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.     

Introduction of Exogenous Epitopes in the Variable Regions of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Effect on Viral Infectivity and the Neutralization Phenotype

In this study we examined whether human immunodeficiency virus type 1 (HIV-1) is equally susceptible to neutralization by a given antibody when the epitope of this antibody is introduced at different positions within the viral envelope glycoprotein (Env). To this end, we introduced two exogenous “epitope tags” at different locations within three major Env regions in two distinct HIV-1 isolates. We examined how the introduction of the exogenous epitopes affects Env expression, Env incorporation into virions, Env fusogenic potential, and viral susceptibility to neutralization. Our data indicate that even within the same Env region, the exact positioning of the epitope impacts the susceptibility of the virus to neutralization by the antibody that binds to that epitope. Our data also indicate that even if the same epitope is introduced in the exact same position on two different Envs, its exposure and, as a result, the neutralization susceptibility of the virus, can be very different. In contrast to the findings of previous studies conducted with HIV-1 isolates other than those used here, but in agreement with results obtained with simian immunodeficiency virus, we observed that tagging of the fourth variable region of Env (V4) did not result in neutralization by the anti-tag antibodies. Our data indicate that epitopes in V4 are not properly exposed within the functional HIV-1 trimeric Env spike, suggesting that V4 may not be a good target for vaccine-elicited neutralizing antibodies.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is expressed as a heavily glycosylated peptide of approximately 160 kDa (gp160), which is cleaved intracellularly into two noncovalently associated subunits: an extracellular subunit (gp120), responsible for CD4 and coreceptor (primarily CCR5 and/or CXCR4) binding, and a transmembrane subunit (gp41) that mediates fusion between viral and host cell membranes. Based on amino acid sequence homology analysis of gp120s derived from diverse HIV-1 isolates, gp120 is divided into five “constant” regions (C1 to C5) and five “variable” regions (also called “loops,” because most of them have cysteines in the N and C termini that form disulfide bonds). Despite their extensive amino acid variability, the variable loops of gp120 play central roles during the entry of the virus into the cell, for instance, by directly or indirectly modulating the interaction of Env with coreceptor molecules on the target surfaces during virus-cell fusion. They also offer protection from neutralizing antibodies (NAbs) by various mechanisms. The variable loops themselves are targets of NAbs, and during infection, the replicating virus accumulates mutations in the variable regions that allow it to escape the action of anti-variable loop-directed NAbs, while at the same time the variable loops are positioned within the Env trimer so that they prevent, or minimize, the binding of NAbs to more-conserved epitopes, such as the receptor and coreceptor binding sites (4, 5, 12, 15, 20, 23, 25, 27, 31).HIV-1 strains display distinct neutralization phenotypes. Some isolates, such as SF162, are generally susceptible to NAbs that bind to many distinct regions of Env, including the variable regions, while other isolates, such as YU2 or JRFL, are generally resistant to neutralization by the same NAbs (1). It has been proposed that irrespective of the overall neutralizing phenotype of HIV-1 isolates, the binding of only a single antibody per Env trimer on the virion surface can lead to neutralization, when all Env trimers present on the virion surface are bound by at least one antibody (32). This important observation also implies that the epitope specificity of an antibody may not be as important for neutralization as its ability to bind to its target within the trimeric Env structure. In fact, antibodies to diverse regions of Env, such as V1, V2, V3, and the receptor and coreceptor binding sites, can all neutralize HIV-1 (1, 3, 6, 8, 10, 18, 20, 23, 25, 27, 29, 30).In many cases, a given isolate will not be equally susceptible to neutralization by NAbs that bind to different Env regions, for example, the V3 loop and the CD4-binding site (CD4-BS). Whether differences in the neutralizing potentials of two antibodies that bind to distinct