The generation of an antibody response capable of neutralizing a broad range of clinical isolates remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Envelope glycoprotein (Env)-based vaccine candidates will also need to take into account the extensive genetic diversity of circulating HIV-1 strains. We describe here the generation of soluble, stabilized, proteolytically cleaved, trimeric forms of Env (SOSIP gp140 proteins) based on contemporary Env subtype A viruses from East Africa. We discuss issues associated with the construction, purification, and characterization of such complex proteins; not all env sequences allow the expression of trimeric proteins. However, stabilized trimers from one such protein, KNH1144 SOSIP gp140, were successfully made. These proteins are now being prepared for preclinical immunogenicity studies.
"Moreover, the immune response to clade C viruses is generally less well documented than that against clade B, despite their wider distribution (Bures et al., 2002). The relatively recent revelation that the inner domain of g120 is flexibly mobile and effectively generates irrelevant antibody responses has focused attention on gp120 molecules that are 'stabilized' as trimers (Beddows et al., 2006, 2007; Iyer et al., 2007; Schulke et al., 2002) or isolated fragments (Chen et al., 2007; Yang et al., 2004). The definition of the responses that are capable of being produced by such fragments is therefore an important dimension of candidate vaccine design. "
[Show abstract][Hide abstract] ABSTRACT: The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.
Journal of General Virology 11/2008; 89(Pt 10):2597-604. DOI:10.1099/vir.0.2008/003491-0 · 3.18 Impact Factor
"(Binley et al., 2000; Sanders et al., 2000). The HIV-1 Env subtype A clone KNH1144 (accession number AF457066) (Beddows et al., 2006) and the subtype B clones JR-FL and Ba-L have been described previously (Binley et al., 2000). In domain-swap experiments, the JR-FL gp41 ectodomain was replaced with the corresponding region of KNH1144 gp41, using EcoRI and HindIII restriction enzymes, followed by repair of the restriction sites and verification of the sequences. "
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 envelope glycoprotein is expressed on the viral membrane as a trimeric complex, formed by three gp120 surface glycoproteins non-covalently associated with three membrane-anchored gp41 subunits. The labile nature of the association between gp120 and gp41 hinders the expression of soluble, fully cleaved, trimeric gp140 proteins for structural and immunization studies. Disruption of the primary cleavage site within gp160 allows the production of stable gp140 trimers, but cleavage-defective trimers are antigenically dissimilar from their cleaved counterparts. Soluble, stabilized, proteolytically cleaved, trimeric gp140 proteins can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP). We have found that SOSIP gp140 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). We now show that the determinants of this enhanced stability are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability. The stabilized trimers retain the epitopes for several neutralizing antibodies (b12, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. The ability to increase the stability of gp140 trimers might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.
[Show abstract][Hide abstract] ABSTRACT: The third variable region, V3, of the gp120 surface envelope glycoprotein is an approximately 35-residue-long, frequently glycosylated, highly variable, disulfide-bonded structure that has a major influence on HIV-1 tropism. Thus the sequence of V3, directly or indirectly, can determine which coreceptor (CCR5 or CXCR4) is used to trigger the fusion potential of the Env complex, and hence which cells the virus can infect. V3 also influences HIV-1's sensitivity to, and ability to escape from, entry inhibitors that are being developed as antiviral drugs. For some strains, V3 is a prominent target for HIV-1 neutralizing antibodies (NAbs); indeed, for many years it was considered to be the "principal neutralization determinant" (PND). Some efforts to use V3 as a vaccine target continue to this day, despite disappointing progress over more than a decade. Recent findings on the structure, function, antigenicity, and immunogenicity of V3 cast new doubts on the value of this vaccine approach. Here, we review recent advances in the understanding of V3 as a determinant of viral tropism, and discuss how this new knowledge may inform the development of HIV-1 drugs and vaccines.
AIDS Research and Human Retroviruses 03/2005; 21(2):171-89. DOI:10.1089/aid.2005.21.171 · 2.33 Impact Factor
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