Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site.

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Prevalence of gp160 polymorphisms known to be related to decreased susceptibility to temsavir in different subtypes of HIV-1 in the Los Alamos National Laboratory HIV Sequence Database.

BACKGROUND: Fostemsavir, a prodrug of the gp120-directed attachment inhibitor temsavir, is indicated for use in heavily treatment-experienced individuals with MDR HIV-1. Reduced susceptibility to temsavir in the clinic maps to discrete changes at amino acid positions in gp160: S375, M426, M434 and M475. OBJECTIVES: To query the Los Alamos National Laboratory (LANL) HIV Sequence Database for the prevalence of polymorphisms at gp160 positions of interest. METHODS: Full-length gp160 sequences (N = 7560) were queried for amino acid polymorphisms relative to the subtype B consensus at positions of interest; frequencies were reported for all sequences and among subtypes/circulating recombinant forms (CRFs) with ≥10 isolates in the database. RESULTS: Among 239 subtypes in the database, the 5 most prevalent were B (n = 2651, 35.1%), C (n = 1626, 21.5%), CRF01_AE (n = 674, 8.9%), A1 (n = 273, 3.6%) and CRF02_AG (n = 199, 2.6%). Among all 7560 sequences, the most prevalent amino acids at positions of interest (S375, 73.5%; M426, 82.1%; M434, 88.2%; M475, 89.9%) were the same as the subtype B consensus. Specific polymorphisms with the potential to decrease temsavir susceptibility (S375H/I/M/N/T/Y, M426L/P, M434I/K and M475I) were found in <10% of isolates of subtypes D, G, A6, BC, F1, CRF07_BC, CRF08_BC, 02A, CRF06_cpx, F2, 02G and 02B. S375H and M475I were predominant among CRF01_AE (S375H, 99.3%; M475I, 76.3%; consistent with previously reported low temsavir susceptibility of this CRF) and 01B (S375H, 71.7%; M475I, 49.5%). CONCLUSIONS: Analysis of the LANL HIV Sequence Database found a low prevalence of gp160 amino acid polymorphisms with the potential to reduce temsavir susceptibility overall and among most of the common subtypes.

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis.

HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens.IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

Fitness landscape of the human immunodeficiency virus envelope protein that is targeted by antibodies.

HIV is a highly mutable virus, and over 30 years after its discovery, a vaccine or cure is still not available. The isolation of broadly neutralizing antibodies (bnAbs) from HIV-infected patients has led to renewed hope for a prophylactic vaccine capable of combating the scourge of HIV. A major challenge is the design of immunogens and vaccination protocols that can elicit bnAbs that target regions of the virus's spike proteins where the likelihood of mutational escape is low due to the high fitness cost of mutations. Related challenges include the choice of combinations of bnAbs for therapy. An accurate representation of viral fitness as a function of its protein sequences (a fitness landscape), with explicit accounting of the effects of coupling between mutations, could help address these challenges. We describe a computational approach that has allowed us to infer a fitness landscape for gp160, the HIV polyprotein that comprises the viral spike that is targeted by antibodies. We validate the inferred landscape through comparisons with experimental fitness measurements, and various other metrics. We show that an effective antibody that prevents immune escape must selectively bind to high escape cost residues that are surrounded by those where mutations incur a low fitness cost, motivating future applications of our landscape for immunogen design.

Comparisons of Human Immunodeficiency Virus Type 1 Envelope Variants in Blood and Genital Fluids near the Time of Male-to-Female Transmission.