epitopes on HIV-1 Env are due to differences in the binding affinities of the two antibodies or whether they occur because the viruses are intrinsically more susceptible to NAbs that bind certain epitopes and not others (i.e., the relative importance of the various regions of Env in Env function and virus neutralization sensitivity differs) is not yet fully understood. One way to address these issues is to introduce small non-HIV Env amino acid sequences (tags) that are targets of known monoclonal antibodies (MAbs) at various positions within the viral Env and to examine how the placement of the same epitope at different positions within Env affects the neutralization phenotype of the virus.Foreign epitopes have been introduced into the variable regions of HIV and simian immunodeficiency virus (SIV) Envs, and their effects on viral neutralization potential have been examined (14, 19, 22, 33). Yang and colleagues (33) introduced the FLAG epitope into the V4 regions of three HIV-1 isolates (YU2, JRFL, and HxB2) displaying distinct neutralization phenotypes in response to anti-HIV NAbs; they found that all three pseudotyped viruses were equivalently neutralized by an anti-FLAG MAb. One important implication of that study is that neutralization-resistant isolates, such as YU2 or JRFL, are not intrinsically more resistant to neutralization than more-susceptible isolates, such as HxB2, so long as the antibody binds to its epitope on the functional virion-associated Env spike. A second implication is that since the FLAG epitope was exposed in the V4 loops of all three isolates, the V4 loop could theoretically be a good target for vaccine-elicited antibodies. In contrast, Pantophlet et al. (19) introduced the HA tag into various regions of the JRCSF (neutralization-resistant) and HxB2 (neutralization-sensitive) isolates and reported that JRCSF was intrinsically more resistant than HxB2 to anti-HA antibodies. This observation implies, therefore, that some HIV-1 strains (primary, neutralization-resistant strains) have developed mechanisms that limit the accessibility of multiple Env regions, including variable regions, to antibodies developed during infection. Laird and Desrosiers (14) introduced the FLAG epitope into two positions within each of the V1, V2, and V4 loops of SIV239 and SIV316. They reported that the functionality of Env was differentially affected by the precise location of the exogenous tag sequence within the variable loops examined. Importantly, and in contrast to what was reported for the HIV-1 isolates mentioned above, the SIV239 variants containing a V4 FLAG epitope were not neutralized by an anti-FLAG MAb. It appeared, however, that the FLAG epitope was not well exposed on the trimeric Env when introduced into the V4 loop of SIV but was exposed when introduced into the V1 loop of the same virus. Potentially, this means that the V4 loop is differentially exposed in the context of the HIV-1 and SIV Envs.The FLAG epitope (DYKDDDDK) is highly charged. Therefore, it is possible that the effect on Env function and epitope exposure could differ if a different exogenous epitope were inserted instead of FLAG. Here we examined the effect of variable loop tagging on the Env functions and viral neutralization phenotypes of two primary HIV-1 clade B isolates, SF162 (CCR5 tropic) and SF33 (CXCR4 tropic), using two exogenous epitopes (FLAG and hemagglutinin [HA] tags) positioned at multiple locations within the V1, V2, and V4 loops. By placing the same tag in several regions within each loop, we investigated the accessibilities of various parts of the same loop to a given NAb. By using two tags that differ significantly in amino acid composition (FLAG tag, DYKDDDDK; HA tag, YPYDVPDYA), we aimed at distinguishing between the effects of amino acid composition and the positioning of the tag on Env function and overall epitope exposure. Finally, identical evaluations of R5 and X4 Envs may provide information about the relative roles played in neutralization by variable loops in Envs displaying distinct coreceptor usage. We report that both the amino acid sequence and the position of the tag within and among the variable loops greatly affected the functionality of Env. In contrast to previous observations made with other HIV-1 Envs (33) but in agreement with what was reported for the SIV239 Env (14), we observed that tagging of the V4 loops of SF162 and SF33 did not render these isolates susceptible to neutralization by the corresponding anti-tag MAbs.     

