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Engineered single human CD4 domains as potent HIV-1 inhibitors and components of vaccine immunogens.

ABSTRACT: Soluble forms of the HIV-1 receptor CD4 (sCD4) have been extensively characterized for more than 2 decades as promising inhibitors and components of vaccine immunogens. However, they were mostly based on the first two CD4 domains (D1D2), and numerous attempts to develop functional, high-affinity, stable soluble one-domain sCD4 (D1) have not been successful because of the strong interactions between the two domains. We have hypothesized that combining the power of structure-based design with sequential panning of large D1 mutant libraries against different HIV-1 envelope glycoproteins (Envs) and screening for soluble mutants could not only help solve the fundamental stability problem of isolated D1, but may also allow improvement of D1 affinity while preserving its cross-reactivity. By using this strategy, we identified two stable monomeric D1 mutants, mD1.1 and mD1.2, which were significantly more soluble and bound Env gp120s more strongly (50-fold) than D1D2, neutralized a panel of HIV-1 primary isolates from different clades more potently than D1D2, induced conformational changes in gp120, and sensitized HIV-1 for neutralization by CD4-induced antibodies. mD1.1 and mD1.2 exhibited much lower binding to human blood cell lines than D1D2; moreover, they preserved a ?-strand secondary structure and stability against thermally induced unfolding, trypsin digestion, and degradation by human serum. Because of their superior properties, mD1.1 and mD1.2 could be potentially useful as candidate therapeutics, components of vaccine immunogens, and research reagents for exploration of HIV-1 entry and immune responses. Our approach could be applied to other cases where soluble isolated protein domains are needed.

SUBMITTER: Chen W

PROVIDER: S-EPMC3165761 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Engineered single human CD4 domains as potent HIV-1 inhibitors and components of vaccine immunogens.

Chen Weizao W Feng Yang Y Gong Rui R Zhu Zhongyu Z Wang Yanping Y Zhao Qi Q Dimitrov Dimiter S DS

Journal of virology 20110629 18

Soluble forms of the HIV-1 receptor CD4 (sCD4) have been extensively characterized for more than 2 decades as promising inhibitors and components of vaccine immunogens. However, they were mostly based on the first two CD4 domains (D1D2), and numerous attempts to develop functional, high-affinity, stable soluble one-domain sCD4 (D1) have not been successful because of the strong interactions between the two domains. We have hypothesized that combining the power of structure-based design with sequ ...[more]

PMID: 21715496

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Project description:Purpose of reviewTo summarize the in-vivo efficacy of neutralizing human monoclonal antibodies against HIV-1, to discuss the recent finding that an engineered human antibody VH domain, domain antibody (dAb), exhibits exceptionally potent and broadly cross-reactive neutralizing activity against HIV-1 primary isolates by targeting a hidden conserved epitope that is not accessible by larger antibodies and to suggest the possibility of developing a novel class of potent HIV-1 inhibitors based on human dAbs.Recent findingsHIV-1 has evolved a number of strategies to evade humoral immunity, including protecting highly conserved and important structures from the access of antibodies generated by the immune system. We have recently demonstrated that a human dAb (size approximately 15 kDa), m36, targets a highly protected structure on the HIV-1 envelope glycoprotein (Env), gp120, and exhibits exceptionally potent neutralizing activity against HIV-1 primary isolates, with potency on average higher than those of the broadly cross-reactive neutralizing human monoclonal antibody, scFv m9, and the inhibitory peptide, C34.SummaryThe efficacy of the anti-HIV-1 therapy is significantly compromised by resistance to the currently used US Food and Drug Administration-approved antiretroviral drugs, which suggests an urgent need to develop novel classes of potent inhibitors. Several broadly cross-reactive neutralizing human monoclonal antibodies are highly effective against HIV-1 infection in vitro, but their administration to HIV-1-infected humans has only resulted in modest antiviral effects. Engineered human antibody fragments, dAbs, could be more potent because of their small size (about 10-fold smaller than that of an IgG), which allows targeting of highly conserved structures on the HIV-1 envelope glycoprotein that are not accessible by full-size antibodies and relatively efficient penetration into the densely packed lymphoid environment in which HIV-1 mostly replicates and spreads.

Project description:BackgroundThe RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine.Methodology/principal findingsIsoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I.Conclusions/significanceMN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.

Project description:BackgroundTen to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant.MethodsBased on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments.ResultsIt was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region.ConclusionsOur results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

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Engineered single human CD4 domains as potent HIV-1 inhibitors and components of vaccine immunogens.

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Engineered single human CD4 domains as potent HIV-1 inhibitors and components of vaccine immunogens.

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