To better understand the transmission of human immunodeficiency virus type 1 (HIV-1), the genetic characteristics of blood and genital viruses from males were compared to those of the imputed founding virus population in their female partners. Initially serodiscordant heterosexual African couples with sequence-confirmed male-to-female HIV-1 transmission and blood and genital specimens collected near the time of transmission were studied. Single viral templates from blood plasma and genital tract RNA and DNA were sequenced across HIV-1 env gp160. Eight of 29 couples examined yielded viral sequences from both tissues. Analysis of these couples' sequences demonstrated, with one exception, that the women's founding viral populations arose from a single viral variant and were CCR5 tropic, even though CXCR4 variants were detected within four males. The median genetic distance of the imputed most recent common ancestor of the women's founder viruses showed that they were closer to the semen viruses than to the blood viruses of their transmitting male partner, but this finding was biased by detection of a greater number of viral clades in the blood. Using multiple assays, the blood and genital viruses were consistently found to be compartmentalized in only two of eight men. No distinct amino acid signatures in the men's viruses were found to link to the women's founders, nor did the women's env sequences have shorter variable loops or fewer N-linked glycosylation sites. The lack of selective factors, except for coreceptor tropism, is consistent with others' findings in male-to-female and high-risk transmissions. The infrequent compartmentalization between the transmitters' blood and semen viruses suggests that cell-free blood virus likely includes HIV-1 sequences representative of those of viruses in semen.IMPORTANCE Mucosal transmissions account for the majority of HIV-1 infections. Identification of the viral characteristics associated with transmission would facilitate vaccine design. This study of HIV strains from transmitting males and their seroconverting female partners found that the males' genital tract viruses were rarely distinct from the blood variants. The imputed founder viruses in women were genetically similar to both the blood and genital tract variants of their male partners, indicating a lack of evidence for genital tract-specific lineages. These findings suggest that targeting vaccine responses to variants found in blood are likely to also protect from genital tract variants.

Evidence for both Intermittent and Persistent Compartmentalization of HIV-1 in the Female Genital Tract.

HIV-1 has been shown to evolve independently in different anatomical compartments, but studies in the female genital tract have been inconclusive. Here, we examined evidence of compartmentalization using HIV-1 subtype C envelope (Env) glycoprotein genes (gp160) obtained from matched cervicovaginal lavage (CVL) and plasma samples over 2 to 3 years of infection. HIV-1 gp160 amplification from CVL was achieved for only 4 of 18 acutely infected women, and this was associated with the presence of proinflammatory cytokines and/or measurable viremia in the CVL. Maximum likelihood trees and divergence analyses showed that all four individuals had monophyletic compartment-specific clusters of CVL- and/or plasma-derived gp160 sequences at all or some time points. However, two participants (CAP177 and CAP217) had CVL gp160 diversity patterns that differed from those in plasma and showed restricted viral flow from the CVL. Statistical tests of compartmentalization revealed evidence of persistent compartment-specific gp160 evolution in CAP177, while in CAP217 this was intermittent. Lastly, we identified several Env sites that distinguished viruses in these two compartments; for CAP177, amino acid differences arose largely through positive selection, while insertions/deletions were more common in CAP217. In both cases these differences contributed to substantial charge changes spread across the Env. Our data indicate that, in some women, HIV-1 populations within the genital tract can have Env genetic features that differ from those of viruses in plasma, which could impact the sensitivity of viruses in the genital tract to vaginal microbicides and vaccine-elicited antibodies.IMPORTANCE Most HIV-1 infections in sub-Saharan Africa are acquired heterosexually through the genital mucosa. Understanding the properties of viruses replicating in the female genital tract, and whether these properties differ from those of more commonly studied viruses replicating in the blood, is therefore important. Using longitudinal CVL and plasma-derived sequences from four HIV-1 subtype C-infected women, we found fewer viral migrations from the genital tract to plasma than in the opposite direction, suggesting a mucosal sieve effect from the genital tract to the blood compartment. Evidence for both persistent and intermittent compartmentalization between the genital tract and plasma viruses during chronic infection was detected in two of four individuals, perhaps explaining previously conflicting findings. In cases where compartmentalization occurred, comparison of CVL- and plasma-derived HIV sequences indicated that distinct features of viral populations in the CVL may affect the efficacy of microbicides and vaccines designed to provide mucosal immunity.

Prediction of VRC01 neutralization sensitivity by HIV-1 gp160 sequence features.

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons.

Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles.

BACKGROUND: We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. RESULTS: Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. CONCLUSIONS: Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.

Similar Epitope Specificities of IgG and IgA Antibodies Elicited by Ad26 Vector Prime, Env Protein Boost Immunizations in Rhesus Monkeys.