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.     

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.     

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.     

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.     

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

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.     

HIV-1 gp120 Determinants Proximal to the CD4 Binding Site Shift Protective Glycans That Are Targeted by Monoclonal Antibody 2G12

HIV-1 R5 envelopes vary considerably in their capacities to exploit low CD4 levels on macrophages for infection and in their sensitivities to the CD4 binding site (CD4bs) monoclonal antibody (MAb) b12 and the glycan-specific MAb 2G12. Here, we show that nonglycan determinants flanking the CD4 binding loop, which affect exposure of the CD4bs, also modulate 2G12 neutralization. Our data indicate that such residues act via a mechanism that involves shifts in the orientation of proximal glycans, thus modulating the sensitivity of 2G12 neutralization and affecting the overall presentation and structure of the glycan shield.The trimeric envelope (Env) spikes on HIV-1 virions are comprised of gp120 and gp41 heterodimers. gp120 is coated extensively with glycans (9, 11, 15) that are believed to protect the envelope from neutralizing antibodies. The extents and locations of glycosylation are variable and evolving (15). Thus, while some glycans are conserved, others appear or disappear in a host over the course of infection. Such changes may result in exposure or protection of functional envelope sites and can result from selection by different environmental pressures in vivo, including neutralizing antibodies.We previously reported that HIV-1 R5 envelopes varied considerably in tropism and neutralization sensitivity (3, 4, 12-14). We showed that highly macrophage-tropic R5 envelopes were more frequently detected in brain than in semen, blood, and lymph node (LN) samples (12, 14). The capacity of R5 envelopes to infect macrophages correlated with their ability to exploit low levels of cell surface CD4 for infection (12, 14). Determinants within and proximal to the CD4 binding site (CD4bs) were shown to modulate macrophage infectivity (3, 4, 5, 12, 13) and presumably acted by altering the avidity of the trimer for cell surface CD4. These determinants include residues proximal to the CD4 binding loop, which is likely the first part of the CD4bs contacted by CD4 (1). We also observed that macrophage-tropic R5 envelopes were frequently more resistant to the glycan-specific monoclonal antibody (MAb) 2G12 than were non-macrophage-tropic R5 Envs (13).Here, we investigated the envelope determinants of 2G12 sensitivity by using two HIV-1 envelopes that we used previously to map macrophage tropism determinants (4), B33 from brain and LN40 from lymph node tissue of an AIDS patient with neurological complications. While B33 imparts high levels of macrophage infectivity and is resistant to 2G12, LN40 Env confers very inefficient macrophage infection and is 2G12 sensitive (12-14).     

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.     

