Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [(35)S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a (35)S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.     

An immunoprecipitin study of the incidence of influenza A antibodies in animal sera in the Ottawa area.

A survey of over 600 'normal' sera from 14 animal species by immunoprecipitin tests in cellulose acetate using viron antigens revealed a high incidence of precipitating activity against a broad range of influenza A virus strains, particularly A2hHong Kong/1/68 and /PR8. However, serum treatments trypsin-heat-periodate, NaIO4, V. cholerae receptor-destroying enzyme (RDE), or kaolin eliminated most precipitating activity, which suggests that it was due to "non-specific" inhibitors of influenze viruses. A resistant minority could not be identified as inhibitor or antibody on this basis. Precipitation of the influenza A major type-specific antigen in virus-soluble antigens by human 7S gamma globulin antibody (IgG), demonstrated to be specific for influenza virus, was established as a reference reaction to identify similar immunoprecipitin reactions occurring between virus-soluble antigens and normal or immune sera. Complement fixation tests provided supplementary evidence for the presence of influenza A antibodies in these sera. Influenza A antibodies were found in only a few sera of six animal species: cat, dog, rabbit, goat, chipmunk, and sheep. Thus the animal species examined in the Ottawa area have not revealed an unequivocal reservoir for human influenza A viruses.     

Proteins of Vesicular Stomatitis Virus: II. Immunological Comparisons of Viral Antigens

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20S and in the leading edge of the 6S region are antigenically related to VP3, the protein of the virus core, whereas the 6S soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6S antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6S antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20S antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.     

Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics

Cohen, Gary H. (University of Pennsylvania, Philadelphia), and Wesley C. Wilcox. Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics. J. Bacteriol. 92:676-686. 1966-Infection of mammalian cells with members of the poxvirus group elicits production of a number of virus-induced, soluble antigens. Immunoelectrophoresis and immunodiffusion techniques employing soluble antigen preparations obtained from vaccinia virus-infected KB cells revealed at least seven well-defined immunoprecipitin bands. On the basis of fractionation and subsequent characterization of the soluble antigen mixture by gel filtration, calcium phosphate chromatography, isoelectric precipitation, disc electrophoresis, and ultracentrifugation studies, two distinct classes of virus-induced antigens differing markedly in molecular weight were recognized. A high molecular weight class (200,000 and greater) contained at least three virus-induced antigens; a low molecular weight class (50,000 to 100,000 range) contained at least four immunoprecipitins. Further separation of the antigens within the two groups was accomplished. The two classes were distinguished also by their ability to stimulate synthesis of virus-neutralizing antibody. Antisera prepared against the high molecular weight class proved effective in neutralizing vaccinia virus. In contrast, the low molecular weight antigens showed little, if any, ability to induce formation of neutralizing antibody.     

Separation of Herpes Simplex Virus-Induced Antigens by Concanavalin A Affinity Chromatography

Biologically active herpes simplex virus (HSV)-induced antigens were selectively removed from extracts of infected BHK cells by affinity chromatography by utilizing an insoluble form of concanavalin A (Con A). Soluble extracts of (3)H-glucosamine-labeled, HSV-infected cells were absorbed to a Con A column. Bound material was eluted with alpha-methyl-d-mannoside (alphaMM) and NaCl. The specific activity of the eluted glycoproteins increased by 10-fold. Two broad groups of viral-induced antigens were isolated from Con A. Group I includes two antigens which bind to Con A by a specific mechanism because the antigens are dissociated by alphaMM. Group II contains three antigens which bind to Con A but apparently by a nonspecific or electrolytic mechanism. One antigen in group I was identified as the glycoprotein antigen, CP-1, described previously.     

Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolk-sac microvilli

Passive Heymann nephritis (PHN) is an animal model of immune-complex-induced renal disease resembling human membranous glomerulonephritis. It was induced in rats by injecting rabbit antiserum directed against glycoprotein antigens isolated from rat embryonic visceral yolk-sac microvilli (VYS-MV). The glycoprotein antigens were isolated by extracting the VYS-MV with detergent Nonidet P-40 followed by gel filtration in Sephacryl S-300 and finally by lectin affinity chromatography with Ricinus communis agglutinin I. In vitro immunofluorescent localization studies demonstrated that the nephritogenic antibodies were localized along the apical region of the visceral yolk-sac endodermal cells and the brush border of the proximal tubular cells of the kidney. Rats injected with a single dose of the antiserum manifested proteinuria. Indirect immunofluorescent studies showed that the injected rabbit IgG was localized in vivo along the capillary walls of the glomerulus in a granular fashion. Electron microscopic examination of the same kidney glomeruli revealed numerous electron-dense deposits along the lamina rara externa of the glomerular basement membrane. Fusion of the epithelial foot processes was also present. These findings represent the typical immunopathological characteristics of Heymann nephritis. Furthermore, with the aid of Ouchterlony analysis, the antiserum against the isolated VYS antigens exhibited an immunoprecipitin band which was in common with that formed by the antiserum against the homogeneous nephritogenic antigen (gp330) of renal brush border origin. Thus, the nephritogenic antigens which have been found to be associated with the brush border of the renal proximal tubules may also be present or cross-reacted in the microvilli of the rat embryonic visceral yolk-sac.     