Vaccine-elicited immunoglobulin G (IgG) has been shown to be important for protection against simian-human immunodeficiency virus (SHIV) infection in rhesus monkeys. However, it remains unclear whether vaccine-elicited IgA responses are beneficial or detrimental for protection. In this study, we evaluated the kinetics, magnitude, breadth, and linear epitope specificities of vaccine-elicited IgG and IgA responses in serum and mucosal secretions following intramuscular immunization with adenovirus 26 (Ad26) prime, Env protein boost vaccination regimens. The systemic and mucosal antibody responses exhibited kinetics similar to those of the serum antibody responses but lower titers than the serum antibody responses. Moreover, the IgG and IgA responses were correlated, both in terms of the magnitude of the responses and in terms of the antibody specificities against linear human immunodeficiency virus type 1 (HIV-1) Env, Gag, and Pol epitopes. These data suggest that IgG and IgA responses are highly coordinated in both peripheral blood and mucosal compartments following Ad26/Env vaccination in rhesus monkeys.IMPORTANCE Vaccine-elicited IgG responses are important for protection against simian-human immunodeficiency virus (SHIV) infection in nonhuman primates. However, much less is known about the role and function of IgA, despite it being the predominant antibody in mucosal sites. There is debate as to whether HIV-1-specific IgA responses are beneficial or detrimental, since serum anti-Env IgA titers were shown to be inversely correlated with protection in the RV144 clinical trial. We thus assessed vaccine-elicited IgG and IgA antibody responses in peripheral blood and mucosal secretions following vaccination with the Ad26/Env vaccine.

High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers.

Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Enhanced Immune Responses to HIV-1 Envelope Elicited by a Vaccine Regimen Consisting of Priming with Newcastle Disease Virus Expressing HIV gp160 and Boosting with gp120 and SOSIP gp140 Proteins.

Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost.

Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia.

UNLABELLED: Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25(+) T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4(+) cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4(+) T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4(+) malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL. IMPORTANCE: Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. HTLV-1 infection is the etiological agent of ATL and, unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In this study, we developed a recombinant strain of vesicular stomatitis virus (VSV) that specifically targets transformed CD4(+) T cells through replacement of the G protein of VSV with a hybrid fusion protein, combining domains from gp160 of HIV-1 and VSV-G. This modification eliminated the normally broad tropism of VSV and restricted infection to primarily the transformed CD4(+) cell population. This effect greatly reduced neurotoxic risk associated with VSV infection while still allowing VSV to effectively target ATL cells.

Broadly neutralizing antibodies developed by an HIV-positive elite neutralizer exact a replication fitness cost on the contemporaneous virus.

Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1(+)) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.

Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection.

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.

Newcastle disease virus expressing human immunodeficiency virus type 1 envelope glycoprotein induces strong mucosal and serum antibody responses in Guinea pigs.

Human immunodeficiency virus type 1 (HIV-1) is transmitted mainly through mucosal sites. Optimum strategies to elicit both systemic and mucosal immunity are critical for the development of vaccines against HIV-1. We therefore sought to evaluate the induction of systemic and mucosal immune responses by the use of Newcastle disease virus (NDV) as a vaccine vector. We generated a recombinant NDV, designated rLaSota/gp160, expressing the gp160 envelope (Env) protein of HIV-1 from an added gene. The gp160 protein expressed by rLaSota/gp160 virus was detected on an infected cell surface and was incorporated into the NDV virion. Biochemical studies showed that gp160 present in infected cells and in the virion formed a higher-order oligomer that retained recognition by conformationally sensitive monoclonal antibodies. Expression of gp160 did not increase the virulence of recombinant NDV (rNDV) strain LaSota. Guinea pigs were administered rLaSota/gp160 via the intranasal (i.n.) or intramuscular (i.m.) route in different prime-boost combinations. Systemic and mucosal antibody responses specific to the HIV-1 envelope protein were assessed in serum and vaginal washes, respectively. Two or three immunizations via the i.n. or i.m. route induced a more potent systemic and mucosal immune response than a single immunization by either route. Priming by the i.n. route was more immunogenic than by the i.m. route, and the same was true for the boosts. Furthermore, immunization with rLaSota/gp160 by any route or combination of routes induced a Th1-type response, as reflected by the induction of stronger antigen-specific IgG2a than IgG1 antibody responses. Additionally, i.n. immunization elicited a stronger neutralizing serum antibody response to laboratory-adapted HIV-1 strain MN.3. These data illustrate that it is feasible to use NDV as a vaccine vector to elicit potent humoral and mucosal responses to the HIV-1 envelope protein.

Escherichia coli-expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen.

BACKGROUND: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. METHODS: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels. RESULTS: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. CONCLUSIONS: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.

Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

Antigenic conservation and immunogenicity of the HIV coreceptor binding site.

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1-infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1-infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

Wide variation in the multiplicity of HIV-1 infection among injection drug users.

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.