations of native and denatured HSV-1 proteins, followed by immunoblotting and microneutralization, it was shown that the majority of neutralizing antibodies are directed against denaturation-sensitive epitopes. Furthermore, preabsorption of sera to proteins of viral ts and deletion mutants revealed that antibodies specific for gB, gC, and gE had a low neutralizing activity. These results suggest a major role of anti-gD in neutralization of viral infectivity. In addition, it was shown that antibodies directed against the gB monomer were distinct from antibodies against the gB homodimers. The latter, however, did not reveal any measurable neutralizing activity.     

Humoral immune response to HSV-1 and HSV-2 viral proteins in patients with primary genital herpes

The humoral immune response to HSV-1 and HSV-2 proteins was examined in patients with primary first-episode genital herpes. Ten patients had culture-proven HSV-1 infections, 37 had HSV-2 infections, and all were seronegative to HSV proteins before developing their infections. Development of serum antibodies to individual HSV proteins and glycoproteins was determined by immunoprecipitation of radiolabeled HSV-1- and HSV-2-infected cell proteins and subsequent gel electrophoresis. In HSV-1 patients, a sequential development of antibodies to HSV-1 proteins was observed with early appearance of antibodies to the nucleocapsid protein p148 and to glycoproteins gB and gC. Seroconversion to gD and to a polypeptide of 88,000 molecular weight (p88) occurred next, and, finally, seroconversion to gE and to a nonglycosylated 66,000 dalton protein p66. In HSV-2 patients, antibodies to HSV-2 proteins p148, gB, and p88 appeared within 1 week of onset of symptoms. Seroconversion to p66, gD, and to a complex of glycoproteins gC and gE ("g80") occurred later, at a mean time of approximately 3 weeks. Seroconversion to HSV-1 gB, p88, and p66 occurred significantly later than seroconversion to the homologous counterparts. Seroconversion within 21 days of onset to HSV-2 gD, g80, and p66 was associated with a longer time to the first recurrence in HSV-2 patients, suggesting a possible role of these antibodies, alone or in combination, in the maintenance of HSV-2 latency in humans.     

Intratypic variation of herpes simplex virus type 2 isolates detected by monoclonal antibodies against viral glycoproteins

Monoclonal antibodies (MAbs) to herpes simplex virus (HSV) glycoproteins gD, gG, gB, and gE were used to analyze antigenic variations of 128 genital HSV-2 isolates by an indirect enzyme-linked immunosorbent assay (ELISA). Isolates were considered significantly different from the standard HSV-2 strain 186 when their optical density (OD) in ELISA was less than half that of strain 186. This criterion gave 30 patterns of reactivity among the genital HSV-2 isolates. The MAbs to gB, gG, and 2 of the gD antibodies reacted with more than 90% of the isolates, suggesting that these MAbs recognized highly conserved epitopes. However, the gE MAb reacted with only 47% of the isolates, and one of the gD antibodies with only 39%. Thus, HSV-2 can readily tolerate modifications in some parts of the gD and gE molecules while remaining infectious.     

Detection of type-specific antibody to herpes simplex virus type 1 by radioimmunoassay with herpes simplex virus type 1 glycoprotein C purified with monoclonal antibody.

Herpes simplex virus type 1 (HSV-1) and HSV-2 specify at least four glycoproteins designated gA/gB, gC, gD, and gE. Previous studies have shown that gC produced by HSV-1 is antigenically distinct from the corresponding HSV-2 glycoprotein. With the exception of gC, the glycoproteins of both serotypes share antigenic sites. Standard serological assays fail to differentiate the antibody to the shared antigenic determinants from the type-specific antibody. In this paper, we describe a procedure for purifying gC from HSV-1-infected cell extracts with an immunoadsorbent prepared with an HCL monoclonal antibody. When used in a solid-phase radioimmunoassay, gC proved to be a type-specific antigen for quantitation of antibody to HSV-1. Among individuals who had no antibody to HSV at the onset of infection, all of those with primary HSV-1 infection developed antibody to gC. Subjects with primary HSV-2 infection failed to develop antibody reactive with gC of HSV-1 (P less than 0.01). Both immunoglobulin G and M antibodies against gC were detected in sera from subjects with either primary or recurrent HSV-1 infection. Higher antibody titers to gC were found in sera from individuals with recurrent infection than in sera from those with primary HSV-1 infection.     

Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses

Summary Recent studies indicate that Ig class and IgG subclass induction varies for different proteins and further that some Ig subclasses, like IgG2a, are more efficient in important biologic processes such as antibody-dependant cell-mediated cytotoxicity (ADCC). Many proteins of herpes simplex virus type 1 (HSV-1) are immunogenic and induce immunoglobulin responses. To determine the distribution of immunoglobulins induced by HSV-1 proteins, we studied immune mouse serum using an Ig isotype specific Elisa assay for antiviral activity. We found by endpoint analysis that the antiviral titer was 1:12,903 for IgG1, 1:5141 for IgG2a, 1:2140 for IgG2b and 1:229 for IgG3. To identify which isotypes were induced by individual glycoproteins and other viral proteins, Western blots containing HSV-1 proteins were probed with immune serum and isotype specific second antibodies. gB, gC, gD and the 42/44KDa nucleocapsid complex induced strong IgG1, IgG2a, IgG2b responses. IgG3 reactivity with viral proteins appeared weaker. Among the IgG3 reactivities detected on immunoblots, gB and gC were the most intense. Other proteins which elicited IgG1, IgG2a and IgG2b responses were 170KDa, 154KDa and gE. IgA responses were induced by 154KDa, gC, gB, gE and gD. Prominent IgM responses included gB, gC, gD and the 42KDa protein. These results indicate that HSV-1 glycoproteins induce prominent responses in all IgG isotypes except IgG3. The biologic implications of the data are discussed.     

Use of monoclonal antibodies for analysis of antibody-dependent immunity to ocular herpes simplex virus type 1 infection.

Monoclonal antibodies specific for the five major glycoproteins of herpes simplex virus type 1 (HSV-1) were tested for their capacity to mediate immunity to ocular HSV-1 infection. The specificity of the immunoglobulin made by each monoclone was determined by immunoprecipitation of [14C]glucosamine-labeled polypeptides from detergent-solubilized HSV-1-infected cells. Of the five monoclonal antibodies studied, two immunoprecipitated glycoproteins gA/B, one immunoprecipitated glycoprotein gC, one immunoprecipitated glycoprotein gD, and one immunoprecipitated glycoprotein gE. All five were effective in passively transferring immunity to mice when they were given 4 to 24 h after HSV-1 infection on an abraded cornea. Four of the monoclonal antibodies were also evaluated for their capacity to neutralize HSV-1 and to promote complement-mediated cell lysis and antibody-dependent cellular cytotoxicity. It was found that none of these in vitro assays correlated with the protective activity of the antibodies in vivo. In fact, one of the monoclonal antibodies was unreactive in all three immunological reactions, even though it was highly effective in promoting recovery from HSV-1 induced ocular disease in vivo. The results suggest that antibodies can interact in vivo with virus-specific glycoproteins gA/B, gC, gD, and gE to initiate recovery from HSV-1-induced ocular disease, and that the therapeutic effectiveness of a specific monoclonal antibody does not correlate with its immunological reactivity in vitro.     

Varicella-zoster virus gpI and herpes simplex virus gE: phosphorylation and Fc binding

gpI, the predominant varicella-zoster virus (VZV) envelope glycoprotein, was shown to be phosphorylated exclusively on serine and threonine residues, and phosphorylated gpI was detected in isolated virions. In cells infected with herpes simplex virus type 1 (HSV-1), a related neurotropic alpha-herpesvirus, HSV gE, the homolog to VZV gpI, and HSV gB, the homolog to VZV gpII, were also phosphorylated. The phosphate on gB and gE was alkali labile and resistant to endo H, suggesting linkage to serine and/or threonine. Although VZV gpI and HSV gE share sequence homology and similar post-translational modifications, no Fc-binding activity similar to that associated with gE was detected for gpI or any of the VZV glycoproteins.     

Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 X 10(3) molecular weight

A type-specific monoclonal antibody that efficiently neutralises HSV-1 immunoprecipitated a glycoprotein of slightly greater electrophoretic mobility than gB from HSV-1 infected cells. Pulse and pulse chase experiments indicate that this glycoprotein is distinct from HSV-1 glycoproteins gB, gC, gD, and gE. This was confirmed by the reactions of LP11 with a series of intertypic recombinants the results of which indicate that the LP11 target gene is located close to the HSV-1 thymidine kinase gene between map positions 0.28 and 0.31. In accordance with the presently agreed convention this glycoprotein should be designated gH-1, and it may correspond to the 110K glycoprotein described by S. D. Showalter, M. Zweig, and B. Hampar (1981), Infect. Immun. 34, 684-692. Antibody LP11 inhibits plaque formation when added to cell monolayers after infection suggesting that gH-1 may play a role in cell-to-cell spread of infectious virus.     

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.     

Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection,reactivation or immunization

The purpose of this study was to compare the antibody responses to varicella-zoster virus (VZV) gE and gB after natural VZV infection and after vaccination with live attenuated OKA vaccine in order to determine the relative importance of these proteins as components of a subunit vaccine. Anti-VZV antibody titers determined by IFA were of the same order of magnitude in sera from individuals with a history of varicella and in vaccinated children but higher in individuals given booster vaccination. The titers of anti-gE and anti-gB antibodies were measured by ELISA using recombinant gE or gB as capture antigen. From these experiments, it appears that the ratio of anti-gE to anti-gB antibody is highly variable from one individual to another but relatively stable over a long period of time for a particular individual, even after a zoster episode. Neutralizing antibodies directed against gE or gB were also measured by subtracting the neutralization titers obtained before and after depletion of the specific antibodies on immobilized recombinant gE, gB, or both. This showed that, with respect to neutralization, anti-gE and anti-gB are equally prevalent in vaccinated children and that anti-gE is generally, but not always, predominant over anti-gB in VZV-infected individuals. Finally, antibodies to these two glycoproteins appear to be predominant among the neutralizing antibodies directed to other VZV antigens. J. Med. Virol. 53:63–68, 1997. © 1997 Wiley-Liss, Inc.     

Development of a competitive ELISA for detection of primates infected with monkey B virus (Herpesvirus simiae)

Two competitive ELISAs (C-ELISAs) are described that allow detection of antibodies against monkey B virus (BV, Cercopithecine herpesvirus 1). The assays utilize monoclonal antibodies (MABs) directed against the BV glycoprotein B (gB). Two of these MABs specifically recognize BV gB while a third MAB also reacts with the gB homologues of other primate alpha-herpesviruses (herpes simplexvirus-1, HSV-1: HSV-2; simian agent-8, SA8; and Herpesvirus papio-2, HVP2). A C-ELISA using the single cross-reactive MAB 3E8 allowed detection of host antibodies against HSV-1, HSV-2, SA8, HVP2 or BV, thus proving to be a sensitive assay for the detection of infection by any of these primate alpha-herpesviruses. The C-ELISA using BV-specific MABs was less sensitive but did allow some discrimination between infection by BV versus other alpha-herpesviruses. It was also shown that a C-ELISA using HVP2 as antigen and the cross-reactive MAB 3E8 was as sensitive for detection of BV antibody in macaque sera as an assay employing BV antigen. This test format allows detection of BV-infected primates without the biohazards associated with preparation and use of BV antigen.     

Role of anti-gB and -gD antibodies in antibody-induced endocytosis of viral and cellular cell surface glycoproteins expressed on pseudorabies virus-infected monocytes

The addition of porcine pseudorabies virus (PrV)-specific polyclonal IgG antibodies to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and major histocompatibility complex (MHC) class I. Using PrV deletion strains, it was shown that gB and gD are essential for the process to occur. The purpose of the current study was to evaluate whether antibodies directed against single viral glycoproteins are able to induce endocytosis. It was shown that monoclonal antibodies directed against viral glycoprotein gB and gD, but not against gC and gE, are able to induce internalization of their respective ligand. Adding a combination of monoclonal antibodies against gB and gD resulted in endocytosis levels, comparable to the endocytosis levels observed when adding porcine PrV-specific polyclonal antibodies. The addition of genistein and tyrphostin 25, two inhibitors of tyrosine kinase activity, abolished endocytosis induced by monoclonal anti-gB and -gD antibodies in a concentration-dependent manner. The addition of similar concentrations of tyrphostin 1, an inactive tyrphostin, had no effect on endocytosis. It was also shown that a mixture of polyclonal, but not monoclonal, antibodies against gB and gD is able to induce cointernalization of MHC class I. This indicates that MHC class I cointernalization results from a passive catching of the molecules rather than from a specific interaction of the MHC class I molecules with one or more viral glycoproteins. In conclusion, it can be stated that antibody-induced crosslinking of gB and gD induces the activation of a tyrosine phosphorylation-dependent signal transduction pathway, leading to their endocytosis. Cointernalization of other viral glycoproteins and MHC class I is most likely caused by a passive catching of these molecules in the gB and gD aggregates.     