Immunochemical examination of soluble antigens in the serum of Balb/c mice infected with Rauscher leukaemia virus.

The serum of Balb/c mice infected with Rauscher leukaemia virus contained soluble antigens characterized by alpha2 and beta globulin electrophoretic mobility; their respective molecular weights, as determined with gel filtration, were 40 000 and 120 000. The antigens differed in specificity and their corresponding determinants were present on the surface of leukaemic cells. For Balb/c mice both antigens, for DBA/1 and C57B1/10Sn mice only the antigen showing alpha2 mobility was immunogenic.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Presence of herpes simplex virus-related antigens in transformed L cells.

Antiserum prepared against herpes simplex virus type 1 (HSV-1)-infected L cells, i.e., lytic antiserum, was shown by an indirect immunofluorescence test to stain 90 percent of HSV-transformed L or HeLa cells. Immunofluorescence in these cells was always most intense in the perinuclear cytoplasmic region. Similar results were obtained with antiserum prepared against HSV-transformed L cells. These data indicate that HSV-transformed cells (both L and HeLa) express HSV-related antigens. Antiserum prepared against HSV-1-transformed L cells, i.e., transformed-cell antiserum, was found to agglutinate purified HSV type 1 virions but failed to neutralize infectivity. This suggests that HSV-1 structural antigens are expressed in HSV-1-transformed L cells. Immunodiffusion studies showed that at least two HSV-related antigens could be demonstrated with antigens from HSV-1-transformed L cells and transformed-cell antiserum. These two antigens were shown to be present in all clonal lines of HSV-1-transformed cells examined, six L cell lines and one HeLa cell line. Therefore, we conclude that transformation of cells by HSV-1, which is known to be associated with acquisition of viral thymidine kinase, must also be associated with the presence of these two antigens. We performed experiments showing that there are species of HSV-related antibody in HSV-transformed cell antiserum that could not be absorbed out with antigens from HSV-infected L cells. Antibodies present in lytic antiserum were completely removed by antigen preparations from cells lytically infected with HSV-1. Also, lytic antiserum failed to block HSV-related staining of transformed L cells in a direct immunofluorescence test. These results are compatible with one of two notions: either (i) certain genes are expressed during transformation that are not expressed during lytic infection, or (ii) these genes are expressed to a much more reduced extent during lytic infection than in transformed cells.     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.     

A human liver alcohol dehydrogenase enzyme-linked immunosorbent assay method specific for class I, II, and III isozymes

A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes.     

Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity

Mammalian cells infected with herpes simplex virus (HSV) express a novel ribonucleotide reductase which is biochemically and immunologically distinct from the uninfected-cell enzyme. Using polyvalent rabbit antiserum raised against partially purified HSV type 2 reductase as well as monoclonal antibodies to HSV type 1 and HSV type 2 early antigens, we have been able to show that in both serotypes reductase activity is associated with phosphoproteins of molecular weights 144,000 and 38,000 encoded between map units 0.566 and 0.602 in the viral genomes. The major antigenic species (144,000) have been tentatively identified as HSV type 1 ICP6 and HSV type 2 ICP10.     

Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibody-dependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given gamma globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LD50 of SF, a virus which is in a different subgroup than SIN. All mice survived if the gamma globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus. Cobra venom factor treatment of normal C3H mice challenged with SF did not reduce the protection, suggesting that complement was not involved. Methyl palmitate (40 mg/mouse) given before gamma globulin and virus challenge suppressed macrophage activity and reduced the level of protection 23% in females and 70% in males. Silica treatment (3 mg/mouse) reduced the protection equally in both males and females by 92%. In vitro experiments were done to test if it were possible that cross-antibody-dependent cellular cytotoxicity (ADCC) could account for the passive cross-protection observed in this system. Cross-ADCC could be demonstrated in vitro at high dilutions of antiserum (1:25,600). On the basis of the in vitro and in vivo results presented, we suggest that cross-ADCC against SF-infected target cells is one of the likely mechanisms to explain the passive cross-protection observed.     

