Postattachment neutralization of a primary strain of HIV type 1 in peripheral blood mononuclear cells is mediated by CD4-specific antibodies but not by a glycoprotein 120-specific antibody that gives potent standard neutralization.

De novo infecting HIV-1 or virus released from an infected cell in vivo attaches relatively quickly to a target cell, but the rate of fusion-entry of such virus is slow, with 50% entry taking > or =2 hr. It is thus desirable that antibodies stimulated by any vaccine or given in immunotherapy are able to neutralize not only free virus, but also virus attached to the target cell. Here we investigated postattachment neutralization (PAN) of a primary HIV-1 strain (JRCSF) in peripheral blood mononuclear cells and of a T cell line-adapted strain (IIIB) in C8166 T lymphoblastoid cells, using the highly potent gp120-specific human monoclonal b12 monoclonal IgG, and monoclonal antibodies specific for the CD4 primary cell receptor. In addition, we improved the experimental protocols of related studies by using a pulse of antibody, thus avoiding the complication of neutralizing progeny virus. We found that b12 IgG PAN was inefficient, with PAN of IIIB needing a 1000-fold greater concentration of antibody than was required for standard neutralization, and PAN of JRCSF being detected erratically only at 4 degrees C and unphysiologically high concentrations (300 microg/ml). Nonetheless, under identical conditions a 10-microg/ml pulse of the CD4-specific MAb Q4120 gave up to 99% PAN of JRCSF, and more than 95% even when added 3 hr after infection at 37 degrees C. Possible mechanisms by which PAN by CD4- specific antibodies is mediated are discussed. We suggest that such anti-CD4 antibodies should be considered as a component of HIV-1 immunotherapy.

Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide.

The ability of five different adjuvants (alum, complete Freund's adjuvant, Quil A, AdjuPrime and Ribi) to stimulate humoral and T-cell mediated immune responses against a purified chimeric virus particle was investigated. Each adjuvant was administered subcutaneously to adult mice together with 10 microg of wildtype (wt) cowpea mosaic virus (CPMV) or a chimeric CPMV displaying the HIV-1 gp41 peptide, residues 731-752. All preparations elicited strong antibody responses to CPMV, but Quil A elicited the highest and most consistent responses to the HIV-1 peptide. This finding was reflected in both ELISA titres with immobilized peptide and in HIV-1-neutralizing antibody. In addition Quil A was also, the only adjuvant to stimulate an in vitro proliferative T-cell response. Surprisingly with all adjuvant formulations a predominately IgG2a anti-gp41 peptide response was observed, indicating a type 1 T-helper cell-like response. Furthermore, the efficiency of the CPMV display system was demonstrated by its ability to induce good levels of peptide specific antibody in the absence of any adjuvant.

Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization.

Control of pandemic human immunodeficiency virus type 1 (HIV-1) infection ideally requires specific mucosal immunity to protect the genital regions through which transmission more often occurs. Thus a vaccine that stimulates a disseminated mucosal and systemic protective immune response would be extremely useful. Here we have investigated the ability of a chimeric plant virus, cowpea mosaic virus (CPMV), expressing a 22 amino acid peptide (residues 731-752) of the transmembrane gp41 protein of HIV-1 IIIB (CPMV-HIV/1), to stimulate HIV-1-specific and CPMV-specific mucosal and serum antibody following intranasal or oral immunization together with the widely used mucosal adjuvant, cholera toxin. CPMV-HIV/1 has been shown previously to stimulate HIV-1-specific serum antibody in mice by parenteral immunization. All mice immunized intranasally with two doses of 10 microg of CPMV-HIV/1 produced both HIV-1-specific IgA in faeces as well as higher levels of specific, predominantly IgG2a, serum antibody. Thus there was a predominantly T helper 1 cell response. All mice also responded strongly to CPMV epitopes. Oral immunization of the chimeric cowpea mosaic virus was less effective, even at doses of 500 microg or greater, and stimulated HIV-1-specific serum antibody in only a minority of mice, and no faecal HIV-1 specific IgA.

Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers.

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic alpha-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.

