Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three amino acid residues.

Two monoclonal antibodies recognizing distinct epitopes the outer boundaries of which are separated by only three amino acid residues, a maximum of 10A, were demonstrated to bind simultaneously to a short synthetic peptide. The affinity of binding of the two monoclonal antibodies and of Fab' fragments derived from them was determined. The stoichiometry of the interaction was analysed by velocity sedimentation and by gel permeation chromatography experiments. The results indicate that the immune complexes formed are composed of two antibody molecules in association with one or two peptide molecules.

Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites.

A number of peptides of the hemagglutinin (HA) of X-31 influenza virus have been synthesised. The amino acid sequences of some of these peptides represent regions of HA which have been postulated [Wiley et al., Nature, Lond. 289, 373-378 (1981)] to form the antigenic sites of this molecule. Animals were immunized with free peptide or peptide conjugated to a carrier and the resulting antisera examined for their capacities to bind to homologous peptide, whole HA, reduced and alkylated HA, and intact virus. Not all peptides examined in this way were immunogenic. Only antibodies raised against the C-terminus of HA1 peptide displayed binding to virus. This antiserum bound to the intact HA but not to the reduced and alkylated form of the molecule. These results raise questions as to the feasibility of using synthetic peptides of the influenza HA in short linear sequences to elicit neutralising antibody.

Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues.

Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well-characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope-directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs.

Delivery of antiviral chemotherapeutic agents to neurons by retrograde axonal transport.

It may be possible to eliminate herpes simplex or zoster viruses from the neurons of carriers by treatment with an antiviral chemotherapeutic agent such as adenine arabinoside, ribavirin or acyclovir, coupled to a compound such as horseradish peroxidase that undergoes retrograde axonal transport.

Antiviral chemotherapy.

Progress in antiviral chemotherapy has taken place as a logical strategy for the design of antiviral agents has emerged. The second-generation nucleoside analogues, led by acyclovir, have proved their worth against herpesviruses and should now become a standard part of medical practice. Meanwhile, recombinant DNA technology has lowered the cost of interferons to the point at which the several human subtypes of these naturally occurring hormones can be subjected individually to controlled clinical trials against the viral diseases in the treatment of which they show promise. Yet, optimism about the future of antiviral chemotherapy must be tempered by the observation that most of the agents discussed in this review are described more accurately as promising rather than proven, and several of these have not yet been released in Australia at the time of writing.

Macrophages bind directly to Semliki Forest virus-infected cells and mediate antibody-dependent cell-mediated cytotoxicity.

Macrophages were found to bind directly to Semliki Forest virus (SFV)-infected, but not uninfected, P815 cells. In the presence of anti-H-2d or anti-BALB/c antibody, macrophages lysed SFV-infected, but not uninfected, P815 cells. It is proposed that macrophage-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) proceeds via two functionally distinguishable initial steps: (1) adhesion of effector to target cell, which may be mediated through antibody or through viral protein, as in the case of SFV-infected target cells suboptimally sensitized with antibody; (2) antibody-dependent initiation of cytolysis.

Further characterization of natural killer cells induced by Kunjin virus.

The natural killer (NK) cell induced one to two days after Kunjin virus infection of BALB/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. It is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. Levels of lysis of non-tumour target cells are dependent on their genotype. Furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible again after culture in vitro. The virus-induced NK cell is elicited as readily in athymic (nude) as in normal mice. X-irradiation inhibits its development if administered prior to infection. It is labile on culture at 37 degrees. The cell carries Fc receptors but its NK activity is not antibody-dependent.

Cytotoxic macrophages: a rapid nonspecific response to viral infection.

In vitro and in vivo assays have been developed to study the relative contributions of various types of immune cytolysis in the destruction of infected cells after Semliki Forest virus infection of BALB/c mice. Highly cytotoxic activated macrophages, not specific for the infecting virus, appear on day 1, peak on day 2 to 3, and disappear within a week. Specifically sensitized T cells appear around day 3, peak on day 6, and disappear within a month. Cytotoxic antibody appears on day 4 and reaches high titers by day 8. Immune spleen cells greatly reduce the yield of virus from cultured cells. Infected cells rapidly disappear after transfer to infected animals.

