Quaternary Epitope Specificities of Anti-HIV-1 Neutralizing Antibodies Generated in Rhesus Macaques Infected by the Simian/Human Immunodeficiency Virus SHIVSF162P4

Monoclonal antibodies (MAbs) that neutralize human immunodeficiency virus type 1 (HIV-1) have been isolated from HIV-1-infected individuals or animals immunized with recombinant HIV-1 envelope (Env) glycoprotein constructs. The epitopes of these neutralizing antibodies (NAbs) were shown to be located on either the variable or conserved regions of the HIV-1 Env and to be linear or conformational. However, one neutralizing MAb, 2909, which was isolated from an HIV-1-infected subject, recognizes a more complex, quaternary epitope that is present on the virion-associated functional trimeric Env spike of the SF162 HIV-1 isolate. Here, we discuss the isolation of 11 anti-HIV NAbs that were isolated from three rhesus macaques infected with the simian/human immunodeficiency virus SHIV_SF162P4 and that also recognize quaternary epitopes. A detailed epitope mapping analysis of three of these rhesus antibodies revealed that their epitopes overlap that of the human MAb 2909. Despite this overall similarity in binding, however, differences in specific amino acid and glycosylation pattern requirements for MAb 2909 and the rhesus MAbs were identified. These results highlight similarities in the B-cell responses of humans and macaques to structurally complex neutralization epitopes on related viruses, HIV-1 and SHIV.HIV-1 infection typically elicits high levels of antibodies directed against the viral surface envelope (Env) glycoprotein, gp160. The initial anti-Env antibody response is nonneutralizing (28), but within 1 or 2 months after infection, neutralizing antibodies (NAbs) emerge which tend to be highly strain specific for the autologous virus and exhibit little or no neutralizing activity against heterologous HIV-1 strains (10, 22). However, several recent reports have indicated that approximately 25% of HIV-1-infected, antiretroviral-naïve patients develop broad cross-neutralizing antibody responses (5, 23, 26). In some cases, these broad neutralizing antibody responses can be mapped to the CD4-binding site of Env while in most cases a single epitope specificity cannot be identified to recapitulate the neutralizing breadth of the corresponding plasma (1, 4, 14, 15, 23, 25). Detailed analyses of the epitope specificities of broad plasma neutralizing antibody responses performed by several groups revealed the presence in HIV-positive (HIV⁺) plasmas of NAbs with as yet undefined epitope specificities (1, 15, 18, 23). It is possible that these undefined specificities include quaternary neutralizing epitopes (QNEs) and/or sugar molecules which coat the HIV Env spike expressed on the surface of viral particles.The human monoclonal antibody (MAb) 2909 recognizes a QNE present on the oligomeric Env spike present on the surface of HIV-1 SF162 virions (8). MAb 2909 can bind and neutralize SF162 virions but does not bind to the corresponding soluble SF162 Env. The binding of MAb 2909 to its QNE depends on the presence of the second and third variable regions of gp120 (the V2 and V3 loops, respectively). One particular amino acid at the amino terminal side of the V2 loop (K at position 158, based on the SF162 numbering, or position 160, based on the strain HxB2 numbering) appears to be critical for its binding (11). MAb 2909 was isolated from a person who was not infected with SF162, but a virus isolated from the donor of MAb 2909 bears a V2 loop with similarities to that of SF162 and, in particular, possesses the same K158 residue (M. K. Gorny, unpublished data). More recently, two additional human MAbs, PG9 and PG16, were isolated from a subject infected with clade A HIV-1 and were shown to bind to a QNE that also includes the V2 and V3 loops (30). In contrast, however, to the narrow neutralizing potential of MAb 2909, MAbs PG9 and PG16 display far broader neutralizing abilities.Similar to the infection of humans by HIV-1, chronic infection of rhesus macaques by simian/human immunodeficiency viruses (SHIVs) or chimpanzees by HIV-1 also results in the elicitation of potent NAbs against the autologous virus and, to a much lesser extent, against heterologous SHIV isolates or HIV-1 viruses (3, 6, 12, 17). Here, we describe a panel of MAbs from SHIV_SF162P4-infected rhesus macaques that demonstrates extremely potent neutralization against the homologous virus (that expresses the same Env as HIV-1 SF162) and that recognizes QNEs present on the surface of intact virions. Similar to the human MAbs 2909, PG9, and PG16, these rhesus macaque monoclonal antibodies (RhMAbs) recognize QNEs that include the V2 and V3 loops. Also, similar to MAb 2909, the RhMAbs neutralize only viruses expressing the SF162 Env. Consequently, we compared the fine epitope specificities of these RhMAbs to the epitope specificity of the human MAb 2909. Our detailed epitope mapping analysis reveals that although the human MAb 2909 and the RhMAbs recognize that same overall Env complex region, their specific requirements for binding differ. Thus, these studies of human and rhesus MAbs indicate that infection of humans and rhesus macaques with viruses expressing distinct Envs can result in the elicitation of antibodies that bind to overlapping conserved quaternary epitopes.     

Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.Human immunodeficiency virus type 2 (HIV-2) infection affects 1 to 2 million individuals, most of whom live in India, West Africa, and Europe (17). HIV-2 has diversified into eight genetic groups named A to H, of which group A is by far the most prevalent worldwide. Nucleotide sequences of Env can differ up to 21% within a particular group and by over 35% between groups.The mortality rate in HIV-2-infected patients is at least twice that of uninfected individuals (26). Nonetheless, the majority of HIV-2-infected individuals survive as elite controllers (17). In the absence of antiretroviral therapy, the numbers of infected cells (39) and viral loads (36) are much lower among HIV-2-infected individuals than among those who are HIV-1 infected. This may be related to a more effective immune response produced against HIV-2. In fact, most HIV-2-infected individuals have proliferative T-cell responses and strong cytotoxic responses to Env and Gag proteins (17, 31). Moreover, autologous and heterologous neutralizing antibodies (NAbs) are raised in most HIV-2-infected individuals (8, 32, 48, 52), and the virus seems unable to escape from these antibodies (52). As for HIV-1, the antibody specificities that mediate HIV-2 neutralization and control are still elusive. The V3 region in the envelope gp125 has been identified as a neutralizing target by some but not by all investigators (3, 6, 7, 11, 40, 47, 54). Other weakly neutralizing epitopes were identified in the V1, V2, V4, and C5 regions in gp125 and in the COOH-terminal region of the gp41 ectodomain (6, 7, 41). A better understanding of the neutralizing determinants in the HIV-2 Env will provide crucial information regarding the most relevant targets for vaccine design.The development of immunogens that elicit the production of broadly reactive NAbs is considered the number one priority for the HIV-1 vaccine field (4, 42). Most current HIV-1 vaccine candidates intended to elicit such broadly reactive NAbs are based on purified envelope constructs that mimic the structure of the most conserved neutralizing epitopes in the native trimeric Env complex and/or on the expression of wild-type or modified envelope glycoproteins by different types of expression vectors (4, 5, 29, 49, 58). With respect to HIV-2, purified gp125 glycoprotein or synthetic peptides representing selected V3 regions from HIV-2 strain SBL6669 induced autologous and heterologous NAbs in mice or guinea pigs (6, 7, 22). However, immunization of cynomolgus monkeys with a subunit vaccine consisting of gp130 (HIV-2BEN) micelles offered little protection against autologous or heterologous challenge (34). Immunization of rhesus (19, 44, 45) and cynomolgus (1) monkeys with canarypox or attenuated vaccinia virus expressing several HIV-2 SBL6669 proteins, including the envelope glycoproteins, in combination with booster immunizations with gp160, gp125, or V3 synthetic peptides, elicited a weak neutralizing response and partial protection against autologous HIV-2 challenge. Likewise, vaccination of rhesus monkeys with immunogens derived from the historic HIV-2ROD strain failed to generate neutralizing antibodies and to protect against heterologous challenge (55). Finally, baboons inoculated with a DNA vaccine expressing the tat, nef, gag, and env genes of the HIV-2UC2 group B isolate were partially protected against autologous challenge without the production of neutralizing antibodies (33). These studies illustrate the urgent need for new vaccine immunogens and/or vaccination strategies that elicit the production of broadly reactive NAbs against HIV-2. The present study was designed to investigate in the mouse model the immunogenicity and neutralizing response elicited by novel recombinant envelope proteins derived from the reference primary HIV-2ALI isolate, when administered alone or in different prime-boost combinations.     

Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC₅₀) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.Immunoglobulin molecules have multiple binding paratopes for antigens; for example, those for IgG1 are bivalent and those for IgM are dodecavalent. It is obvious that multivalent binding is required for the distinct mechanism of neutralization by cross-linking multiple virions to form virus aggregates (reviewed in references 7 and 67). Despite the potential of antibodies for multivalent binding, structural evidence indicates that neutralizing antibodies often bind to an individual virion in a monovalent fashion (19, 20, 27, 29, 50, 53; reviewed in references 12 and 22). Bivalent binding of an antibody to a virion has been documented with clear structural evidence in only one case, in which monoclonal antibodies (MAbs) 17-IA and 8F5 bind to virions of human rhinovirus 14 (HRV14) and HRV2 (19, 43). Even in this unique case, binding bivalency appears to contribute to the neutralization potency of 17-IA but not to that of 8F5 (19, 42, 43). Moreover, these MAbs bind to two hydrophobic canyon structures formed by viral proteins VP1 and VP2 and not to antigenic epitopes within individual viral capsid protomers; thus, this case may represent an exception to the common form of antibody/antigen interactions in which the antibodies bind to individual antigens. Therefore, it is not clear what role antibody-binding multivalency plays in antibody-mediated neutralization of viruses at the level of interaction between antibody molecules and individual virions.The binding affinity of an antibody to its target is defined by intrinsic affinity and avidity (reviewed in reference 16). Intrinsic affinity is the force of monovalent binding between an antibody paratope and an antigenic epitope, often measured by binding a Fab fragment to an antigen. Avidity is the additive or synergistic force of engaging multiple antibody paratope/antigen epitope pairs between one antibody and one antigen. In other words, avidity is a functional consequence of antibody-binding multivalency. The effect of avidity on affinity is readily demonstrated in biochemical reactions such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), in which high-density antigenic sites are available without distinct spatial restrictions. It is commonly assumed that both affinity and avidity have functional consequences in antibody-mediated neutralization of viruses (reviewed in references 7 and 67). At the level of individual virions, the contribution of antibody-binding avidity to neutralization efficiency is often based on two types of experiments. In one, results from a side-by-side comparison between an antibody and its Fab fragment are often reported as evidence supporting a role of antibody-binding multivalency in virus neutralization. However, the interpretation of this type of experiment is complicated by the size difference between an antibody and a Fab fragment, since steric hindrance is a major mechanism of neutralization (reviewed in references 6 and 23). In a second type of experiment, a correlation between neutralization efficiency and the ability of the antibody/virus complex to resist chemical stress without dissociation in the presence of a high concentration of salt in solution is interpreted to support a contributing effect from antibody-binding avidity to neutralization efficiency (2, 21, 36, 49, 51). Data from this type of experiment are limited mostly to measuring binding affinity that is below the affinity required for virus neutralization. Furthermore, these studies often do not distinguish between avidity effects caused by an antibody binding to two (or more) epitopes on one antigen or to multiple epitopes from different molecules on the virion. Therefore, like the situation with antibody-binding multivalency, it remains unclear whether binding avidity contributes to antibody-mediated neutralization of viruses at the level of individual virions.The envelope glycoproteins (Envs) of human immunodeficiency virus type 1 (HIV-1) exist on the virion or cell surface as trimers of gp120 and gp41 heterodimers (13, 30, 62, 65). High-resolution structural information for a native HIV-1 Env trimer is critically important for understanding the function of HIV-1 Envs as well as for guiding the development of an effective immunogen to elicit broad and potent neutralizing antibody responses. X-ray crystal structures of the gp41 ectodomain fragments in the postfusion conformation have been resolved; however, a high-resolution structure of gp41 in the prefusion conformation is still unavailable and likely will be more informative for understanding the function of HIV-1 Env trimers (9, 47, 52). Two X-ray crystal structures of the gp120 core in both the CD4-liganded and unliganded conformations have been solved, but the biological meanings of these structures, especially how they are related to the native, functional Env trimer, are still being debated (10, 26). Several low-resolution structures of the Env trimers from HIV-1 or the closely related simian immunodeficiency virus (SIV) have been determined using cryoelectron microscopy (cryo-EM) tomography (4, 30, 62, 64, 65, 66). The predicted structures for the Env trimer are in general quite different between the two studies, and the difference is particularly dramatic around the gp41 membrane-proximal external region (MPER). A high-resolution structure of the native HIV-1 Env trimer is needed to resolve these differences. In the meantime, a distinctive standard needs to be developed for evaluating the biological relevance of structural information of an HIV-1 Env trimer.Our previous studies of the stoichiometry of antibody-mediated neutralization of HIV-1 Env indicated that MAbs b12, 2G12, and 2F5 neutralize by a stoichiometry designated T=1, i.e., one antibody binds to and neutralizes one HIV-1 Env trimer (57). Furthermore, when an artificial epitope (FLAG) was inserted in the V4 region of HIV-1 gp120, an epitope-specific anti-FLAG MAb achieved neutralization by the mechanism of steric hindrance (37, 61). Using the well-defined 2F5 neutralizing epitope as a model system (35, 39, 45), we constructed HIV-1 Env proteins carrying one 2F5 epitope in the gp120 V4 region and another 2F5 epitope in the gp41 MPER. Here, we investigated whether binding bivalency leads to enhancement in neutralization efficiency. By studying the detailed requirement for binding bivalency, we also probed the structure of the native, functional HIV-1 Env trimer, aiming to establish a standard that can be employed to evaluate the biological relevance of structural information on the HIV-1 Env trimer.     

Breadth of Neutralizing Antibodies Elicited by Stable,Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.The development and evaluation of novel HIV-1 Env immunogens are critical priorities of the HIV-1 vaccine field (2, 10, 25). The major antigenic target for neutralizing antibodies (NAbs) is the trimeric Env glycoprotein on the virion surface (4, 18, 30). Monomeric gp120 immunogens have not elicited broadly reactive NAbs in animal models (5, 13, 28, 29) or humans (16, 31), and thus several groups have focused on generating trimer immunogens that better mimic the native Env spike found on virions (3, 7, 14, 15, 20, 22, 27). It has, however, proven difficult to produce stable and conformationally homogeneous Env trimers. Strategies to modify Env immunogens have therefore been explored, including the removal of the cleavage site between gp120 and gp41 (3, 7, 23, 39, 40), the incorporation of an intramolecular disulfide bond to stabilize cleaved gp120 and gp41 moieties (6), and the addition of trimerization motifs such as the T4 bacteriophage fibritin “fold-on” (Fd) domain (8, 17, 39).Preclinical evaluation of candidate Env immunogens is critical for concept testing and for the prioritization of vaccine candidates. Luciferase-based virus neutralization assays with TZM.bl cells (21, 24) have been developed as high-throughput assays that can be standardized (26). However, the optimal use of this assay requires the generation of standardized virus panels derived from multiple clades that reflect both easy-to-neutralize (tier 1) and primary isolate (tier 2) viruses (21, 24). A tiered approach for the evaluation of novel Env immunogens has been proposed, in which tier 1 viruses represent homologous vaccine strains and a small number of heterologous neutralization-sensitive viruses while tier 2 viruses provide a greater measure of neutralization breadth for the purpose of comparing immunogens (24).We screened a large panel of primary HIV-1 isolates for Env stability and identified two viruses, CZA97.012 (clade C) (32) and 92UG037.8 (clade A) (17), that yielded biochemically homogeneous and stable Env trimers with well defined and uniform antigenic properties (17). The addition of the T4 bacteriophage fibritin “fold-on” (Fd) trimerization domain further increased their yield and purity (17). In the present study, we assessed the immunogenicity of these stable clade A and clade C gp140 trimers in guinea pigs. Both trimers elicited high-titer binding antibody responses and cross-clade neutralization of select tier 1 viruses as well as low-titer but detectable NAb responses against select tier 2 viruses from clades A, B, and C. These data demonstrate the immunogenicity of these stable gp140 trimers and highlight the utility of standardized virus panels in the evaluation of novel HIV-1 Env immunogens.     

Alterations in the Immunogenic Properties of Soluble Trimeric Human Immunodeficiency Virus Type 1 Envelope Proteins Induced by Deletion or Heterologous Substitutions of the V1 Loop

