Effect of Antibody to Neuraminidase on the Maturation and Hemagglutinating Activity of an Influenza A2 Virus

Antiserum to a recombinant between an A(o) and an A(2) influenza virus had no detectable antibody against an A(2) virus in standard hemagglutination-inhibition tests, and inhibited 95% of viral neuraminidase activity at a 1 to 400 dilution. However, on mixing virus with antiserum, a drop of up to 90% in hemagglutinin titer was observed. The effects of ultrasonication and direct electron microscopic examination indicated that the antiserum caused aggregation of virus particles. When antiserum was added to A(2) virus-infected chick embryo fibroblasts, release of virus appeared markedly inhibited. After ultrasonication to disrupt aggregates, an increase in released hemagglutinin was observed, but the resulting level was considerably lower than that in control cultures containing normal rabbit serum. In thin sections of infected cells, similar numbers of virus profiles were observed in control and antiserum-treated cultures. A marked increase in release of hemagglutinin was noted if receptor-destroying enzyme was added to antiserum-treated cultures. The results indicate that antibody to neuraminidase does not exert a direct effect on viral maturation, but inhibits the detachment of viral progeny from cell surface receptors.     

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG₁ antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.     

Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.     

Applications of a Synthetic Neuraminidase Substrate

A rapid and precise assay for neuraminidase using 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid (MPN) is described. It is proposed that this substrate be used for the standardization of activity of neuraminidases from viral, bacterial, and mammalian sources. MPN is also used as a chromogenic substrate to localize influenza and parainfluenza virus foci in tissue culture. This technique permits the recovery of infective virus from these stained "plaques." It has also been demonstrated that immunoprecipitin lines containing neuraminidase complexes with antibody in the Ouchterlony test can be observed by a similar staining procedure. No enzyme inhibition occurs in the presence of anti-neuraminidase antibodies or concanavalin A when MPN is used as a substrate in contrast to the results with high-molecular-weight substrates such as fetuin.     

Isolation of Hemagglutinin and Neuraminidase Subunits of Hemagglutinating Virus of Japan

When purified hemagglutinating virus of Japan (HVJ) was treated with trypsin, two major surface antigens were released from the virus. The "hemagglutinin" subunits obtained by this method were reactive with homologous hemagglutination-inhibition antibody and could be detected by an antibody-blocking test. They adsorbed to but did not agglutinate red cells and thus appeared to be "monovalent." The neuraminidase subunits were obtained in fully active form and did not adsorb to red cells. This finding suggests that these two activities of HVJ are associated with different subunits of the virus particle. The hemagglutinin and neuraminidase subunits could be partially separated by zonal rate centrifugation or gel filtration on Sephadex G-200. The molecular weights estimated for these subunits were approximately 124,000 and 114,000, respectively. After treatment with trypsin, virus-associated hemagglutinin and neuraminidase activities were both reduced significantly. The electron micrographs of such trypsinized virus particles showed complete or partial loss of surface projections. These results suggested that the subunits obtained by this method seemed to be those projections liberated from the virus by the action of trypsin.     

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.     

小鼠对重组痘苗病毒表达流感病毒血凝素的免疫反应

实验比较了小鼠对表达流感病毒A/NJ/11/76(H1N1)和A/Jap/305/57(H2N2)血凝素基因的痘苗病毒重组株HSW2和VInf1的免疫反应,两株重组病毒经静脉或静脉加鼻腔免疫小鼠后都产生相应血凝抑制抗体;用不同剂量的痘苗病毒重组株HSW2皮内接种家兔也产生相应抗体,且抗体滴度与接种的病毒量成正比关系,但用痘苗病毒野毒株免疫的家兔未测到抗体,这两株痘苗病毒重组株免疫的小鼠,能保护小鼠对流感病毒母株的攻击,HSW2和VInf1的保护指数分别为3.3和3.9个对数,但这两株病毒未能诱导细胞毒性T细胞反应。     

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA₁ genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA₁ had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.     

