Scientific Publications

The HIV-1 envelope (Env) spike (gp120(3)/gp41(3)) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a 'ground state' for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from 'snapping' into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.

Failure to elicit broadly neutralizing (bNt) antibodies (Abs) against the membrane-proximal external region of HIV-1 gp41 (MPER) reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal Abs (MAbs) (2F5, 4E10, and Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR) or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1 and yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Replacement of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating that the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure.

The broadly neutralizing antibody 2G12 binds a fairly conserved cluster of oligomannose sugars on the HIV surface glycoprotein gp120, which has led to the hypothesis that these sugars pose potential vaccine targets. Here, we present the chemical analysis, antigenicity, and immunogenicity of a bacterial lipooligosaccharide (LOS) comprised of a manno-oligosaccharide sequence analogous to the 2G12 epitope. Antigenic similarity of the LOS to oligomannose was evidenced by 2G12 binding to the LOS and the inability of sera elicited against synthetic oligomannosides, but incapable of binding natural oligomannose, to bind the LOS. Immunization with heat-killed bacteria yielded epitope-specific serum antibodies with the capacity to bind soluble gp120. Although these sera did not exhibit specific anti-HIV activity, our data suggest that this LOS may find utility as a template for the design of glycoconjugates to target HIV.

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.

The primary objective was to measure HIV incidence in two prospective cohorts of HIV-negative women. Secondary objectives included measuring pregnancy rates and participant retention rates.

To identify and describe populations at risk for HIV in 3 clinical research centers in Kenya and South Africa.

The extent of HIV-1 diversity was examined among patients attending a rural district hospital in a coastal area of Kenya. The pol gene was sequenced in samples from 153 patients. Subtypes were designated using the REGA, SCUEAL, and jpHMM programs. The most common subtype was A1, followed by C and D; A2 and G were also detected. However, a large proportion of the samples was found to be recombinants, which clustered within the pure subtype branches. Phylogeographic analysis of Kilifi sequences compared with those from other regions of Africa showed that while many sequences were closely related to sequences from Kenya, others were most closely related to known sequences from other parts of Africa, including West Africa. Overall, these data indicate that there have been multiple introductions of HIV-1 into this small rural town and surroundings with ongoing diversity being generated by recombination.

Several CC-motif chemokine ligands (CCLs) can block HIV-1-binding sites on CC-motif chemokine receptor 5 (CCR5) and inhibit viral entry. We studied single-nucleotide polymorphisms (SNPs) in genes encoding three CCR5 ligands (CCL3 (MIP-1a), CCL4 (MIP-1b)and CCL5 (RANTES)) along with an adjacent gene encoding a CCR2ligand (CCL2 (MCP-1)) to identify candidate markers for HIV-1 infection and pathogenesis. Analyses of 567 HIV-1 serodiscordant Zambian couples revealed that rs5029410C (in CCL3 intron 2) was associated with lower viral load (VL) in seroconverters, adjusted for gender and age (regression β=-0.57 log(10), P=4x10(-6)). Inaddition, rs34171309A in CCL3 exon 3 was associated with increased risk of HIV-1 acquisition in exposed seronegatives(hazard ratio=1.52, P=0.006 when adjusted for VL of the initially seropositive partner and genital ulcer/inflammation). SNPrs34171309 encodes a conservative Glu-to-Asp substitution. Fiven eighboring SNPs in tight linkage disequilibrium with rs34171309all showed similar associations with HIV-1 acquisition. How these multiple CCL3 SNPs may alter the occurrence or course of HIV-1 infection remains to be determined [corrected].

The male genital tract is of major importance in the transmission and acquisition of HIV-1. Studying cellular immunity in the male genital tract is important in development of HIV-1 vaccines protective at mucosal sites. Semen is the primary HIV-1 containing fluid released from the male genital tract and reducing virus levels in semen would also reduce HIV-1 spread. Characterizing lymphocytes from semen requires the isolation of viable T cells that can be analyzed by downstream applications such as flow cytometry. The aims of this study were to investigate the influence of various parameters on CD3(+) T cell yields from semen and to compare isolation methods to maximize CD3(+) T cell yields for the purpose of functional characterization by flow cytometry.

A major priority in HIV vaccine research is the development of an immunogen to elicit broadly neutralizing antibodies (NAbs). Monoclonal antibody (mAb) b12 is one of now several broadly neutralizing mAbs that bind epitopes overlapping the CD4-binding site (CD4bs) on HIV-1 gp120 and that serve as templates to engineer effective immunogens. We are exploring a strategy whereby extra glycans are incorporated onto gp120 to occlude the epitopes of non-neutralizing mAbs while maintaining exposure of the b12 site. Immunizing with these so-called hyperglycosylated gp120s is hypothesized to preferentially elicit b12-like NAbs. Here, the effects of two adjuvants, monophosphoryl lipid A (MPL) and Quil A, on eliciting b12-like responses when formulated with a new hyperglycosylated mutant, ΔN2mCHO(Q105N), is presented. Sera from ΔN2mCHO(Q105N)_MPL immunized animals bound the homologous antigen ΔN2mCHO(Q105N) with greater preference than sera from ΔN2mCHO(Q105N)_QuilA immunized animals, demonstrating the modulation of antibody fine specificity by these two adjuvants. We also found that sera from ΔN2mCHO(Q105N)_QuilA immunized animals bound best to a resurfaced HIV gp120 core protein on which non-CD4bs epitopes are substituted with non-HIV residues, suggesting that these sera contain a relatively larger fraction of CD4bs-specific antibodies. Consistent with these data, inhibition assays revealed epitope overlap with the binding sites of the CD4bs-specific antibodies b12, b13 and VRC03. Unexpectedly, these sera did not exhibit significant neutralizing activity against a set of HIV-1 primary strains. Our results show that although formulating mutant ΔN2mCHO(Q105N) with Quil A promotes the elicitation of CD4bs-directed antibodies relative to wild-type gp120, tweaking of the immunization regimen is needed to yield robust, CD4bs-focused NAbs.

Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.

To identify predictors of promotion of couples' HIV voluntary counseling and testing (CVCT) in Kigali, Rwanda.