[Show abstract][Hide abstract] ABSTRACT: An immunization regimen was evaluated in rabbits consisting of the soluble, oligomeric form of envelope glycoprotein of HIV-1, strain R2 (gp140(R2)), or the surface component of the same envelope (Env), gp120(R2), in the adjuvant AS02A. The gp140(R2) was selected based on its unusual CD4-independent phenotype and the exceptionally broad neutralizing response in the infected donor. The gp140(R2) immunogen induced antibodies that achieved 50% neutralization of 48/48, and 80% neutralization of 43/46 primary strains of diverse HIV-1 subtypes tested. The strains tested included members of standard panels of subtype B and C strains, and other diverse strains known to be neutralization resistant. The gp120(R2) induced antibodies that neutralized 9/48 of the same strains. Neutralization was IgG-mediated and HIV-1-specific. These results demonstrate that induction of truly broad spectrum neutralizing antibodies is an achievable goal in HIV-1 vaccine development.
Proceedings of the National Academy of Sciences 07/2007; 104(24):10193-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we tested the hypothesis that donors with broadly cross-reactive HIV-1 neutralizing (BCN) sera are infected with viruses encoding envelope glycoproteins (Envs) with unusual immunogenic properties. Cloned env genes were from samples of donors previously identified as having BCN antibodies (BCN donors) and from other donors not known to have such antibodies (non-BCN donors). Neutralization properties of viruses pseudotyped with BCN and non-BCN Envs were determined using BCN, non-BCN sera and broadly cross-neutralizing monoclonal antibodies (Mabs). BCN sera neutralized with higher frequency and geometric mean titers than non-BCN sera. Viruses pseudotyped with BCN Envs were mostly resistant to neutralization by anti-gp120 Mabs but tended to be more sensitive to the anti-gp41 Mabs, 2F5 and 4E10 than non-BCN Env-pseudotyped viruses. Sequence analysis of clones obtained from sequential samples of two BCN donors revealed respective 2F5 epitope mutations T662A and K665T. The K665T mutation evolved as the predominant genotype in the respective donor, consistent with an escape mutation event. The A662T mutation reduced sensitivity to 4E10, as well as 2F5 and homologous sera, consistent with neutralization escape mutation and targeting of the 2F5 epitope region by the serum. Our study suggests that viruses infecting these BCN donors encoded Envs that may have been unusually competent for induction of antibodies against the membrane proximal epitope region (MPER) of gp41, and these Envs may be useful vaccine components.
[Show abstract][Hide abstract] ABSTRACT: We studied the capacity of active immunization of rhesus monkeys with HIV-1 envelope protein (Env) to induce primary virus cross-reactive neutralizing antibodies to prevent infection following intravenous challenge with simian-human immunodeficiency virus (SHIV). Monkeys were immunized with the human immunodeficiency type 1 (HIV-1) strain R2 Env. Initially, the Env was expressed in vivo by an alphavirus replicon particle system, and then it was administered as soluble oligomeric gp140. Concurrently, groups of monkeys received expression vectors that encoded either simian immunodeficiency virus (SIV) gag/pol genes or no SIV genes in vivo to test the additional protective benefit of concurrent induction of virus-specific cell-mediated immune (CMI) responses. Groups of control monkeys received either the gag/pol regimen or sham immunizations. The antibodies induced by the Env immunization regimen neutralized diverse primary HIV-1 strains. Similarly, potent CMI responses were induced by the gag/pol regimen, as measured by gamma interferon enzyme-linked immunospot assays. Differences in the responses among groups of monkeys strongly suggested that there was interference between the Env and gag/pol immunization regimens. Complete protection of some of the monkeys against infection after intravenous challenge with the partially pathogenic SHIV(DH12R (Clone 7)) was associated independently with both neutralizing antibody and CMI responses. Protection was associated with SHIV(DH12 (Clone 7)) serum neutralizing antibody titers of > or =1:80 or with cellular immune responses corresponding to >2,000 spot forming cells per 10(6) peripheral blood mononuclear cells. Immunization was also associated with a reduction in the magnitude and duration of virus load. Induction of cross-reactive, primary HIV-1-neutralizing antibodies is feasible and, when potent, may result in complete protection against infection with a heterologous challenge virus strain.
Journal of Virology 04/2005; 79(6):3358-69. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.
Journal of Virology 10/2004; 78(17):9233-42. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Resistance to neutralization is an important characteristic of primary isolates of human immunodeficiency virus type 1 (HIV-1) that relates to the potential for successful vaccination to prevent infection and use of immunotherapeutics for treatment of established infection. In order to further elucidate mechanisms responsible for neutralization resistance, we studied the molecular mechanisms that determine the resistance of the primary virus isolate of the strain HIV-1 MN to neutralization by soluble CD4 (sCD4). As is the case for the global neutralization resistance phenotype, sCD4 resistance depended upon sequences in the amino-terminal heptad repeat region of gp41 (HR1), as well as on multiple functional interactions within the envelope complex. The functional interactions that determined the resistance included interactions between the variable loop 1 and 2 (V1/V2) region and sequences in or near the CD4 binding site (CD4bs) and with the V3 loop. Additionally, the V3 loop region was found to interact functionally with sequences in the outer domain of gp120, distant from the CD4bs and coreceptor-binding site, as well as with a residue thought to be located centrally in the coreceptor-binding site. These and previous results provide the basis for a model by which functional signals that determine the neutralization resistance, high-infectivity phenotype depend upon interactions occurring across the surface of the gp120 core structure and involving variable loop structures and gp41. This model should be useful in efforts to define epitopes that may be important for primary virus neutralization.
Journal of Virology 08/2003; 77(14):8061-71. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160deltaCT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160deltaCT was released by cells in the form of membrane-bound vesicles. gp160deltaCT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virus-neutralizing activity by vaccination may be realized.
Journal of Virology 04/2003; 77(5):3119-30. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.
Journal of Virology 02/2002; 76(2):644-55. · 4.65 Impact Factor