HIV-1 gp140 envelope immunogens express conserved epitopes that are targeted by broadly cross-reactive neutralizing antibodies, but they fail to elicit similar antibodies upon immunization. The poor immunogenicity of conserved epitopes on gp140 could be linked to the high immunogenicity of variable Env regions on such constructs. Previous studies have shown that the first hypervariable region (V1 loop) is immunogenic on soluble gp140s but elicits type-specific antibodies. To address issues related to the high immunogenicity of the V1 loop, two conceptually opposite approaches were tested. In the first approach, we eliminated the V1 loop from our gp140 construct and examined how V1 deletion altered the immunogenic properties of other Env regions. In the second approach, we took advantage of the high immunogenicity of the V1 loop and engrafted four diverse V1 loops onto a common gp140 Env “scaffold.” These four scaffolds were used as a cocktail of immunogens to elicit diverse anti-V1 antibodies, under the hypothesis that eliciting diverse anti-V1 antibodies would expand the neutralizing breadth of immune sera. Our study indicates that three of four heterologous V1 loops were immunogenic on the common Env backbone “scaffold,” but heterologous anti-V1 neutralizing responses were observed in only one case. Both types of V1 modification dampened the immunogenicity of the V3 loop, differentially altered the immunogenicity of the transmembrane gp41 subunit, and altered the relative immunogenicities of unknown Env regions, including potentially the CD4-binding site (CD4-bs) and trimer-specific targets, which elicited cross-reactive neutralizing antibodies but of limited breadth.An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will need to incorporate an envelope-derived immunogen capable of eliciting potent and broadly cross-reactive neutralizing antibody responses against diverse primary HIV-1 isolates. The target of anti-HIV neutralizing antibodies (NAbs), the viral envelope (Env) glycoprotein, is expressed as a single transmembrane polypeptide precursor (gp160) that is glycosylated and cleaved into an extracellular subunit (gp120) and a transmembrane subunit (gp41) during intracellular processing (10, 20, 21, 54). The functional Env form on virion surfaces is a trimer composed of three noncovalently associated gp120-gp41 heterodimers. Soluble forms of the trimeric Env have been generated by introducing stop codons immediately upstream of the transmembrane domain of gp41. These constructs are commonly referred to as gp140 proteins and have been tested extensively as immunogens to elicit anti-HIV-1 NAbs. Soluble gp140s express epitopes that are targets of NAbs, including cross-reactive NAbs such as b12, 4E10, and 2G12 (5, 17, 34, 45, 47, 49, 50, 52, 57). Immunization with gp140 immunogens nonetheless does not result in a broadly cross-reactive neutralizing antibody response (2, 3, 17, 18, 26, 56, 58).Epitope mapping analyses of the Abs elicited by soluble trimeric gp140 immunogens revealed that a large fraction of the gp140-induced neutralization response targets the first hypervariable region of gp120 (the V1 loop). In our hands, ∼40 to 70% of the neutralizing activity of sera from animals immunized with SF162 gp140 constructs is due to anti-V1 antibodies (17). In a study by Li et al. with YU2 gp140 (30) and a study by Wu et al. with HxB2/BaL gp145 (56), ∼10 to 80% of anti-YU2 neutralizing activity and 100% of anti-HxB2 neutralizing activity, respectively, were due to anti-V1 Abs. These anti-V1 Abs, however, are not cross-reactive. Previously, we also demonstrated that the diverse positionings of the V1 across heterologous strains limit access of broadly cross-reactive monoclonal antibodies (MAbs) to their targets (12).Here, taking into consideration the V1 loop''s high immunogenicity, we employed two opposing approaches aimed at the elicitation of cross-reactive neutralizing antibody responses to HIV-1. In the first approach, we deleted the V1 loop on our soluble trimeric gp140 construct (ΔV1SF162 gp140) and examined whether and how this modification altered the immunogenic properties of other Env regions. In the second approach, we substituted the V1 loop on our SF162 gp140 construct with the V1 loops from four heterologous HIV-1 viruses (89.6, YU2, JRFL, and HxB2) that differ in their amino acid compositions and in the number of potential N-linked glycosylation sites (PNGs). These four heterologous viruses display various neutralization phenotypes (7) and coreceptor utilization profiles (15, 35, 36, 48, 51). A total of four SF162 Env-based gp140 “scaffolds” expressing four different V1 loops were created and used as immunogens in a cocktail to test as a “proof of principle” the hypothesis that if diverse V1 loops are presented to the immune system simultaneously, the elicitation of anti-V1 NAbs with diverse specificities would broaden the overall neutralizing activity of immune sera. We also immunized animals with each of the four V1 chimeric scaffolds individually to ensure that all V1 loops were immunogenic when presented on the heterologous SF162 Env background.All immunogens (including wild-type [WT] SF162 gp140 and ΔV1SF162 gp140) elicited homologous anti-SF162 NAbs. All immunogens except the scaffold construct expressing the YU2 V1 also elicited heterologous NAbs against the sensitive lab-adapted strain HxB2. The heterologous YU2, 89.6, and HxB2 V1 loops, but not the JRFL V1 loop, were immunogenic on the background of the SF162 Env scaffold. However, only anti-V1 neutralizing activity against the HxB2 virus was observed. Although neither approach resulted in the development of broad anti-HIV-1 cross-neutralizing antibody responses, cross-neutralizing antibody responses of narrow breadth were elicited. These responses were not due to antibodies that target to variable regions of gp120 but were due to antibodies that target either epitopes of the CD4-binding site (CD4-bs) or epitopes that are not present on monomeric gp120. These observations have implications for guiding rational Env-based immunogen design and for potentially eliciting broadly cross-reactive NAb responses.     

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.The external glycoprotein gp120 and the membrane-anchored glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1), collectively referred to as the envelope glycoproteins (Env), mediate viral entry and are the sole virally encoded targets for neutralizing antibodies (NAbs). Prior to binding the primary host cell receptor, CD4, the trimeric Env spike may sample multiple conformations on the surface of the virus. Which of these potential conformations display neutralizing Ab epitopes and are recognized by broadly reactive NAbs is currently unclear. A substantial conformational change occurs when the functional Env spike interacts with CD4, leading to the exposure and the formation of the bridging sheet, a highly conserved and immunogenic structure spanning the inner and outer domains of gp120 that contributes to coreceptor interaction (6, 14, 25, 30). CD4 binding is also thought to lead to the displacement of variable region 3 (V3) from a less exposed conformation in the packed functional spike to a more protruding conformation. Exposure of V3 is necessary for viral entry, as it also contributes to Env interaction with coreceptor (21). Additional or concurrent rearrangements of the functional spike structure may occur upon CD4 binding, as suggested by cryotomography (38), However, these rearrangements are less well understood due to the absence of a high-resolution structure of the static or CD4-liganded trimeric spike.In attempts to elicit broadly reactive NAbs against HIV-1 through vaccination, a range of recombinant Env variants were designed and tested (reviewed in references 15, 26, 49, and 50). The capacity of such immunogens to elicit broadly reactive NAbs is often determined using standardized in vitro neutralization assays (34). However, the ability of HIV-1 Env vaccine-elicited B cell responses to mediate actual protective and functional responses against in vivo virus challenge is evaluated less frequently, since this requires the use of nonhuman primates (NHPs) and infection with chimeric simian-human immunodeficiency viruses (SHIVs). A series of SHIVs was developed, including those based on the HIV-1 Env glycoproteins from SF162 (40), 89.6 (54), ADA (45), BaL (48), DH12 (59), and 1157i (27). So far, few of these models, if any, fully mimic HIV-1 infection in humans. Currently, serially passaged CCR5-using SHIV-SF162 (SHIV-SF162P), which establishes transient or more prolonged viremia in macaques, represent a frequently used model to evaluate the protective effect of Env-based immunogens (2-5, 19, 20, 23, 24, 29, 53, 67). Depending on the number and nature of passages that this virus has been exposed to, the SHIV-SF162P stocks are more or less neutralization resistant (19, 62), allowing one to test the efficacy of a given vaccine candidate against a more or less rigorous form of viral challenge. Protection against mucosal SHIV-SF162P4 challenge after homologous SF162ΔV2 Env protein immunization of rhesus macaques was recently reported (2, 3). However, the nature and specificities of the vaccine-induced immune responses that mediate this effect remain incompletely defined.We recently showed that Abs against the HIV-1 gp120 coreceptor binding site (CoRbs) are elicited as a consequence of in vivo interactions between Env and primate CD4 during immunization with soluble CD4 (sCD4)-binding-competent Env trimers (14). We subsequently showed that rhesus macaques inoculated with CD4-binding competent and CD4-binding defective soluble YU2-derived gp140-F trimers in adjuvant generate comparable levels of Env-specific binding Abs and T cell responses but that CoRbs-directed Abs are elicited only in animals immunized with wild-type (wt) CD4-binding competent Env trimers (13). So far, the impact of Env-CD4 in vivo interactions during Env immunization and the role of CoRbs-directed Abs in protection against SHIV infection remain incompletely understood. A majority of the well-characterized CoRbs-directed monoclonal Abs (MAbs) lack the capacity to neutralize primary viruses in vitro (7, 31). However, it has been suggested that Abs directed against this region may contribute to the neutralizing Ab response seen in some HIV-1-infected individuals (18, 35, 58) and to the protection observed in some SHIV challenge experiments (12).The distinct difference in the capacity of the CD4-binding competent and CD4-binding defective Env trimers to elicit CoRbs-directed Abs described in our previous study presented an opportunity to evaluate the protective effect of CoRbs-directed Abs in the SHIV model. The availability of animals immunized with these Env immunogens also allowed us to ask the more general question about whether in vivo interactions between soluble Env trimers and CD4-expressing host cells would influence the outcome of heterologous SHIV-SF162P4 infection. We show here that Env trimer-immunized animals displayed improved control of SHIV-SF162P4 viremia compared to unimmunized control animals, independent of whether they were inoculated with CD4-binding-competent or CD4-binding-defective trimers. These results suggest that the capacity of soluble Env trimers to interact with CD4 in vivo and to stimulate the production of CoRbs-directed Abs did not measurably influence the protective effect of the vaccine-elicited immune responses in this SHIV challenge model.     