Conformational changes in influenza virus haemagglutinin and its monomer detected by monoclonal antibodies.

Exposure of influenza virus haemagglutinin to pH 5 results in conformational changes occurring in the molecule which are accompanied by antigenic modifications. Furthermore, isolated haemagglutinin (HA) at a concentration of 0.1 nM undergoes dissociation from the trimeric to a monomeric form when exposed to pH 5. Whether present on intact virus or as the isolated monomer, each form of haemagglutinin from pH 5 exhibits similar alterations in antigenic characteristics. These forms of HA show modifications in the antigenic sites located in the hinge (site C), tip (site B) and subunit interface (site D) regions. Whereas binding of monoclonal antibodies recognizing the tip and interface is abrogated or diminished, binding of antibodies to the hinge region is greatly enhanced following exposure of virus or the monomeric form of HA to pH 5.

Influenza viruses are T cell-independent B cell mitogens.

UV-inactivated influenza virus A strains of subtypes H1, H2, H3, and H6 were shown to be mitogenic for unprimed splenic lymphocytes from BALB/c mice. Representative viruses of these four subtypes all behaved as T cell-independent B cell mitogens. The magnitude of the proliferative response was determined by the subtype of the hemagglutinin molecule: H2 and H6 viruses were the most potent mitogens, and H3 viruses were moderately mitogenic, whereas H1 viruses induced only low, but significant, levels of proliferation. Mitogenesis was inhibited by antiviral sera and by monoclonal antibodies directed against hemagglutinin.

Comparison of natural killer cells induced by Kunjin virus and Corynebacterium parvum with those occurring naturally in nude mice.

Natural killer (NK) cells are rapidly elicited in the spleen and peritoneal cavity of mice inoculated intravenously or intraperitoneally with live Kunjin virus, and more slowly in the peritoneal cavity of mice inoculated intraperitoneally with Formalin-inactivated Corynebacterium parvum. NK cells induced by either agent display cytotoxicity for a similar spectrum of syngeneic, allogeneic, and xenogeneic cultured cell lines. By contrast, the cells occurring naturally in the spleen of congenitally athymic (nude) mice show substantially lower NK activity and are cytotoxic for a more restricted range of target cell lines. The distinction suggests that there may be more than one type of NK cell or that activation enhances the cytotoxicity and perhaps broadens the range of target specificity of endogenous NK cells.

Conservation of determinants for class II-restricted T cells within site E of influenza virus hemagglutinin and factors influencing their expression.

The determinants recognized by helper T cells specific for the site E region of H3 subtype influenza virus hemagglutinin (HA) have been defined by examining the reactivity of T-cell clones with sets of overlapping peptides of various lengths covering the site. Two overlapping sequences, TLIDALLG and LIDALLGDP, were identified as the minimal determinants for four of five representative clones. These sequences are located within a loop of the molecule closed by a disulfide bond and presumably require cleavage of this bond for interaction with the class II major histocompatibility molecule. In contrast, the determinant recognized by the fifth clone was dependent on the presence of an intact disulfide bond for its expression and could not be represented by a synthetic peptide homolog of the linear sequence. Both TLIDALLG and LIDALLGDP are conserved within all field strains of the H3 subtype. Nevertheless, recognition of these sequences by the T-cell clones is affected by the glycosylation pattern of the hemagglutinin and by residues lying outside the minimal determinant. Three distinct clones directed towards the sequence LIDALLGDP were remarkably similar in their pattern of response to a set of synthetic analogs of the determinant, suggesting that residues of the T-cell receptor other than those contacting the minimal determinant may be responsible for the different specificities observed for these clones with different field strains of virus.

Two cytotoxic cells in peritoneal cavity of virus-infected mice: antibody-dependent macrophages and nonspecific killer cells.

Two new types of cell-mediated immune cytolysis of togavirus-infected cells are compared. The peritoneal cavity of mice 2 days after infection contains a nonadherent, non-phagocytic, non-O-bearing, trypsin-resistant, EDTA-sensitive cell displaying broadly specific cytotoxicity for uninfected or virus-infected syngeneic or xenogeneic cell lines. Peritoneal macrophages from normal mice are cytotoxic to infected target cells sensitized with minute amounts of homologous antiviral IgG.

