We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp4l invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp4l which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp4l monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp4l in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp4l multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.

Three of 16 human monoclonal antibodies (hu-mAbs) enhanced human immunodeficiency virus type 1 (HIV-1) infection of MT-2 target cells by means ofa mechanism that is dependent on complement. Enhanced infections are characterized by an increase in cytopathic effects and antigen synthesis as well as an increase in the production of progeny virus as detected by release of reverse transcriptase activity and infectious virus into the culture medium. Analyses by radioimmunoprecipitation, Western blot, and ELISA using the pENV9 envelope fragment localize the antigenic specificities of these three hu-mAbs to the N-terminal two-thirds of the transmembrane protein gp4l. Competitive binding experiments indicate that the hu-mAbs are reactive with immuno-

Four of eight human monoclonal antibodies (huMAbs) to gp4l were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp4l transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp4l which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection.

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.The principal mechanism by which effective viral vaccines confer protective immunity is by induction of antibodies capable of neutralizing prevalent strains of virus (21). Efforts to develop a vaccine to protect against human immunodeficiency virus type 1 (HIV-1) infection are complicated by the fact that virus strains in infected patients tend to be highly resistant to neutralization. Studies that clarify the mechanisms responsible for this neutralization resistance may provide important leads regarding possible methods for the induction of potent neutralizing antibody responses capable of neutralizing these primary isolates. Previously, we described the selection and characterization of a neutralization-resistant mutant of the MN strain of HIV-1, which generally resists neutralization by human sera. The resistance phenotype was found to be determined by polymorphisms located in the C-terminal region of gp120 and N-terminal region of gp41 (16,17). There were two residues in gp120 and four in gp41 involved. Genetic analysis demonstrated t...

Two human monoclonal antibodies specific for the envelope glycoprotein (gp), gp4l, of the human immunodeficiency virus were conjugated to deglycosylated ricin A chain. These immunotoxins killed human immunodeficiency virus-infected H9 (T cell) and U937 (monocyte) cell lines but were nontoxic to the uninfected cell lines or to class II-positive Daudi cells. Specific killing of infected H9 cells could be completely blocked by recombinant gpl6O and partially blocked by unconjugated anti-gp4l antibody but was not blocked by recombinant gpl20 or human IgG demonstrating specificity for gp4l. The specific toxicity of the immunotoxins for infected U937 cells was markedly potentiated by chloroquine.