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1⁺) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1⁺ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.The development of an HIV-1 vaccine that can elicit protective humoral and cellular immunity is one of the highest priorities in the global fight against HIV/AIDS (2, 44). Data from lentiviral animal models suggest that antibodies capable of neutralizing primary strains of HIV-1 may have the capacity to prevent HIV-1 infection (1, 28, 30, 35). However, the ability to design immunogens that can elicit such broadly reactive neutralizing antibodies (NAbs) has proven to be a formidable obstacle, due in part to the extensive genetic diversity of HIV-1 and the complex escape mechanisms employed by the envelope gp120 and gp41 glycoproteins that form the trimeric viral envelope spike (Env) (20, 34, 45). As improved vaccine immunogens enter the stage of detailed preclinical analysis, the in vitro assays used for evaluating vaccine sera will need to detect incremental advances in the magnitude, breadth, and durability of NAb responses (37). Such data can then be used to distinguish and prioritize among antibody-based vaccine immunogens. Furthermore, highly reproducible and quantitative data on vaccine-elicited NAbs can enhance our understanding of the relationship between Env immunogen design and the resulting antibody response generated.Current recommendations for evaluating candidate vaccine sera for NAb activity include the use of standard reference panels of molecularly cloned HIV-1 Env pseudoviruses and a tiered algorithm of testing (27). Reference virus panels should represent genetically and geographically diverse subsets of viruses with neutralization phenotypes that are generally representative of primary isolate strains that a vaccine would need to protect against. As such, standard reference panels for HIV-1 subtypes B and C have been described (22, 23), and efforts continue toward the creation of virus reference panels representing additional genetic subtypes. For tiered evaluation of NAb activity, vaccine sera are first tested against homologous Env pseudoviruses and/or a small number of isolates that are known to be highly sensitive to antibody-mediated neutralization (commonly referred to as tier 1 viruses). A more rigorous assessment of the potency and breadth of vaccine-induced NAbs entails testing against more resistant reference panel viruses (commonly referred to as tier 2 viruses) that are either matched or mismatched in genetic subtype to the vaccine immunogen (second and third tiers of testing, respectively). This tiered approach for testing candidate HIV-1 vaccine sera is advantageous in that it provides increasingly stringent levels for assessing the potency and breadth of NAbs, uses standardized panels of reference viruses for consistency and reproducibility, and allows for the generation of comparative data sets for evaluating different candidate vaccine regimens.While the tiered algorithm for evaluating vaccine sera has gained acceptance in the field, a major limitation has been the lack of objective data to characterize HIV-1 Env pseudoviruses according to their overall sensitivity or resistance to antibody-mediated neutralization. The category of sensitive, tier 1 viruses arose in part from the observation that HIV-1 isolates passaged through T-cell lines often become highly sensitive to antibody-mediated neutralization (33). Compared to these laboratory-adapted viruses, most primary isolate strains are moderately resistant to NAbs. Yet, even among recently isolated circulating viral Envs, there is a wide spectrum of neutralization sensitivity. Some HIV-1 isolates have a neutralization phenotype closer to that of tier 1 viruses, while others appear to be quite neutralization resistant (6, 19, 22, 23). Overall, there are few data from which to understand or categorize the viral neutralization phenotypes of HIV-1 strains. As a result, we have a limited ability to assess the potential potency of vaccine-elicited NAbs or to estimate the percentage of circulating HIV-1 isolates that would be neutralized. Further categorization of isolates into distinct subgroups based on sensitivity to NAbs may reveal patterns of neutralization that could provide a greater understanding of the NAb response generated by current and future vaccine immunogens. In addition, the structure-based design of novel immunogens may be facilitated by an ability to monitor the types of viruses neutralized and to specifically map the viral epitopes targeted by vaccine-elicited NAbs.In this study, we assembled a diverse panel of 109 HIV-1 Env pseudoviruses, including multiple representatives from clades A, B, and C and circulating recombinant forms (CRFs) CRF07_BC and CRF02_AG-related. These were tested for their sensitivities using HIV-1-positive (HIV-1⁺) plasma samples representative of clades A, B, and C and CRF01_AE and CRF02_AG. Clinical, demographic, and viral genetic sequence data were collected for each virus. The neutralization phenotype of each virus was assessed with a panel of seven clade-specific HIV-1⁺ plasma pools. Viruses were rank ordered according to average neutralization sensitivity, and k-means clustering was utilized to identify four subgroups of viruses with neutralization phenotypes ranging from highly sensitive to resistant. Together, these results will improve the ability to rigorously evaluate antibody-based HIV-1 vaccines and will facilitate the interpretation of assay results to identify immunogens with improved capacity to elicit broadly cross-reactive NAbs.     

Comparative Immunogenicity of Subtype A Human Immunodeficiency Virus Type 1 Envelope Exhibiting Differential Exposure of Conserved Neutralization Epitopes

Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.The ability to elicit broadly cross-reactive neutralizing antibodies (NAbs) is likely to be an important component of an effective vaccine to human immunodeficiency virus type 1 (HIV-1). Unfortunately, the HIV-1 envelope (Env)-based vaccines developed to date do not elicit such antibodies. Initial vaccines based on soluble, monomeric gp120 generated antibodies capable of only weakly neutralizing the homologous virus, with a very narrow breadth of cross-reactivity (13, 30, 53). Subsequent modifications to the Env immunogens, including variable loop deletions (15, 20, 31, 34, 35, 61, 64-66), alterations in the glycosylation pattern (4, 10, 11, 14, 30, 43, 55, 56), epitope repositioning (39, 46), the use of consensus Envs (22, 36, 37, 47), and the use of soluble trimeric gp140 molecules as immunogens (1-3, 5, 14, 16, 20, 21, 24, 25) have led to only modest enhancements in NAb breadth or potency. These modified Env immunogens have failed to redirect NAbs from the variable loops to more conserved regions of Env (reviewed in reference 33).Differences in Env structure between HIV-1 subtypes may further hinder efforts to elicit broadly cross-reactive antibodies capable of protecting against transmitted strains worldwide. Most immunogens tested to date have been derived from subtype B Envs. However, there are clear antigenic differences between subtype B strains and the subtype A and C strains that account for most infections worldwide (6, 8, 27, 28, 40, 42). For instance, most transmitted subtype A Envs are resistant to the monoclonal antibodies 2G12, b12, 2F5, and 4E10, either because of alterations in the epitopes for these monoclonal antibodies (MAbs) or because the epitopes are shielded in these Envs (6, 8). It is therefore possible that even NAbs specific for a conserved region of subtype B Envs, such as the CD4 binding site, would not be able to access and neutralize a similar epitope on a subtype A Env.In order to evaluate the immunogenicity of subtype A Envs, which account for ∼25% of global HIV-1 infections (12), we previously investigated the types of antibody responses elicited following gp160 priming and gp140 boosting with immunogens derived from four subtype A Envs in comparison to the subtype B Env SF162 (38). These experiments were also designed to explore whether deriving immunogens from HIV-1 Envs isolated from early in infection would better target NAbs to transmitted strains. Although all of the subtype A-based immunogens and the SF162 immunogen elicited anti-V3 NAbs capable of neutralizing the easy-to-neutralize SF162 pseudovirus, only one of the four immunogens generated homologous NAbs (38). Even immunogens with shorter variable loops or fewer potential N-linked glycosylation sites (PNGS) did not lead to enhanced breadth of neutralization against heterologous subtype A or B Envs (38). However, the four subtype A Envs used in these immunizations were generally neutralization resistant to both plasma samples from HIV-1-infected individuals and to monoclonal antibodies (6), raising the possibility that the poor breadth observed could be related to the shielding of conserved epitopes within these Envs.In order to determine whether using subtype A Env immunogens that do not shield conserved epitopes could improve neutralization breadth, here we performed immunizations with pairs of Env immunogens derived from two individuals acutely infected with subtype A HIV-1. The Envs in each pair were very similar in their amino acid sequences yet differed dramatically in their neutralization phenotype (6, 9) (Fig. ​(Fig.1A).1A). The pair from subject Q461 had a neutralization-resistant Env, {"type":"entrez-protein","attrs":{"text":"Q461e2","term_id":"123931369","term_text":"Q461E2"}}Q461e2 (termed {"type":"entrez-protein","attrs":{"text":"Q461e2","term_id":"123931369","term_text":"Q461E2"}}Q461e2^R to indicate neutralization resistance), and a neutralization-sensitive Env, {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 (termed {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1^S to indicate neutralization sensitivity), which was sensitive to neutralization by plasma, 2F5, 4E10, b12, and soluble CD4 (sCD4). We previously demonstrated that the neutralization sensitivity of the {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1^S Env is mediated entirely by two amino acid substitutions in gp41, one in the first heptad repeat and one in the membrane proximal external region (MPER) (9). These mutations led to enhanced exposure of both the CD4 binding site and the MPER (9). From subject Q168, the Env Q168b23^S was sensitive to autologous and heterologous plasma and to the MPER antibodies 2F5 and 4E10 but resistant to b12 and sCD4, while {"type":"entrez-protein","attrs":{"text":"Q168a2","term_id":"123361966","term_text":"Q168A2"}}Q168a2^R was weakly neutralized by the MPER antibodies, less sensitive to neutralization by autologous plasma, and resistant to heterologous plasma (6). The {"type":"entrez-protein","attrs":{"text":"Q168a2","term_id":"123361966","term_text":"Q168A2"}}Q168a2^R and Q168b23^S Envs contain identical sequences in the MPER region yet have >500-fold differences in neutralization sensitivity to 2F5 and 4E10, indicating that the exposure of the MPER region, rather than the sequence, likely accounts for the enhanced neutralization of the Q168b23^S Env.Open in a separate windowFIG. 1.Analysis of {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1^S gp140 used for immunizations. (A) SDS-PAGE analysis of final preparation of {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1^S gp140 from the GNA capture and DEAE and CHAP columns. Lane 1 contains molecular weight standards, lane 2 the concentrated DEAE flowthrough, and lane 3 the final concentrated protein. The purified {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1^S gp140 protein is indicated by an arrow. The sizes of the molecular weight markers (in thousands) are indicated on the left. (B) Binding of purified gp140 subtype A to CD4 as determined by a high-pressure liquid chromatography (HPLC)-based assay. The bottom line represents the protein obtained after the GNA column, and the top line represents purified protein after all three steps. The trimer and monomer peaks are marked. (C) Summary of neutralization characteristics of all four HIV-1 subtype A Env variants used in the immunizations, adapted from reference 6. The pseudovirus is shown in the far left column. IC₅₀ values for plasma sample (left) and monoclonal antibodies (right) are displayed. The autologous plasma samples were taken 3.7 ypi for subject Q461 and 2.6 ypi for subject Q168. The Kenya pool was derived by pooling plasma from 30 HIV-1-infected individuals in Kenya and has been described previously (6).Thus, to directly test whether using Env immunogens that expose conserved epitopes could enhance neutralization breadth immunization, here we immunized with these pairs of related Envs, in which one variant exposes conserved regions, while the other does not. We also compared the specificity of the NAb responses following immunization with these Envs with the specificities of the NAbs that developed during natural infection in the individuals from whom these variants were cloned.     

Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicate in vivo under relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.Development of an effective vaccine will be critical in the efforts to control the human immunodeficiency virus type 1 (HIV-1) pandemic. Unfortunately, vaccines evaluated in completed human efficacy trials have shown moderate to no protective effects, and, clearly, much more work is needed to define the correlates of lentivirus immune protection. Although these correlates are still not entirely known, vaccine strategies that elicit antibodies with broad neutralizing activity are currently of considerable interest, and it is widely believed that HIV-1 envelope glycoproteins that induce broadly neutralizing antibodies (NAbs) will be critical components of a protective vaccine (21, 28, 53, 63).Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that causes persistent infection in horses worldwide and serves as an important large-animal translational model in which to dissect basic correlates of protective lentiviral immunity (9, 31, 33, 38, 57). EIAV is a naturally occurring lentivirus, and infection results in a predictable course of recurrent episodes of plasma viremia and clinical disease. As with HIV-1 and simian immunodeficiency virus (SIV), EIAV infection is not cleared. However, infected horses eventually control viral replication and clinical disease to remain persistently infected inapparent carriers. Adaptive immune responses, including NAbs, are required for EIAV control since young horses (foals) with severe combined immunodeficiency (SCID), unlike normal foals, fail to eliminate the initial viremia following challenge (46). Equine SCID is caused by a frameshift mutation in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (55, 60) and has an autosomal recessive mode of inheritance (47). The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints (55). Adoptive transfer of EIAV-specific T and B lymphocytes to a SCID foal results in functional cytotoxic T lymphocytes (CTL) and NAb activity and is protective against homologous EIAV challenge (33).During acute EIAV infection, each recurrent episode coincides with the emergence of an antigenically distinct EIAV variant as defined by type-specific NAb, which neutralizes virus isolated during early disease episodes but not virus isolated during subsequent disease episodes (2, 20, 22, 43, 52). Amino acid variation primarily occurs within hypervariable regions V1 to V8 of the envelope gp90 surface unit (SU) and particularly within the V3/principal neutralizing domain (PND) region (1, 19, 24, 25, 57). Our work with EIAV-infected SCID foals indicates that significant envelope diversification does not occur in the absence of NAbs but that rapid envelope diversification occurs when adaptive immune responses are reconstituted (35). Thus, adaptive immunity, including NAb, drives selection of EIAV envelope variants during acute infection. Amino acid changes occur primarily within the V3 to V7 hypervariable SU regions, and many changes affect potential N-linked glycosylation sites (PNLGS) (35). Importantly, however, CTL also target the SU, and variants that escape CTL recognizing an EIAV V3/PND epitope have been identified (37, 38). Thus, both NAbs and CTL are capable of contributing to the selection of EIAV SU variants, but the relative contributions of each to such selection are not known.Recently, SU variation was evaluated in an immunocompetent pony experimentally inoculated with the virulent wild-type Wyoming strain of EIAV (57). Seventy-one distinct V3 variants that partitioned into five major nonoverlapping groups were identified and designated PND1 to PND5. Neutralization assays using chimeric infectious molecular clones containing these PNDs suggested a transition from type-specific NAb responses toward more broadly reactive immune responses during the course of infection and indicated that genetic changes conferring resistance to broadly NAbs lead to recrudescence of clinical disease following a lengthy clinically quiescent period (57). Thus, the NAb response broadens significantly during long-term persistent EIAV infection, and broadly NAbs play a critical role in EIAV immune control.Studies of nonhuman primates have provided important information regarding the protective effects of NAbs. Passive immunization of macaques with purified immunoglobulin from chimpanzees infected with several different HIV-1 isolates results in complete protection from homologous chimeric simian/human immunodeficiency virus (SHIV) infection when the immunoglobulin is given 24 h prior to challenge (54). Passive transfer of a triple combination of broadly neutralizing human monoclonal antibodies directed against the envelope of a primary HIV-1 isolate results in complete protection against SHIV infection in some macaques while others become infected but exhibit decreased plasma viremia (29). The contribution of T cells to partial protection in these studies is not clear, and the presence or absence of viral escape variants in the unprotected macaques has not been evaluated. In neonatal macaques, various combinations of broadly neutralizing human monoclonal antibodies directed against conserved HIV envelope epitopes administered before and after SHIV challenge result in protection against persistent systemic infection in some animals, but clinical disease develops in others (12-14). Virus-specific T-cell proliferative responses are detected in some of the protected animals, indicating that cellular immune responses occur and likely contribute to protection by eliminating infected cells (13).Despite the fact that NAbs can block experimental SHIV infection, selection pressure exerted by NAbs plays a critical role in HIV-1 and SIV envelope evolution during infection, and evasion of NAb responses is an important mechanism of HIV-1 and SIV persistence (11, 16, 27, 48, 59). The maturation of a type-specific NAb response in SIV-infected rhesus macaques significantly correlates with diversification in the V1/V2 region of the SIV envelope (50). In HIV-1, NAbs are detectable within the first 2 months postinfection and result in an early and significant selection force on the virus population (49). Escape from NAbs involves many amino acid substitutions with little cross-neutralization between closely related strains, and NAb responses drive the diversification of the HIV-1 envelope during the early stages of infection (16). The early appearance of NAbs in patients with acute HIV-1 infection results in the replacement of neutralization-sensitive virus by successive populations of resistant virus, and virus escape primarily involves changes in N-linked glycosylation (59). Thus, overcoming neutralization escape constitutes a significant barrier to the ultimate efficacy of any NAb-eliciting HIV-1 vaccine.Because the SCID defect occurs naturally in the horse, it provides a powerful and unique opportunity to finely dissect the protective effects of immune interventions against a naturally occurring lentivirus independent of other de novo adaptive immune responses. This level of dissection is not possible in other lentivirus model systems. The goal of the current study was to determine if broadly NAbs could protect against lentivirus challenge in the complete absence of T lymphocytes and other adaptive immune responses. We hypothesized that convalescent immune plasma from a long-term persistently infected inapparent carrier horse containing antibodies capable of neutralizing homologous and several heterologous EIAV SU PND variants would provide complete protection when infused into SCID foals before experimental virus inoculation. This plasma was administered to four SCID foals 24 h prior to challenge, and four control SCID foals received normal horse plasma. Clinical outcome, plasma viral load, and serum neutralization activity were analyzed in all foals. Although complete protection was achieved in one treated foal, infection occurred in the others. In foals that became viremic, the SU sequence and neutralization phenotype of the breakthrough virus were determined. As part of these experiments, blood containing this virus was inoculated into a naive immunocompetent horse, and the adaptive immune responses associated with its control were further evaluated.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.The envelope glycoprotein complex of the human immunodeficiency virus type 1 (HIV-1) is involved principally in virion attachment to target cell surfaces and in the entry process (15, 18, 27, 29, 52). Envelope glycoproteins (Env) are initially translated as a gp160 precursor glycoprotein, which is then processed during its trafficking through the secretory pathway, to yield a surface subunit gp120 noncovalently attached to a transmembrane subunit gp41. During HIV-1 assembly, Env proteins are incorporated at the surface of the viral particle as a trimeric structure consisting of three gp120/gp41 dimers (59, 62).The gp41 consists of an ectodomain, a hydrophobic transmembrane anchor, and a cytoplasmic tail (CT). Lentiviruses, including HIV-1 and simian immunodeficiency virus (SIV), are unusual in having a transmembrane subunit with much longer CTs (∼150 amino acids) than most other retroviruses (20 to 50 amino acids) (27). Early studies with T-cell laboratory-adapted HIV-1 mutants showed that the gp41 CT region played an important role in regulating Env functions, the incorporation of Env into virus particles and, consequently, viral replication (16, 21, 35, 63). The integrity of the gp41 CT thus appears to be crucial for replication in primary T cells, macrophages, and in many transformed T-cell lines (1, 44). Viral variants with truncated gp41 are rarely isolated from infected patients. One study reported the isolation of a CD4-independent variant harboring a sharply truncated CT (64). However, this atypical isolate existed as a minority variant in the original quasispecies of the patient (54). SIV variants with truncated CTs obtained in cell culture in vitro have also been shown to revert rapidly (to full-length CT) when introduced into macaques (39). These observations indicate that the long CTs of lentiviruses, such as HIV-1 and SIV, have functions specific to viral replication and persistence in vivo.Two groups of conserved sequence motifs have been identified in the gp41 CT that are likely to be involved in its functions. The first group, involved in regulating the intracellular trafficking of Env, includes a membrane-proximal tyrosine-based endocytic motif, Y₇₁₂SPL, (9, 47); a diaromatic motif, Y₈₀₂W₈₀₃, implicated in the retrograde transport of Env to the trans-Golgi network (8), and a C-terminal dileucine motif recently identified as a second endocytic motif (7, 10, 60). We have also provided evidence for the existence of additional as-yet-unidentified signals in studies of primary HIV-1 (34). The second group of motifs consists of three structurally conserved amphipathic α-helical domains: lentivirus lytic peptides 1, 2, and 3 (LLP-1, LLP-2, and LLP-3) (11, 17, 33). LLP domains have been implicated in various functions, including Env fusogenicity and the incorporation of Env into HIV-1 particles (28, 32, 43, 45, 50, 61).Several lines of evidence suggest that Env incorporation requires direct or indirect interactions between the matrix domain of the structural protein precursor Pr55^Gag (matrix) and the gp41 CT during HIV-1 assembly. This possibility was first suggested by the observation that HIV-1 Env drives the basolateral budding of Gag in polarized cells (37, 48). A direct interaction between the matrix and a glutathione S-transferase fusion protein containing Env CT was subsequently observed in vitro (13). Synthetic peptides corresponding to various domains of the gp41 CT have also been shown to interact directly with Pr55^Gag molecules (26). Furthermore, effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by studies based on T-cell laboratory-adapted virus mutants (19, 40, 43). Finally, the cellular protein TIP47 was recently implicated in Env incorporation, based on its ability to bind both the matrix protein and the gp41 CT (38).In a previous study describing the evolutionary dynamics of the glycan shield of HIV-1 Env, we identified a patient (patient 153) for whom the 15 env clones obtained during primary infection (early stage) encoded full-length Env, whereas the 15 env sequences from the HIV-1 present 6 years later (late stage) encoded truncated gp41 CTs (14). These late-stage sequences contained a deletion introducing an in-frame stop codon, resulting in a 20-amino-acid truncation of the Env. Note that, unlike a point mutation, this deletion cannot easily revert to the full-length form. Such a deletion affecting various known motifs of the gp41 CT would be expected to impair viral replication. However, the plasma viral load measured in patient 153 demonstrated that the virus had retained its ability to replicate.In the present study, we explored the molecular mechanisms by which a primary HIV-1 maintained its capacity to replicate efficiently in this patient and demonstrated for the first time the occurrence of matrix and Env coevolution in vivo, providing insight into the ability of HIV-1 to overcome major structural alterations.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.Few of the hundreds of known neutralizing anti-HIV monoclonal antibodies (MAbs) display broad cross-reactive activities (4). Of those derived from clade B-infected patients, b12 binds to the gp120 subunit of the HIV envelope protein (Env), to an epitope that overlaps the CD4 binding site, and neutralizes approximately 50% of virus isolates tested, including non-clade B viruses (27). 2G12 binds to N-linked carbohydrates on gp120 (32, 34) and neutralizes 41% of isolates tested, although not clade C or E isolates. 447-52D also binds to the gp120 subunit, to an epitope within the V3 loop, and potently neutralizes up to 45% of clade B isolates but rarely non-clade B isolates. 4E10 and 2F5 recognize adjacent epitopes located at the membrane-proximal external region (MPER) of the gp41 Env subunit (9, 22, 24, 28, 42). Two neutralizing antibodies (NAbs) isolated from a clade A-infected patient (PG9 and PG16) show broad and potent neutralizing activity by recognizing epitopes consisting of conserved regions of the V2 and V3 loops of gp120, preferentially on native trimers (40).4E10 is capable of neutralizing all isolates tested at some level (4), although there is evidence for the existence of rare viruses that are resistant to 4E10 neutralization (30). The exact structure of the epitope recognized by 4E10 within the trimeric, functional HIV Env is unknown, but structural studies have shown that an isolated peptide spanning the epitope adopts a helical conformation, a short 3₁₀ segment followed by a 4₁₃ (or true α-helical) segment, with an extended structure at the N terminus when bound to 4E10 (9). It has also been reported that 4E10 interacts with a variety of lipids and membrane components, particularly the phospholipid cardiolipin (15), suggesting that difficulties in eliciting 4E10-like broadly neutralizing antibodies by immunization and the apparent rarity of 4E10-like antibody responses in HIV-1-infected subjects (19, 33) are linked to this polyspecificity to autoantigens, contributing to their elimination through tolerance mechanisms. However, subsequent studies have shown that the measurable, but quite weak, affinity of 4E10 for certain lipids is comparable to that of some antiphospholipid antibodies elicited during many infections, suggesting that 4E10 is not remarkably autoreactive (35). Therefore, it is still unclear whether lipid binding properties are linked to the rarity of 4E10-like specificities. It has also been proposed that the neutralizing activity of 4E10 may partly depend on lipid binding, either through interactions with viral membrane lipids that disturb the membrane-bound structure of the MPER on the trimeric, virion-associated Env spike (39) or through an encounter model. In the latter, initial interactions with membrane components align 4E10 with its protein epitope or allow 4E10 to gain proximity to its epitope (1), perhaps partially alleviating steric occlusion effects (for example, see reference 17). We sought to determine whether specific interactions exist between 4E10 and membrane lipid components and whether such interactions meaningfully contribute to neutralization by any mechanism.     

