Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits.

Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.

Covalent binding of human two-domain CD4 to an HIV-1 subtype C SOSIP.664 trimer modulates its structural dynamics.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates host cell infection by binding to the cellular receptor CD4. Recombinant Env bound to CD4 has been explored for its potential as an HIV vaccine immunogen as receptor binding exposes otherwise shielded, conserved functional sites. Previous preclinical studies showed an interchain disulphide linkage facilitated between Env and 2dCD4S60C generates an immunogenic complex that elicits potent, broadly neutralizing antibodies (bNAbs) against clinically relevant HIV-1. This study investigated conformational dynamics of 2dCD4WT and 2dCD4S60C bound to an HIV-1C SOSIP.664 Env trimer using hydrogen-deuterium exchange mass spectrometry. The Env:2dCD4S60C complex maintains key contact residues required for MHCII and Env/gp120 binding and the residues encompassing Ibalizumab's epitope. Important residues remaining anchored, with an increased flexibility in surrounding regions, evidenced by the higher exchange seen in flanking residues compared to Env:2dCD4WT. While changes in Env:2dCD4S60C dynamics in domain 1 were moderate, domain 2 exhibited greater variation. Lack of stability-inducing H-bonds in these allosteric sites suggest the improved immunogenicity of Env:2dCD4S60C result from exposed CD4 residues providing diverse/novel antigenic targets for the development of potent, broadly neutralizing Ibalizumab-like antibodies.

Correction: Features of Recently Transmitted HIV-1 Clade C Viruses that Impact Antibody Recognition: Implications for Active and Passive Immunization.

[This corrects the article DOI: 10.1371/journal.ppat.1005742.].

Potent and broad HIV-neutralizing antibodies in memory B cells and plasma.

Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Antibody 10E8, reactive with the distal portion of the membrane-proximal external region (MPER) of HIV-1 gp41, is broadly neutralizing. However, the ontogeny of distal MPER antibodies and the relationship of memory B cell to plasma bnAbs are poorly understood. HIV-1-specific memory B cell flow sorting and proteomic identification of anti-MPER plasma antibodies from an HIV-1-infected individual were used to isolate broadly neutralizing distal MPER bnAbs of the same B cell clonal lineage. Structural analysis demonstrated that antibodies from memory B cells and plasma recognized the envelope gp41 bnAb epitope in a distinct orientation compared with other distal MPER bnAbs. The unmutated common ancestor of this distal MPER bnAb was autoreactive, suggesting lineage immune tolerance control. Construction of chimeric antibodies of memory B cell and plasma antibodies yielded a bnAb that potently neutralized most HIV-1 strains.

Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity.

Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

Features of Recently Transmitted HIV-1 Clade C Viruses that Impact Antibody Recognition: Implications for Active and Passive Immunization.

The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following infection. Viruses collected pre-seroconversion were significantly more resistant to serum neutralization compared to post-seroconversion viruses (p = 0.001). Over 13 years of the study as the epidemic matured, HIV-1 diversified (p = 0.0009) and became more neutralization resistant to monoclonal antibodies VRC01, PG9 and 4E10. When tested at therapeutic levels (10ug/ml), VRC01 only neutralized 80% of viruses in the panel, although it did exhibit potent neutralization activity against sensitive viruses (IC50 titres of 0.42 µg/ml). The Gp120 amino acid similarity between the clade C panel and candidate C-clade vaccine protein boosts (Ce1086 and TV1) was 77%, which is 8% more distant than between CRF01_AE viruses and the RV144 CRF01_AE immunogen. Furthermore, two vaccine signature sites, K169 in V2 and I307 in V3, associated with reduced infection risk in RV144, occurred less frequently in clade C panel viruses than in CRF01_AE viruses from Thailand. Increased resistance of pre-seroconversion viruses and evidence of antigenic drift highlights the value of using panels of very recently transmitted viruses and suggests that interventions may need to be modified over time to track the changing epidemic. Furthermore, high divergence such as that observed in the older clade C epidemic in southern Africa may impact vaccine efficacy, although the correlates of infection risk are yet to be defined in the clade C setting. Findings from this study of acute/early clade C viruses will aid vaccine development, and enable identification of new broad and potent antibodies to combat the HIV-1 C-clade epidemic in southern Africa.

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

Structure and immune recognition of trimeric pre-fusion HIV-1 Env.