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.The HIV-1 Envs mediate the entry of the virus into target cells and are the only virally encoded proteins exposed on the surface of the virus. HIV-1 Env is the sole target for neutralizing antibodies (Abs) and therefore is an important component of a vaccine designed to elicit protective antibody responses (4, 20). The viral spike is a trimer comprised of three heterodimers of the exterior envelope glycoprotein, gp120, noncovalently attached to the transmembrane protein, gp41. The gp120 subunit binds the primary receptor, CD4 (7), to form or to expose the gp120 coreceptor binding elements, which interact with the viral coreceptor, primarily CCR5 (1, 9, 12, 45). The highly conserved coreceptor binding site (CoRbs) overlaps the gp120 bridging sheet and also contains both proximal and distal elements of V3 (18, 32, 43, 45).In attempts to mimic the native trimeric structure of Env present on the virus, various forms of soluble Env trimers were designed (reviewed in reference 14). One design consists of cleavage-defective trimers derived from the primary R5 isolate YU2 that possess a heterologous trimerization motif derived from T4 bacteriophage fibritin (F; YU2 gp140-F) (3, 21, 34, 40, 50, 51). We recently demonstrated that the immunization of monkeys, but not rabbits, with gp140-F trimers resulted in the generation of Abs directed against the CoRbs of gp120 capable of cross-neutralizing HIV-2 (15). CoRbs-directed Abs (also referred to as CD4-induced, or CD4i, Abs) also were elicited in rabbits transgenic for human CD4 (15). Taken together, these data strongly suggest that Env interacts with high-affinity primate CD4 in vivo, resulting in the formation, or exposure, of a highly immunogenic gp120 determinant that overlaps the CoRbs. Early in infection, the frequency of HIV-1-infected individuals with significant antibody responses against the CoRbs is high (8, 33), and CoRbs-directed antibody responses are elicited abundantly in humans inoculated with Env-based immunogens (15). Collectively, these data suggest that the recognition of the HIV-1 CoRbs by naïve B cells is greatly increased when Env is presented in complex with high-affinity primate CD4, leading to a productive Ab response against this epitope (41). With rare exceptions, the majority of CoRbs-directed monoclonal antibodies (MAbs) do not neutralize HIV-1 primary viruses in vitro, bringing into question the utility of this region as a relevant neutralization target (23, 31, 47, 49). Strategies aimed to diminish vaccine-elicited B-cell responses to the CoRbs, and shift responses toward more accessible neutralization targets, represent one approach to improve the design of Env-based vaccine candidates. The selective manipulation of Env immunogens to decrease their CD4 binding capacity may reduce the elicitation of CoRbs-directed Abs and circumvent potential occlusion effects of the conserved CD4 binding site caused by CD4 itself.In addition to the potential effects of in vivo Env-CD4 interactions on the Ab repertoire elicited by Env-based immunogens, interactions between Env and CD4 also may have consequences on CD4⁺ T-cell responses. CD4 is an important costimulatory molecule expressed on several subsets of T cells and antigen-presenting cells, and interactions with Env were shown to alter the function of CD4-expressing T cells in a number of in vitro systems (13, 37, 44). The elimination of the Env-CD4 interaction in the context of vaccination may be beneficial to improve the elicitation of helper T-cell responses and effective neutralizing Ab responses. In vivo evaluation in subjects possessing high-affinity CD4 (i.e., rhesus macaques or humans) of CD4 binding-competent and CD4 binding-deficient Env immunogens so far have not been described.To address these questions, we designed Env trimer variants rendered CD4 binding defective through two distinct mechanisms. In the first variant, the interaction between CD4 and HIV-1 Env was directly disrupted by the introduction of a mutation (368D/R) in the CD4 binding loop of the gp120 outer domain (29). This alteration abolishes the initial binding of CD4 and most CD4 binding site (CD4bs)-directed MAbs (42) to variant forms of gp120 and would be expected to do the same in the soluble stable timer context. The aim of the second variant was to decrease the CD4 binding affinity while preserving the antigenicity of the CD4bs (48). This variant was generated in the soluble gp140-F trimers by the introduction of three point mutations, 423I/M, 425N/K, and 431G/E, in the β20 strand region of gp120. These mutations were suggested to favor a helix rather than the β20/21 antiparallel strands visible in the gp120 structure (23, 31, 47, 49). In the monomeric context, mutations in the β20 strand region of gp120 abolish binding by CoRbs-directed Abs, presumably because the bridging sheet cannot form (48). The introduction of the 423I/M, 425N/K, and 431G/E mutations in the trimer context therefore should disrupt the normally high-affinity gp120-CD4 interaction, while recognition by CD4bs Abs would not be affected. Indeed, a recent study provides a mechanistic basis for the impact of these mutations on CD4 binding (52). This study revealed that CD4 interacts with gp120 by a two-step binding mechanism in which the first step involves a direct, but low-affinity, CD4 interaction with the gp120 outer domain, while the second step requires a conformational change in gp120 to fully stabilize the high-affinity gp120-CD4 interaction.Here, we exploit this two-step model to generate novel CD4 binding-defective soluble trimers that, unlike the 368D/R trimers, possess a CD4bs surface that retains recognition by well-described CD4bs Abs. By immunizing rhesus macaques with the wild-type (WT) and CD4 binding-defective trimer variants, we demonstrate that similar levels of Env-specific Ab and T-cell responses were elicited in the three groups, suggesting that in vivo interactions between CD4 binding-competent (WT) Env and CD4 do not measurably affect T-cell responses against Env in this immunization regimen. However, the quality of the Ab response was markedly different between the groups. As hypothesized, CoRbs-directed Abs were elicited only in animals inoculated with WT trimers and not in those inoculated with 368D/R or 423I/M, 425N/K, and 431G/E trimers (hereafter referred to as 368 and 423/425/431 trimers, respectively). Importantly, we show that the 423/425/431 trimers retain the capacity to elicit binding and neutralizing CD4bs-directed Abs. In conclusion, the results generated in this study suggest that CD4 engagement by the WT soluble Env trimers did not impair the overall magnitude of the elicited Env-specific antibody or T-cell responses. Furthermore, our data provide new insights into the characteristics of Env that impact immunogenicity. The data also provide a potential path forward for the design of Env immunogens that have the capacity to elicit neutralizing Abs against the conserved gp120 CD4 binding surface while eliminating both the elicitation of nonneutralizing CoRbs-directed Abs and the potential occlusion of the CD4 binding surface of gp120 by the engagement of the primary virus receptor, CD4.     