A human IgG1 (b12) specific for the CD4 binding site of HIV-1 neutralizes by inhibiting the virus fusion entry process, but b12 Fab neutralizes by inhibiting a postfusion event.

The human b12 IgG1, specific for the CD4 binding site of the gp120 of HIV-1, was prepared by recombinant DNA technology. It had a high neutralization rate constant (-3.5 x 10(5) M(-1) sec(-1)), although this is about 10-fold less than the values for the best poliovirus or influenza A virus MAbs. The recombinant b12 Fab neutralized well, with about one-tenth of the activity of b12 IgG. The mechanisms by which b12 IgG1 and its Fab neutralize HIV-1 IIIB on C8166 cells have been investigated. Neither inhibited attachment of virus to the target cell as judged by FACS, immunofluorescence, and ELISA data. This was controlled using MAb F105, another human IgG1, that did neutralize by inhibiting attachment under our conditions. The interactions of b12 IgG- and Fab-neutralized virions with target cells were compared with those of nonneutralized virus using a number of different techniques (fluorescence dequenching of R18-labeled virions, immunofluorescence of virion gp41 and p24 antigens, and acquisition of resistance to removal of virions from the cell by protease). These and the inhibition of HIV-1-mediated cell-cell fusion all demonstrated that b12 IgG neutralized by inhibiting the primary fusion-uncoating mechanism. However, the interactions of b12 Fab-neutralized and nonneutralized virions with C8166 cells were indistinguishable. Thus b12 Fab did not inhibit fusion uncoating, and by inference inhibited a stage of infection that occurs after the entry of the virion core into the cytoplasm. It is therefore possible that b12 IgG kills HIV-1 twice over, by fusion-inhibition and by inhibiting the postentry event proposed for the Fab. The mechanism of neutralization of b12 Fab and of other MAbs that neutralize in a similar way and why b12 Fab and IgG neutralize by different mechanisms are discussed.

Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1.

Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.li had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 degrees C and 35 degrees C. However, ICR41.1i gave PAN at 21 degrees C but not at 35 degrees C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.

Analysis of the interaction between a synthetic peptide of influenza virus hemagglutinin and monoclonal antibodies using an optical biosensor.

The interaction between two monoclonal antibodies and their corresponding Fab' fragments with a synthetic peptide, corresponding to the C-terminal 23 residues of the HA1 chain of influenza virus hemagglutinin against which they were generated, has been examined using an optical biosensor employing the detection principal of surface plasmon resonance (Pharmacia BIAcore). The data obtained has been analysed in detail by linear transformation of the primary data and nonlinear regression analysis, as well as by analysis of equilibrium binding data. The 2/1 antibodies and their Fab' fragments displayed higher affinity than the corresponding 1/1 proteins. The IgGs were found to have equilibrium association constants (KA) 10-20-fold higher than the corresponding Fab' fragments. This appears largely to be due to differences in the dissociation rate constant (kd) and probably reflects increased avidity due to bivalent binding.

The assembly and immunological properties of non-linear synthetic immunogens containing T-cell and B-cell determinants.

For the rational design of synthetic vaccines, a potential immunogen must contain the appropriate helper T-cell and B-cell determinants to elicit a strong and relevant immune response. In this study we describe a method for the assembly of antigenic determinants from influenza virus hemagglutinin onto a lysine-based support, resulting in dimeric and trimeric constructs bearing both T-cell and B-cell determinants. A panel of synthetic immunogens was constructed incorporating peptides representing: (i) the B-cell determinant TLKLATG and the T-cell determinant PKYVKQNTLKLA which overlaps this sequence in the heavy chain (HA1) of the hemagglutinin; and (ii) the same B-cell determinant with an alternate T-cell determinant ALNNRFQIKGVELKS from the light chain (HA2). With these peptides we were able to investigate the effects of altering the source of T-cell help, increasing the copy number of B-cell determinants as well as comparing the presentation of determinants in either linear tandem or branched geometries. In general, peptides incorporating the non-native helper T-cell determinant in a branched conformation were superior immunogens, eliciting higher titres of both peptide-specific and virus-specific antibody. Increasing the copy number of the B-cell determinant also proved to be an advantage in terms of increasing antibody titres. Other evidence was obtained indicating that presentation of determinants to T cells may be different for linear peptide constructs compared to branched immunogens bearing the same determinants.