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu_RD, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.The gammaretrovirus gibbon ape leukemia virus (GaLV) has been widely used for gene therapy because of its wide host cell tropism and nonpathogenicity (1, 6, 10, 12, 13, 20). The host cell receptor for GaLV Env has been cloned and identified as a sodium-dependent phosphate transporter protein (25, 26). Like other retroviruses, GaLV encodes a single transmembrane surface glycoprotein (GaLV Env), which is cleaved into surface (SU) and transmembrane (TM) subunits (Fig. ​(Fig.1).1). The TM domain of GaLV Env contains a short 30-amino-acid C-terminal cytoplasmic tail. Although GaLV Env functions well when coupled (pseudotyped) with murine leukemia virus (MLV)-based retroviral vectors, it has been shown to be completely incompatible with HIV-1 (4, 35). When GaLV Env is expressed with HIV-1, essentially no infectious HIV-1 particles are produced (4, 35). The mechanism for this infectivity downmodulation is unknown, but the component of GaLV Env responsible for the restriction has been mapped to the cytoplasmic tail. Replacing the cytoplasmic tail of GaLV Env with the equivalent sequence from MLV Env ameliorates the restriction. Likewise, replacing the cytoplasmic tail of MLV Env with that from GaLV Env confers the restriction (4).Open in a separate windowFIG. 1.Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.Vpu is an 81-amino-acid HIV-1 accessory protein produced from the same mRNA as the HIV-1 Env gene. The N terminus of Vpu contains a membrane-spanning domain, followed by a 50-amino-acid cytoplasmic domain. Vpu is unique to HIV-1 and a few closely related SIV strains. The best-characterized roles for Vpu in the HIV-1 life cycle are modulation of host proteins CD4 and tetherin (also known as BST-2, CD317, and HM1.24) (24, 38, 39). Vpu promotes the degradation of CD4 in the endoplasmic reticulum through a proteasome-dependent mechanism (29). The cytoplasmic tail of Vpu physically interacts with the cytoplasmic tail of CD4 and recruits the human β-transducing repeat-containing protein (β-TrCP) and E3 ubiquitin ligase components to polyubiquitinate and ultimately trigger the degradation of CD4 (18). Two serine residues at positions 52 and 56 of Vpu are phosphorylated by casein kinase-2 and are required for CD4 degradation (31, 32). The membrane-spanning domain of Vpu is not specifically required for CD4 degradation. A mutant protein containing a scrambled membrane-spanning sequence, Vpu_RD, is still able to trigger the degradation of CD4 (32). The region of CD4 that is targeted by Vpu is approximately 17 to 13 amino acids from the C terminus in the cytoplasmic tail (Fig. ​(Fig.1)1) (2, 15).In addition to degrading CD4, Vpu has also long been known to result in enhanced viral release (EVR) in certain cell lines (14, 36). Recently, the type I interferon-induced host protein tetherin was identified as being responsible for this Vpu-modulated restriction (24, 38). In the absence of Vpu, tetherin causes particles to remain tethered (hence the name) to the host cell postfission. Although Vpu counteracts the function of tetherin, the exact mechanism has not been fully elucidated. However, the mechanism for tetherin antagonism appears to be distinct from that for modulating CD4. Mutation of the serines 52 and 56 of Vpu abolish CD4 degradation, but only reduce EVR activity (5, 17, 21, 32). Some EVR activity remains even when much of the Vpu cytoplasmic tail is deleted (30). In addition, many mutations in the membrane-spanning domain, such as Vpu_RD, do not affect CD4 degradation and yet completely abolish EVR activity (27, 30, 37). The critical residues in tetherin for recognition by Vpu appear to be in the membrane-spanning domain and not the cytoplasmic tail (9, 19, 28). Although β-TrCP is required for complete EVR activity, there is no consensus whether the degradation of tetherin is proteasome or lysosome mediated (5, 7, 21) or whether degradation is required at all. In some cases there can be some EVR activity in the absence of tetherin degradation (17, 22).We demonstrate here that Vpu is responsible for the incompatibility between HIV-1 and GaLV Env. Glycoproteins containing the cytoplasmic tail from GaLV Env are prevented from being incorporated into HIV-1 particles by Vpu, effectively reducing infectious particle production by 50- to 100-fold. The serines at positions 52 and 56 are required for this restriction, but the membrane-spanning domain is not. Although the mechanism for this restriction appears similar to CD4 degradation, there are apparent differences. Vpu does not prevent surface expression, and it does not prevent its incorporation into MLV particles. Therefore, the mechanism of restriction appears to involve a system that does not rely directly on global protein degradation.     