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.

A fusion intermediate gp41 immunogen elicits neutralizing antibodies to HIV-1.

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41(int)-Cys) and show that it folds into an elongated ∼ 12-nm-long extended structure based on small angle x-ray scattering data. Gp41(int)-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41(int)-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140(CA018) in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140(CA018) was higher than that induced by gp41(int)-Cys, the majority of animals immunized with gp41(int)-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.

Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.

Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.

Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape.

Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful, a minority of individuals naturally develop these antibodies after many years of infection. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.

UCLA1, a synthetic derivative of a gp120 RNA aptamer, inhibits entry of human immunodeficiency virus type 1 subtype C.

Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC(50)s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor.

Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals.

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 "tier 2" viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.

The neutralization breadth of HIV-1 develops incrementally over four years and is associated with CD4+ T cell decline and high viral load during acute infection.

An understanding of how broadly neutralizing activity develops in HIV-1-infected individuals is needed to guide vaccine design and immunization strategies. Here we used a large panel of 44 HIV-1 envelope variants (subtypes A, B, and C) to evaluate the presence of broadly neutralizing antibodies in serum samples obtained 3 years after seroconversion from 40 women enrolled in the CAPRISA 002 acute infection cohort. Seven of 40 participants had serum antibodies that neutralized more than 40% of viruses tested and were considered to have neutralization breadth. Among the samples with breadth, CAP257 serum neutralized 82% (36/44 variants) of the panel, while CAP256 serum neutralized 77% (33/43 variants) of the panel. Analysis of longitudinal samples showed that breadth developed gradually starting from year 2, with the number of viruses neutralized as well as the antibody titer increasing over time. Interestingly, neutralization breadth peaked at 4 years postinfection, with no increase thereafter. The extent of cross-neutralizing activity correlated with CD4(+) T cell decline, viral load, and CD4(+) T cell count at 6 months postinfection but not at later time points, suggesting that early events set the stage for the development of breadth. However, in a multivariate analysis, CD4 decline was the major driver of this association, as viral load was not an independent predictor of breadth. Mapping of the epitopes targeted by cross-neutralizing antibodies revealed that in one individual these antibodies recognized the membrane-proximal external region (MPER), while in two other individuals, cross-neutralizing activity was adsorbed by monomeric gp120 and targeted epitopes that involved the N-linked glycan at position 332 in the C3 region. Serum antibodies from the other four participants targeted quaternary epitopes, at least 2 of which were PG9/16-like and depended on the N160 and/or L165 residue in the V2 region. These data indicate that fewer than 20% of HIV-1 subtype C-infected individuals develop antibodies with cross-neutralizing activity after 3 years of infection and that these antibodies target different regions of the HIV-1 envelope, including as yet uncharacterized epitopes.

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.

Comparative analysis of expression and secretion of placental leptin in mammals.

Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mechanisms by which leptin levels are increased may be species specific, however, with some species upregulating adipose leptin production and others expressing leptin in the placenta. Placental leptin expression was examined in representative species of the two most abundant mammalian orders, Rodentia and Chiroptera, and in cultured human choriocarcinoma (BeWo) cells. Leptin mRNA was expressed in BeWo cells and in placentas of Myotis lucifugus (little brown bat), Eptesicus fuscus (big brown bat), and Rattus norvegicus (laboratory rat), but not the common laboratory mouse Mus musculus. cAMP stimulated secretion of leptin from BeWo cells and also stimulated leptin mRNA expression in the cells. In addition to adipose and placental tissue, leptin transcript in M. lucifugus was detectable in heart, spleen, and liver, but not in lung, brain, and kidney. Hepatic expression was also observed in E. fuscus, but not in mice or rats, and did not appear to result from hepatic fat deposition. Leptin cDNA was cloned and sequenced from M. lucifugus placenta and shared up to 95% homology with other mammalian leptin cDNAs. It is concluded that 1) placental leptin expression and secretion are species-specific traits, 2) placental leptin production represents one of three major mechanisms for achieving high circulating maternal leptin levels during pregnancy, the others being upregulation of adipose leptin production and production of circulating leptin-binding proteins, and 3) hepatic leptin expression in pregnant insectivorous bats may be an adaptation resulting from the presence of extremely low amounts of subcutaneous fat during pregnancy and lactation in these species.