Immunogenic contaminants in mouse nerve-growth factor.

Experiments are described that confirm the presence of gamma-globulin in standard preparations of mouse nerve growth factor (7-S complex and beta subunit) and show that antibodies to this protein are present in horse antisera to the growth factor. These antibodies may be partially removed by adsorption with soluble antigen and totally removed by affinity chromatography on columns of insolubilised antigen. These procedures do not affect the potency of the antiserum in vitro or in vivo. Contaminating globulin may be removed from samples of the isolated beta subunit by gel filtration to give an immunochemically pure preparation which will permit more meaningful studies on nerve growth factor and its antiserum.     

Identification and purification of the specific antigen of Paracoccidioides brasiliensis responsible for immunoelectrophoretic band E.

A new purified antigen (E2) of Paracoccidioides brasiliensis mycelial growth phase was isolated by immunoadsorption from a crude metabolic soluble extract of the fungus. The antiserum prepared in a rabbit by inoculation of E2 antigen developed only one immunodiffusion line with the crude metabolic extract. Findings on immunological analysis showed that E2 antigen is the antigenic component of immunoelectrophoretic band E. The isolated antigens did not possess detectable alkaline phosphatase activity. It reacted in immunodiffusion tests with all the sera (14/14) from P. brasiliensis infected patients containing precipitating antibodies.     

Enzyme-linked immunosorbent assay of antigens from Candida albicans circulating in infected mice and rabbits: The role of mannan

Various antisera raised either to antigens ofCandida albicans or to sub-lethal infections of blastospores (convalescent sera) were tested for their efficacy in diagnosing systemic disease in artifically infected animals. Globulin from convalescent serum, when conjugated with alkaline phosphatase and used in enzyme-linked immunosorbent assays (ELISA), was the only antiserum type which detected circulatingCandida-related antigen in the serum of infected animals. Conjugates made from anti-mannan, anti-blastospore or antimycelial globulin did not detect antigen. Mannan did not appear to be related to an antigen produced in sera of experimentally infected mice. The significance of these results in the diagnosis of systemic candidosis is discussed.     

The use of Staphylococcus aureus rich in protein A in the detection of herpes simplex virus antigens

Herpes simplex virus (HSV) type 1 antigens were detected in infected human embryonic lung cells with the aid of specific antiserum and Staphylococcus aureus rich in protein A. When such staphylococci carrying specific anti-HSV IgG on their surface were interacted with various suspension of virus, a reduction in the initial virus titre of about 65% was obtained. However, no direct coagglutination was observed between cell-free supernatants of HSV or HSV-infected cells and sensitized staphylococci. When monolayers or suspended cells infected with the virus were treated with dilutions of specific anti-HSV antiserum followed by non-sensitized staphylococci (indirect method), an "aureola" of the bacteria was detected around the cells expressing the viral antigens. A similar picture was observed when infected cells were interacted directly with sensitized staphylococci. Viral antigens were detected already 12 hours post infection, well before the appearance of cytopathic effect. The sensitivity of the indirect method was found to be higher than that of the direct one and dependent on the multiplicity of infection and the serum dilution used. The method is proposed as a rapid means of identifying viral antigens in diagnostic and experimental virology.     

Interaction of Vi antigen with proteins

Whiteside, Roberta E. (Boston University School of Medicine, Boston, Mass.), and Edgar E. Baker. Interaction of Vi antigen with proteins. J. Bacteriol. 92:1597-1603. 1966.-Purified Vi antigen (Vi) mixed in equal amounts with bovine serum albumin (BSA) or human gamma globulin (HGG) at pH values above 4.7 formed a complex which was not precipitated by trichloroacetic acid or tungstic acid. At pH values below 4.7, the interaction between Vi and either BSA or HGG produced insoluble complexes except when excess Vi antigen was present. When sufficient Vi was present at the lower pH values, the soluble complex was not precipitated by trichloroacetic acid. Other acid polysaccharides tested did not form trichloroacetic acid-soluble complexes with BSA. When subjected to immunoelectrophoresis, the Vi-BSA complex migrated in agar at a rate different from that of either BSA or Vi alone. The complex reacted with both Vi and BSA antiserum. The addition of either BSA or Vi antiserum to a Vi-BSA complex resulted in dissociation of the complex and precipitation of either Vi or BSA, depending upon the antiserum used. Vi antigen mixed with purified O antigen from Salmonella typhosa formed a complex which migrated in agar at a rate different from that of either component alone when subjected to immunoelectrophoresis.     

Developmental Sequence and Intracellular Sites of Synthesis of Three Structural Protein Antigens of Influenza A2 Virus

Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.