Protection of mice against cancer by immunization with membranes but not purified virions from virus infected cancer cells.

The life span of C57/Bl mice inoculated with Lewis lung carcinoma cells was prolonged if the mice were pre-immunized with membranes from these cells infected in vitro with influenza virus. Likewise, BALB/c mice were protected against the malignant tumour WEHI-11 by prior immunization with extracts of cultured WEHI-11 cells which had been infected with influenza virus or Semiliki Forest virus (SFV). Partially purified SFV grown in WEHI-11 cells also protected mice from cancer grafts but neither highly purified SFV nor the glycoprotein from the envelope of this virus protected the mice. It is concluded that SFV-induced immunopotentiation against cancer is not due to covalent linkage of tumour specific transplantation antigen (TSTA) to viral envelope protein but more probably is due to the apposition of viral glycoprotein and cellular TSTA in the plasma membrane of the cancer cell.

Antigenic determinants of influenza virus haemagglutinin. VI. Antigenic characterization of the oligosaccharide sidechains form HA1 of influenza virus haemagglutinins.

The oligosaccharide sidechains attached to the major polypeptide, HA1 of the haemagglutinin of influenza virus were examined for antigenic activity using a solid-phase radioimmunoassay. Cross-reactivity between the HA1 of the different human subtypes was clearly demonstrable with IgG raised against purified virus but was abrogated if anti-carbohydrate antibodies were first removed by passage of the IgG through an immunoadsorbent column containing haemagglutinin (HA) from an unrelated avian influenza strain. Antibodies eluted from the column were found to cross-react with the HA1 of all subtypes tested. 'Host antigen' extracted from chick chorioallantoic membrane and coupled to Sepharose was also able to remove cross-reactive antibodies from antiviral sera, while antibodies raised against host antigen bound to the HA1 isolated from each subtype tested. It is concluded that, although there are qualitative and quantitative differences between the oligosaccharide sidechains of influenza haemagglutinins, the antigenically active sidechains are cross-reactive.

Mitogenic activity of influenza virus and haemagglutinin.

Influenza A viruses behave as T cell-independent B cell mitogens in vitro. The magnitude of the proliferation induced varies with the haemagglutinin subtype of the virus, the order being H2 greater than H6 greater than H3 greater than H1 for Balb/c mice. H3 viruses are equally mitogenic for all strains of mice tested. In contrast, the mitogenic response to H2 and H6 viruses is controlled by the I-E subregion of the major histocompatibility complex. These viruses are mitogenic only for strains of mice that express surface I-E antigen (haplotypes a, d, k, p, r), and not for haplotypes b, f, q, s, which fail to synthesize a normal E alpha chain and do not express surface I-E antigen. Mitogenesis by H2 and H6 viruses may involve direct interaction of virus with I-E molecules on the B lymphocyte or an accessory cell, whereas mitogenesis by H3 viruses does not appear to involve I-E.

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.

Murine helper T lymphocyte response to influenza virus: recognition of haemagglutinin by subtype-specific and cross-reactive T cell clones.

Helper T cel lines specific for influenza virus were established by continuous culture of Mem 71-Bel (H3) virus-immune spleen cells in the presence of virus and antigen-presenting cells and their specificity assessed in proliferation experiments. A line stimulated in vitro with Mem 71-Bel virus was able to proliferate in response to viruses of the same, and also of different, type A haemagglutinin (HA) subtypes as the immunizing virus but not to a type B influenza virus. A component of this cross-reactivity was due to recognition of the HA molecule. Lines stimulated in vitro with purified disrupted H3 or H2 viruses showed a higher degree of cross-reactivity. Of nine clones isolated from these lines, seven were directed against the HA molecule and recognized the HA1 chain. The HA-specific T cell clones were either subtype-specific T cell clones were either subtype-specific, recognizing only viruses of the H3 subtype, or cross-reactive, also recognizing viruses of the H2 subtype of type A (but not type B). Subtype-specific and cross-reactive T cell clones were shown to function as helper T cells in vitro. In addition to collaborating with H3 virus-primed B cells responding to H3 virus in culture, the cross-reactive T cell clone could also provide help for H2 virus-primed B cells making anti-HA antibody in response to H2 virus.