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Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures, we carefully characterized these particles, concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays, Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay, pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis, fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly, the sequence of one of these novel human antibodies, identified during cloning of single HIV-specific B cells and designated 2C6, exhibited homology to mAb 47e, a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120, but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays, yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271–276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration. [ABSTRACT FROM AUTHOR]

HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specificmemory B cells encoding Abs that mediate ADCC froma subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activitywhen compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise fromunique B-cell lineages in the same individual. Further, thesemAbsmediate potent plasma IgG-specificADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner. [ABSTRACT FROM AUTHOR]

Summary: HIV-1 assembly and release occur at the plasma membrane in T lymphocytes, while intracellular sites of virus assembly or accumulation are apparent in macrophages. The host protein tetherin (BST-2) inhibits HIV release from the plasma membrane by retaining viral particles at the cell surface, but the role of tetherin at intracellular HIV assembly sites is unclear. We determined that tetherin is significantly upregulated upon macrophage infection and localizes to an intracellular virus-containing compartment (VCC). Tetherin localized at the virus-VCC membrane interface, suggesting that tetherin physically tethers virions in VCCs. Tetherin knockdown diminished and redistributed VCCs within macrophages and promoted HIV release and cell-cell transmission. The HIV Vpu protein, which downregulates tetherin from the plasma membrane, did not fully overcome tetherin-mediated restriction of particle release in macrophages. Thus, tetherin is essential for VCC formation and may account for morphologic differences in the apparent HIV assembly sites in macrophages versus T cells. [ABSTRACT FROM AUTHOR]

Abstract: HIV pseudovirion or virus-like particle vaccines represent a promising approach for eliciting humoral and cellular immune responses. Pseudovirions present the envelope glycoprotein complex in its authentic trimeric form, and thus have the potential to generate neutralizing antibodies against relevant virion-associated epitopes that may be lacking in protein subunit vaccines. The development of pseudovirion particles as a viable vaccine approach for progression to clinical testing has been limited by a number of factors, including shedding of particle-associated gp120, practical limitations to large-scale production and purification, and the generation of antibodies against cellular proteins incorporated on the particle surface that confound the analysis of HIV-specific neutralizing antibody responses. Here, we review methods that address each of these challenges, with a focus on production methods for generating non-infectious Gag–Env pseudovirions. Mammalian cell lines that inducibly express HIV Gag and Env can overcome production limitations, and produce pseudovirions that retain gp120 following purification. Baculovirus production systems have the potential to provide higher quantities of particles, but cleavage of gp160 remains a current limitation. Anti-cellular antibody responses can be diminished by adsorption with cell lysates or whole cells. These technical advances should facilitate the further development of pseudovirion vaccine approaches in preclinical testing and future clinical trials. [Copyright &y& Elsevier]

Human immunodeficiency virus type-1 (HIV-1) particles incorporate a trimeric envelope complex (Env) made of gp120 (SU) and gp41 (TM) heterodimers. It has been previously established that soluble CD4 (sCD4) interaction leads to shedding of gp120 from viral particles, and that gp120 may also be easily lost from virions during incubation or particle purification procedures. In the design of HIV particle or pseudovirion-based HIV vaccines, it may be important to develop strategies to maximize the gp120 content of particles. We analyzed the gp120 retention of HIV-1 laboratory-adapted isolates and primary isolates following incubation with sCD4 and variations in temperature. NL4-3 shed gp120 readily in a temperature- and sCD4-dependent manner. Surprisingly, inactivation of the viral protease led to markedly reduced shedding of gp120. Gp120 shedding was shown to vary markedly between HIV-1 strains, and was not strictly determined by whether the isolate was adapted to growth on immortalized T cell lines or was a primary isolate. Pseudovirions produced by expression of codon-optimized gag and env genes also demonstrated enhanced gp120 retention when an immature core structure was maintained. Pseudovirions of optimal stability were produced through a combination of an immature Gag protein core and a primary isolate Env. These results support the feasibility of utilizing pseudovirion particles as immunogens for the induction of humoral responses directed against native envelope structures. [Copyright &y& Elsevier]

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.

[Display omitted] • Wild-type HIV-1 intasomes are assembled into several distinct multimeric species in vitro. The high-resolution structures of these intasomes are presented. • The different multimeric intasome species have the same arrangement of integrase domains around the viral DNA. • All the multimeric species are similarly active in vitro. • Assembly of multiple intasome species in an intrinsic property of HIV integrase in vitro. Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis. [ABSTRACT FROM AUTHOR]

Abstract: The process of HIV assembly requires extensive homomultimerization of the Gag polyprotein on cellular membranes to generate the nascent particle bud. Here we generated a full-length, monomeric Gag polyprotein bearing mutations that eliminated multimerization in living cells as indicated by fluorescence resonance energy transfer (FRET). Monomeric Gag resembled non-myristoylated Gag in its weak membrane binding characteristics and lack of association with detergent-resistant membranes (DRMs or lipid rafts). Monomeric Gag failed to assemble virus-like particles, but was inefficiently rescued into particles by wildtype Gag through the influence of the matrix domain. The subcellular distribution of monomeric Gag was remarkably different than either non-myristoylated Gag or wildtype Gag. Monomeric Gag was found on intracellular membranes and at the plasma membrane, where it highlighted plasma membrane extensions and ruffles. This study indicates that monomeric Gag can traffic to assembly sites in the cell, where it interacts weakly with membranes. [Copyright &y& Elsevier]