Analysis of antibody-antigen and antibody-anti-(idiotypic antibody) cross-reactivity using synthetic peptide probes.

The extent, nature and structural basis of immunological cross-reactivity of an anti-synthetic peptide monoclonal antibody (MAb) with the parent antigen (influenza virus haemagglutinin) from which the peptide was derived, and with a paratope-directed anti-idiotypic (anti-Id) antibody was investigated. Use of synthetic homologs and analogs of the peptide indicated that the anti-peptide MAb utilizes a common binding site to complex with peptide, haemagglutinin (HA) and anti-Id antibody, and the affinity constants for the binding of the anti-peptide MAb to peptide and to the anti-Id MAb were found to differ only by three fold. Determination of the amino acid sequence of the heavy chain variable domain (VH) of the anti-Id MAb did not reveal any obvious sequence homology with the peptide. Consideration of the spatial arrangement of residues, however, disclosed a region within the framework of the anti-Id VH with similarity to the epitope recognized by the anti-peptide MAb. This region, formed from antiparallel chains, contains amino acid residues arranged in a conformation similar to that assumed by amino acid residues comprising the epitope within the intact HA.

Equilibrium analysis of the interaction between a synthetic peptide of influenza virus hemagglutinin and monoclonal antibodies.

The affinity of interaction between two monoclonal antibodies and a synthetic peptide representing the C-terminal 23 residues of the heavy chain (HA1) of influenza virus hemagglutinin were determined using an air-driven ultracentrifuge. The technique makes use of common laboratory equipment and is based on sound theoretical principles. Because the method does not rely on the solid-phase immobilisation of one of the interacting species, it circumvents problems associated with ELISA-like assays, which, in the case of peptides, may involve the immobilisation of ligand through association of amino acid residues necessary for recognition by antibody. The technique should be applicable to the study of a wide range of ligand-acceptor systems. Because only one of the reagents needs to be pure to allow labelling, prior purification of the biological receptor is not necessary. The method also lends itself to inhibition experiments in which the effects of various homologs on the binding event can be examined in a way which permits an evaluation of potential agonists and antagonists.

Binding affinity of influenza virus N9 neuraminidase with Fab fragments of monoclonal antibodies NC10 and NC41.

Sedimentation equilibrium centrifugation has been applied to determine the affinity and stoichiometry of the interaction between Fab fragments, derived from monoclonal antibodies NC10 and NC41, with influenza virus neuraminidase N9 isolated from either tern or whale. Although the two neuraminidase epitopes recognized by NC10 and NC41 Fab overlap, crystallographic studies have shown that the modes of binding of each Fab are different. The sedimentation equilibrium experiments described here reveal that the binding affinities are also different, with NC10 Fab binding more strongly to each neuraminidase. Furthermore, comparison of the affinity of binding of each antibody fragment reveals a stronger interaction with tern neuraminidase than with whale neuraminidase. Although the respective epitopes recognized by each antibody on the two antigens are similar, this technique shows that they do nevertheless possess sufficient differences to affect significantly the binding of antibody.

Quantitative analysis of the interaction between lysozyme and monoclonal antibody D1.3.

The method of sedimentation equilibrium has been used to determine the stoichiometry and binding constant for the interaction between hen egg white lysozyme and monoclonal antibody D1.3. The procedures described allow the relative binding affinities of 125I-labelled lysozyme and unlabelled lysozyme to be compared. The data indicate that labelled and unlabelled lysozyme bind to monoclonal D1.3 with similar affinity (binding constant, K = 1.6 x 10(9)/M). Using solid-phase methods estimates obtained for the binding constant were lower and dependent both on the amount of antigen coated on the plate and the dilution of primary antibody (D1.3). These data were not consistent with a simple equilibrium binding model, suggesting kinetic or orientation effects. In contrast sedimentation equilibrium experiments provide a rapid and accurate method for determining both the stoichiometry and binding constants for the interaction between antigens and antibodies.