Antagonism to and Intracellular Sequestration of Human Tetherin by the Human Immunodeficiency Virus Type 2 Envelope Glycoprotein

Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to HIV-2 does not. It has been previously reported that the HIV-2 envelope glycoprotein has a Vpu-like function in promoting virus release. Here we demonstrate that the HIV-2 Rod envelope glycoprotein (HIV-2 Rod Env) is a tetherin antagonist. Expression of HIV-2 Rod Env, but not that of HIV-1 or the closely related simian immunodeficiency virus (SIV) SIVmac1A11, counteracts tetherin-mediated restriction of Vpu-defective HIV-1 in a cell-type-specific manner. This correlates with the ability of the HIV-2 Rod Env to mediate cell surface downregulation of tetherin. Antagonism requires an endocytic motif conserved across HIV/SIV lineages in the gp41 cytoplasmic tail, but specificity for tetherin is governed by extracellular determinants in the mature Env protein. Coimmunoprecipitation studies suggest an interaction between HIV-2 Rod Env and tetherin, but unlike studies with Vpu, we found no evidence of tetherin degradation. In the presence of HIV-2 Rod Env, tetherin localization is restricted to the trans-Golgi network, suggesting Env-mediated effects on tetherin trafficking sequester it from virus assembly sites on the plasma membrane. Finally, we recapitulated these observations in HIV-2-infected CD4⁺ T-cell lines, demonstrating that tetherin antagonism and sequestration occur at physiological levels of Env expression during virus replication.Various stages of the replication cycle of primate lentiviruses can be targeted by host antiviral restriction factors (reviewed in reference 49). In addition to the well-characterized antiviral effects of members of the APOBEC3 family of cytidine deaminases, particularly APOBEC3G and -3F, and species-specific variants of tripartite motif family 5α, the release of nascent retroviral particles has recently been shown to be a target for a novel restriction factor, tetherin (CD317/bone marrow stromal cell antigen 2 [BST-2]) (31, 46). Tetherin is an interferon-inducible gene that was originally shown to impart a restriction on the release of mutants of human immunodeficiency virus type 1 (HIV-1) that lack a vpu gene (31, 46). In tetherin-positive cells, mature Vpu-defective HIV-1 particles are retained on the cell surface, linked to the plasma membrane (PM) and each other via protease-sensitive tethers, and can be subsequently endocytosed and accumulate in late endosomes (30, 31). Tetherin is not HIV specific and restricts the release of virus-like particles derived from all retroviruses tested (18), as well as those of filoviruses and arenaviruses (18, 19, 39).Tetherin is a small (181-amino-acid) type II membrane protein with an unusual topology that exists mainly as a disulfide-linked dimer (34). It consists of an N-terminal cytoplasmic tail, a transmembrane anchor, an extracellular domain that includes three cysteine residues important for dimerization, a putative coiled-coil, and finally a glycophosphatidyinosityl-linked lipid anchor (22) that is essential for restriction (31). Tetherin localizes to retroviral assembly sites on the PM (18, 31), and this unusual structure is highly suggestive that tetherin restricts virion release by incorporation into the viral membrane and cross-linking virions to cells. Such a mechanism would make tetherin a powerful antiviral effector that can target an obligate part of most, if not all, enveloped virus assembly strategies. Moreover, since tetherin restriction has no specific requirement for virus protein sequences, to avoid its action, mammalian viruses have evolved to encode several distinct countermeasures that specifically inhibit tetherin''s antiviral function.The Vpu accessory protein antagonizes tetherin-mediated restriction of HIV-1 (31, 46). In the presence of Vpu, tetherin is downregulated from the cell surface (2, 46) and is targeted for degradation (10, 13, 14), although whether these processes are required for antagonism of tetherin function is unclear (27). HIV-1 Vpu displays a distinct species specificity in that it is unable to target tetherin orthologues from rhesus macaques or African green monkeys (14, 25). This differential sensitivity maps to the tetherin transmembrane domain, particularly residues that are predicted to have been under high positive selection pressure during primate evolution (14, 16, 25). This suggests that tetherin evolution may have been driven in part by viral countermeasures like Vpu. Vpu, however, is only encoded by HIV-1 and its direct simian immunodeficiency virus (SIV) lineage precursors. The majority of SIVs, including the SIVsm, the progenitor of both HIV-2 and SIVmac, do not encode a Vpu protein (21). In some of these SIVs, tetherin antagonism was recently shown to map to the nef gene (16, 51). SIV Nef proteins, however, are generally ineffective against human tetherin because they target a (G/D)DIWK motif that was deleted from the human tetherin cytoplasmic tail sometime after the divergence of humans and chimpanzees (51). This raises the question of how HIV-2 is able to overcome human tetherin, as recent data show chronically HIV-2-infected CEM T cells have reduced tetherin levels on their surface (10).Interestingly, it has long been known that the envelope glycoprotein of certain HIV-2 isolates can stimulate the release of Vpu-defective HIV-1 virions from cells we now know to be tetherin positive (5, 6, 43). HIV and SIV Envs form trimeric spikes of dimers of the surface subunit (SU-gp105 in HIV-2/SIVmac and gp120 in HIV-1) that bind CD4 and the chemokine coreceptor and gp41 (the transmembrane [TM] subunit that facilitates fusion with and entry into the target cell). Envelope precursors (gp140 or gp160) are synthesized in the endoplasmic reticulum, where they become glycosylated and are exported to the surface via the secretory pathway (8). During transit through the Golgi apparatus and possibly in endosomal compartments, the immature precursors are cleaved by furin-like proteases to form mature spikes (15, 29). Multiple endocytosis motifs in the gp41 cytoplasmic tail lead to only minor quantities of Env being exposed at the cell surface at any given time (7, 40). Recent data demonstrated that the conserved GYxxθ motif, a binding site for the clathrin adaptor protein AP-2 (3), in the membrane-proximal region of HIV-2 gp41 is required to promote Vpu-defective HIV-1 release from HeLa cells (1, 32). Based on experiments with HIV-1/HIV-2 chimeric envelopes, an additional requirement in the extracellular component was suggested (1). In this study we set out to examine the Vpu-like activity of HIV-2 envelope in light of the discovery of tetherin. We demonstrate that the HIV-2 Env is a tetherin antagonist, and we provide mechanistic insight into the basis of this antagonism.     

Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.Antibodies capable of neutralizing a subject''s own virus, called autologous neutralizing antibodies (ANAbs), have been the subject of recent studies redefining the timing and character of this response. ANAbs develop early in essentially all seropositive subjects and increase in titer during the first few months and years of infection (15, 30). Previously published data were obtained using an assay that measures ANAbs against the complete quasispecies without an analysis of the individual envelope protein (Env) sequences to which these ANAb responses were directed (10). The contemporaneous virus pool was poorly neutralized, leading to an assumption that contemporaneous ANAbs are ineffective in controlling viremia. In chronic infection, ANAbs generally have been difficult to detect (3, 29, 31, 40), but there is ample evidence for selection by NAb and resulting virus env evolution in the host (12, 30, 38). The titers of ANAbs measured against clinical or autologous isolates cultured in peripheral blood mononuclear cells typically have been low in chronic infection (31, 40), while other studies indicated the presence of strong ANAbs (2). Although ANAbs may be ineffective in subjects with high virus loads due to the continuous generation of escape variants, their role in maintaining low viral loads in human immunodeficiency virus (HIV) controllers is not known.NAbs that recognize heterologous isolates to which the subject has never been exposed, called heterologous NAbs (HNAbs), are found later in infection, and not all subjects develop this broadening of the response (5). In studies that utilized easy-to-neutralize laboratory or primary viruses, titers of HNAbs can be high (5, 6, 26, 29). Early work had shown that polyclonal HNAbs in HIV-infected subjects are directed to conserved conformational determinants on gp120 (32), including the CD4-binding site (CD4bs) (22). Several human neutralizing monoclonal antibodies with broad activity also are directed to conserved conformational determinants on Env proteins, such as the CD4bs (4) and V3 (17). However, the mechanisms that lead to the development of broad HNAbs are unknown. Their development likely is dependent upon the specific autologous Env proteins to which the subject is exposed, and these proteins are variants of the original infection in these subjects, except for cases of superinfection. Thus, we reasoned that a detailed analysis of the neutralization of individual autologous variants in subjects with broad responses and viral control could be informative.The purpose of this study was to examine the autologous neutralizing responses against autologous viral variants in the plasma of HIV-positive subjects that were controlling infection for many years. These subjects have moderate HNAbs against the quasispecies of other subjects (27). We compared longitudinal samples from five chronically infected, antiretroviral treatment-naive adults late in infection. Despite the short time frame between the sample time points, the amount of env variation was surprisingly high, indicating continuous viral evolution in controllers; contemporaneous ANAbs were present and maintained in all except one elite controller. We cloned individual env gp160 plasma variants and analyzed sequence changes related to the autologous neutralization sensitivity or resistance. We systematically examined the ANAb response directed to individual variants using contemporaneous and noncontemporaneous plasma samples and observed patterns that have not been previously reported. Mutations that were significantly associated with sensitivity or resistance to ANAbs were found on parts of the envelope that are exposed and thus may be accessible to antibodies, consistently with a role in escape and containment by NAbs.     

Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120₃/gp41₃). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.Identification of conserved regions accessible on the HIV-1 envelope and design of immunogens that elicit broadly neutralizing antibodies against these sites continue to be major challenges in the development of an effective HIV-1 vaccine. The HIV-1 viral spike—composed of three exterior gp120 subunits and three transmembrane gp41 subunits—is highly protected, but a limited number of these conserved regions exist on the spike, identified primarily by the broadly neutralizing antibodies that target them. One region is quaternary in nature and appropriately formed only on the assembled viral spike (gp120₃/gp41₃). This region is targeted by a recently discovered (14) and fast expanding class of monoclonal antibodies (36, 40) that recognize epitopes with quaternary structural constraints, which are composed of portions of two gp120-variable loops, V2 and V3 (reviewed in reference 49). These quaternary structure-specific (or quaternary-specific) antibodies (also called quaternary-neutralizing epitope or “QNE” antibodies) are found in the sera of selected HIV-1-infected individuals who have broadly neutralizing serum antibodies (41); individual members of the class, however, vary greatly in their breadth of neutralization.Initial evidence for the existence of quaternary-specific antibodies arose in simian/human immunodeficiency virus-infected rhesus macaques and HIV-1-infected chimpanzees (6, 9, 13). Characterization of polyclonal sera from these infected animals suggested the presence of antibodies targeting a conformational epitope involving the variable loop regions of the gp120 viral envelope.Antibody 2909 was the first human monoclonal antibody against HIV-1 to be characterized as being specific for an epitope dependent on the quaternary interaction of envelope glycoproteins (14). It was identified by direct screening for neutralization activity against a pseudovirus derived from strain SF162 of HIV-1. It recognizes a quaternary epitope on the surface of native virions and infected cells but does not bind soluble gp120/gp140 envelope proteins or cell surface-expressed gp120 monomers (14, 20). Competition analysis and virological assays indicate that the 2909 epitope includes portions of the V2 and V3 loops of gp120 (14, 16), with the V2-V3 elements originating either from within a gp120 monomer or between gp120 protomers in the trimer context. Mapping of 2909 recognition identifies a particular anomaly in its recognition (16); neutralization by 2909 depends on the presence of a rare lysine at position 160 in the V2 loop rather than the conserved N-linked site of glycosylation found at this position in most HIV-1 isolates (providing a residue-specific explanation for the neutralization specificity of 2909 for the SF162 virus, which contains this rare lysine).Other strain-specific monoclonal antibodies like 2909 have been isolated from rhesus macaques infected with a chimeric simian/human immunodeficiency virus that contained an SF162 isolate-derived viral spike (SHIV_SF162P4) (36). These rhesus monoclonal antibodies exhibit properties similar to those of 2909 in their potent neutralization of SF162 and their recognition of V2-V3 only in the context of the functional viral spike (e.g., on virus particles) (36). Details from epitope mapping indicate that these rhesus antibodies and human antibody 2909 recognize overlapping epitopes, with some differences in requirements for V2 N-linked glycosylation (36).The somatically related human monoclonal antibodies, PG9 and PG16, were also identified by a direct screen for neutralization (40). They target a quaternary-specific V2-V3 epitope, but unlike 2909, they neutralize an extraordinary 70 to 80% of circulating primary HIV-1 isolates and appear to have some reactivity for monomeric gp120 (40). Much of their increased breadth of neutralization arises from their ability to recognize an N-linked glycan at position 160 in the V2 loop, a motif which is found in greater than 90% of HIV-1 group M isolates (25).Despite substantial differences in their neutralization breadth, antibodies 2909 and PG9/PG16 may be closely related. Notably, an N160K mutation in the V2 loop of typical primary HIV-1 isolates like YU2 and JR-FL can recover 2909 activity (16). Conversely, isolate SF162 can be converted to a PG9- and PG16-sensitive pseudovirus by the K160N mutation (40). Thus, a single N or K at position 160 appears to control much of the neutralization difference between 2909 and PG16. Together the results suggest that 2909 and PG9/PG16 antibodies recognize distinct immunotypes of a similar quaternary epitope.To gain insight into how antibodies achieve recognition of this epitope, we determined the crystal structure of the antigen-binding fragment (Fab) of 2909 at a 3.3-Å resolution and compared this structure to the previously determined structure of PG16 (31, 33). Mutational analysis was used to confirm structural hot spots, and chimeric analysis of domain swaps between 2909 and other quaternary-specific antibodies was used to refine assessments of functional similarity. By identifying structural features—shared between 2909 and PG16 but otherwise highly uncommon in antibodies—the results provide insight into conserved solutions by human antibodies for recognition of an important vaccine target on HIV-1.     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

Autologous Neutralizing Antibodies to the Transmitted/Founder Viruses Emerge Late after Simian Immunodeficiency Virus SIVmac251 Infection of Rhesus Monkeys

While the simian immunodeficiency virus (SIV)-infected rhesus monkey is an important animal model for human immunodeficiency virus type 1 (HIV-1) infection of humans, much remains to be learned about the evolution of the humoral immune response in this model. In HIV-1 infection, autologous neutralizing antibodies emerge 2 to 3 months after infection. However, the ontogeny of the SIV-specific neutralizing antibody response in mucosally infected animals has not been defined. We characterized the kinetics of the autologous neutralizing antibody response to the transmitted/founder SIVmac251 using a pseudovirion-based TZM-bl cell assay and monitored env sequence evolution using single-genome amplification in four rhesus animals that were infected via intrarectal inoculations. We show that the SIVmac251 founder viruses induced neutralizing antibodies at 5 to 8 months after infection. Despite their slow emergence and low titers, these neutralizing antibodies selected for escape mutants that harbored substitutions and deletions in variable region 1 (V1), V2, and V4 of Env. The neutralizing antibody response was initially focused on V4 at 5 to 8 months after infection and then targeted V1/V2 and V4 by 16 months. These findings reveal a striking delay in the development of neutralizing antibodies in SIVmac-infected animals, thus raising questions concerning the suitability of SIVmac251 as a challenge strain to screen AIDS vaccines that elicit neutralizing antibodies as a means to prevent virus acquisition. They also illustrate the capacity of the SIVmac quasispecies to modify antigenic determinants in response to very modest titers of neutralizing antibodies.While neutralizing antibodies (Nabs) mediate protection in humans against a diversity of viral pathogens (38, 53, 72), it is unclear how they impact human immunodeficiency virus type 1 (HIV-1) infection. One reason that the contribution of neutralizing antibodies to the control of HIV-1 remains uncertain is that HIV-specific neutralizing antibodies develop relatively late in natural infection. High titers of HIV-specific autologous neutralizing antibodies usually emerge as late as 2 to 3 months after infection and continue to evolve throughout the first years of infection (18, 25, 57, 66, 74). Such neutralizing antibodies have been shown to influence HIV-1 evolution within a host and to be responsible for viral escape mutations (47, 48, 58, 59). A better understanding of why there is a prolonged time associated with the maturation of the neutralizing antibody response in HIV-1 infection and whether conserved viral epitopes exist that could mediate antibody protection is important for the development of effective HIV-1 vaccine strategies.The simian immunodeficiency virus (SIV)/rhesus macaque model of AIDS provides an important system to study AIDS immunopathogenesis and to evaluate HIV-1 vaccine strategies. SIVmac251, an uncloned, pathogenic, CCR5-tropic virus isolate comprised of a swarm of quasispecies that are closely related (33), and SIVmac239, an infectious molecular clone derived from SIVmac251, are the two most commonly used rhesus monkey SIV challenge viruses utilized in AIDS vaccine research in the nonhuman primate (NHP) model. SIVmac239 has been shown to be relatively resistant to antibody-mediated neutralization by both autologous antibodies and a wide range of monoclonal antibodies (29, 30). The env sequence evolution in SIVmac239-infected rhesus monkeys and SIVMne-CL8-infected pigtailed macaques has been well described (8, 50, 51). Some of these changes in Env have been shown to result in viral escape from neutralizing antibodies (7, 10, 34, 60). In particular, a recent study by Sato et al. characterized SIVmac239 env sequence changes that were associated with viral escape in a rhesus monkey with an unusually high titer of neutralizing antibodies after intravenous infection (67). However, the antibody-mediated neutralization of SIVmac251 has not been tested rigorously using standardized assays that are currently being used to measure neutralization of HIV-1, thereby precluding a direct comparison of the neutralization sensitivities of HIV-1 and SIV. Furthermore, it is also unclear whether more typical titers of neutralizing antibodies against SIV239/251 exert selection pressure on the viral population in animals that acquire infection mucosally.The aims of this study were to elucidate the kinetics of the neutralizing antibody response against the transmitted viruses and the sequence evolution of env in association with humoral immunity in mucosally infected rhesus macaques. We hypothesized that a low titer of SIVmac Env-specific neutralizing antibodies exerts potent selection pressure on the viral quasispecies. To test this hypothesis, we utilized a pseudovirion-based TZM-bl reporter gene neutralization assay and single genome amplification (SGA) in order to characterize the humoral immune pressures driving viral sequence evolution in four rhesus monkeys that were infected with SIVmac251 via intrarectal inoculations.