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

Requirements for transport of HSV-1 glycoproteins to the cell surface membrane of human fibroblasts and Vero cells

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.     

Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types

Summary The effect of specific pseudorabies virus (PRV) antibodies on the enlargement of plaques produced by PRV were studied in monolayers of different cell types. The plaque size was used as parameter for the efficacy of the cell-associated spread (CAS) of PRV. First, the effect of anti-PRV hyperimmune serum on the plaque growth was examined in monolayers of the continuous cell lines ST, SK-6 and MDCK and monolayers of the primary cultures of porcine fibroblasts, endothelial cells and endometrial cells. A tenfold increase in the serum concentration did reduce the plaque size with 50% in both SK-cells and fibroblasts and with 40, 28 and 16% in MDCK, endothelial and endometrial cells, respectively. In ST cells, no change in size was observed with increasing antibody concentrations. Secondly, the effects of monoclonal antibodies (mAbs) directed against PRV glycoproteins gB, gC, gD and gE and polyclonal antibodies against gC were evaluated in SK-6 cells. MAbs against gB, gD and gE were able to reduce the CAS with a cumulative effect between mAbs against gD and either mAbs against gB or mAbs against gE. Monoclonal and polyclonal antibodies against gC did not change the plaque size.     

A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate

Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T-C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).     

Analysis of the intracellular maturation of the herpes simplex virus type 1 glycoprotein gH in infected and transfected cells.

We have expressed the HSV-1 glycoprotein, gH, in transiently transfected COS-1 cells. The expressed protein was retained intracellularly, contained unprocessed carbohydrate, and was unrecognized by the monoclonal antibody, LP11. In addition, the protein was aggregated. These properties suggest that unlike other HSV glycoproteins, gH is misfolded in transfected cells. Pulse-chase studies of HSV-1-infected cells indicate that the kinetics of processing of gH are comparable to those of gB, gC, and gD. Rescue studies suggest that gH may interact with another protein during maturation in infected cells. However, we were unable to detect any stable interaction, although analysis of gH on neutral sucrose gradients shortly after synthesis indicated a possible transient association with a high molecular weight molecule or complex. The processing and cell surface expression of gH were also analyzed in HSV-1 virus mutants lacking gB, gC, or gD. Our results indicate that the maturation and cell surface transport of gH did not require the presence of these HSV-1 glycoproteins. In addition, three truncation mutants were constructed by linker insertion mutagenesis. Each of the three truncated proteins was synthesized, but the proteins were aggregated, contained only endo H-sensitive carbohydrate, and none were secreted.     

Immunogenicity of influenza and HSV-1 mixed antigen ISCOMs in mice

Summary Immunostimulating complexes (ISCOMs) were prepared with mixtures of antigens from influenza A virus (A/PR/8/34 or A/Sichuan/2/87) and herpes simplex virus type 1 (HSV-1), and were characterised by enzyme linked immunosorbent assay (ELISA) and electron microscopy using double-labelling immunogold techniques employing monoclonal antibodies to influenza or HSV-1 glycoproteins. The immunogenicity of the mixed antigen ISCOMs was evaluated in mice, following administration by the subcutaneous route, by measuring the total and subclass IgG antibody responses. Protection of these animals against challenge with live influenza A/Sichuan virus or live HSV-1, was compared with that induced by immunization with aqueous mixed antigen preparations. It was found that relatively high humoral responses to both influenza and HSV antigens, and increased levels of protection to both influenza and HSV viruses were elicited in mice receiving the mixed antigen ISCOM preparation compared to those observed in animals receiving the mixed aqueous subunit preparation. The findings also indicate that antigens from more than one virus can be used in an ISCOM formulation to produce immunity and protection.