Two isozymes of the Na,K-ATPase have distinct antigenic determinants

Two isozymes of the Na,K-ATPase were purified from rat renal medulla and rat brainstem axolemma, and antisera were raised in rabbits. When antibody titers were measured, two sera showed specificity for either the kidney or axolemma Na,K-ATPases and had limited cross-reactivity which could be removed by cross-adsorption. In blots of polyacrylamide gels, these sera reacted with only the alpha or alpha (+) Na,K-ATPase catalytic subunits, while they cross-reacted with both types of beta subunits. Two other sera each recognized both alpha and alpha (+), indicating that the catalytic subunit isozymes have additional shared antigenic determinants. A comparison of the Na,K-ATPases from the brains of different vertebrate species indicates that birds and fish differ from mammals and amphibians in the manifestation of Na,K-ATPases isozymes. Neither neuraminidase nor endoglycosidase F treatment eliminated specific antibody reaction or affected the electrophoretic mobilities of the alpha and alpha (+) subunits, although endoglycosidase F increased the mobilities of the two types of beta subunits to similar final apparent molecular weights. Blots of the peptide fragments produced by incomplete papain and trypsin digests of the alpha and alpha (+) subunits were stained with the specific sera, and the patterns of immunoreactive fragments were found to be markedly different. The results suggest that the antigenic differences reside in differences in the primary protein sequences of the two isozymes.     

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

The application of different cell lines and virus strains for detection of immunofluorescence rubella antibodies.

Fluorescence antibodies (FA) were titrated in human sera against antigens synthesized in four different infected cell lines: SIRC, RK-13, Vero and BHK-21. The cells infected in suspension by rubella virus attained the optimal concentration of fluorescence staining antigen earlier, fluorescence was more intensive and the titers of the tested human sera were slightly higher than in monolayer cell cultures. FA titers were high and they were correlated with the antibody titers obtained by hemagglutination-inhibition test. Some practical implications of these findings are discussed.     

Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing).

The mammalian reoviruses have provided a valuable model for studying the pathogenesis of viral infections of the central nervous system (CNS). We have used this model to study the effect of antibody on disease produced by the neurally spreading reovirus type 3 (Dearing) (T3). Polyclonal and monoclonal antibodies protect mice from fatal infection with T3 after either footpad or intracerebral virus challenge. Protection occurs with monoclonal antibodies directed against the viral cell attachment protein sigma 1, and with polyclonal antisera without T3 sigma 1 binding activity. In vivo protection occurs with both neutralizing and nonneutralizing monoclonal antibodies. Antibody-mediated protection does not require serum complement and, under specific circumstances, can occur via Fc-independent mechanisms. Antibody can protect mice when transferred up to 5 days after intracerebral challenge and up to 7 days after footpad challenge, times when high titers of virus are present in the CNS. Thus, antibody mediated protection against this neurally spreading virus does not require neutralizing antibody or serum complement and occurs even in the face of established CNS infection.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A.

Human lysosomal N-acetyl-alpha-neuraminidase is deficient in two lysosomal storage disorders, sialidosis, caused by structural mutations in the neuraminidase gene, and galactosialidosis, in which a primary defect of protective protein/cathepsin A (PPCA) leads to a combined deficiency of neuraminidase and beta-D-galactosidase. These three glycoproteins can be isolated in a high molecular weight multi-enzyme complex, and the enzymatic activity of neuraminidase is contingent on its interaction with PPCA. To explain the unusual need of neuraminidase for an auxiliary protein, we examined, in transfected COS-1 cells, the effect of PPCA expression on post-translational modification, turnover and intracellular localization of neuraminidase. In pulse-chase studies, we show that the enzyme is synthesized as a 46 kDa glycoprotein, which is poorly phosphorylated, does not undergo major proteolytic processing and is secreted. Importantly, its half-life is not altered by the presence of PPCA. However, neuraminidase associates with the PPCA precursor shortly after synthesis, since the latter protein co-precipitates with neuraminidase using anti-neuraminidase antibodies. We further demonstrate by subcellular fractionation of transfected cells that neuraminidase segregates to mature lysosomes only when accompanied by wild-type PPCA, but not by transport-impaired PPCA mutants. These data suggest a novel role for PPCA in the activation of lysosomal neuraminidase, that of an intracellular transport protein.     

Murine natural killer cells limit coxsackievirus B3 replication

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.     

Antibodies to phospholipids and liposomes: binding of antibodies to cells

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.