Roopa Kalyanaraman

We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.

The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schülke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.

OBJECTIVES: To engineer Lactobacillus spp. to secrete HIV-1 fusion inhibitors with potent neutralizing activity against primary HIV-1 isolates. METHODS: HIV-1 fusion inhibitors (FI-1, FI-2, and FI-3) were introduced into the previously developed shuttle vector pTSV2 and transformed in L. plantarum and L. gasseri. The signal peptide Usp45 from L. lactis was used to achieve high secretion efficiency of peptides into the bacterial supernatant. The antiviral activity of lactobacillus-derived HIV-1 fusion inhibitors was tested against a panel of primary HIV-1 isolates and a chimeric simian/HIV (SHIV-162P3) using the TZM infection assay. TZM-bl cells are engineered HeLa cells that express CD4, CCR5, and CXCR4 and contain integrated reporter genes for firefly luciferase and beta-galactosidase under the control of an HIV-1 long terminal repeat. The amount of secreted fusion inhibitor FI-3 was determined by Western blot analysis and the antiviral specificity verified by antibody-mediated depletion of peptide FI-3 and HIV-1 infection with VSV-G envelope pseudotyped virions. RESULTS: Viral infectivity of primary HIV-1 isolates and SHIV-162P3 was neutralized by up to 98% and 72%, respectively, by 10% (v/v) lactobacillus supernatant containing fusion inhibitor FI-3. The antiviral activity of the lactobacillus-derived fusion inhibitor FI-3 was clearly shown to be attributable to the secreted fusion inhibitor peptide. CONCLUSION: The development of recombinant lactobacilli expressing HIV-1 fusion inhibitors with potent neutralizing activity represents an important step toward the development of a live microbial (topical) microbicide against HIV-1 transmission.