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.Pathogenic viruses often have evolved mechanisms to neutralize host defenses that act at the cellular level to interfere with the virus life cycle. Such cellular restriction factors have been most extensively characterized for HIV-1 (38) and include the interferon-inducible membrane protein BST-2/HM1.24/CD317/tetherin (28, 40). If unchecked, tetherin blocks the release of newly formed HIV-1 particles from cells by physically tethering them at the cell surface (7, 28, 32, 40). In addition, tetherin has been shown to act against a broad range of enveloped viral particles, including retroviruses, filoviruses, arenaviruses, and herpesviruses (17, 18, 23, 35). In turn, certain viruses that are targeted by tetherin appear to have evolved counteracting activities, and anti-tetherin factors so far identified include HIV-1 Vpu; HIV-2 Env; simian immunodeficiency virus (SIV) Nef, Vpu, and Env proteins; Ebola virus GP; and Kaposi''s sarcoma-associated herpesvirus (KSHV) K5 (11, 16, 18, 20, 23, 28, 36, 40, 44, 45).Tetherin is a homodimeric type II integral membrane protein containing an N-terminal cytoplasmic tail (CT), a single-pass transmembrane domain (TM), an ectodomain-containing predicted coiled-coil regions, two glycoslyation sites, three conserved cysteines, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (2, 19, 31). This unusual topology, with two independent membrane anchors, has led to the suggestion that the retention of virions at the cell surface arises from tetherin''s ability to be inserted simultaneously in both host and viral membranes (28, 32, 41) or, alternatively, that dimers or higher-order complexes of tetherin conferred by the ectodomain mediate this effect (39). Interestingly, an artificial tetherin containing the same structural features as the native protein but constructed from unrelated sequences was able to restrict both HIV-1 and Ebola virus particles (32). This suggests that the viral lipid envelope is the target of tetherin and provides an explanation for tetherin''s broad activity against diverse enveloped viruses.A fraction of tetherin is present at the plasma membrane of cells (9, 14), and it has been proposed that viral anti-tetherin factors function by removing this cell surface fraction (40). This now has been shown to occur in the presence of HIV-1 Vpu (5, 7, 15, 26, 34, 40, 44), HIV-2 Env (5, 20), SIV Env (11), SIV Nef (15), and KSHV K5 (3, 23). In addition, certain anti-tetherin factors also may promote the degradation of tetherin, as has been observed for both HIV-1 Vpu (3, 5, 7, 10, 22, 26, 27) and KSHV K5 (3, 23), although Vpu also appears able to block tetherin restriction in the absence of degradation (8), and no effects on tetherin steady-state levels have been observed in the presence of either the HIV-2 or SIVtan Env (11, 20). Simply keeping tetherin away from the cell surface, or targeting it for degradation, may not be the only mechanism used by anti-tetherin factors, since it also has been reported that Vpu does not affect the levels of surface tetherin or its total cellular levels in certain T-cell lines (27).The interactions between tetherin and viral anti-tetherin factors show evidence of species specificity, suggesting ongoing evolution between viruses and their hosts. HIV-1 Vpu is active against human and chimpanzee tetherin but not other primate tetherins (10, 25, 34, 36, 44, 45), while SIV Nef proteins are active against primate but not human tetherins (16, 36, 44, 45). This suggests that, unlike tetherin restriction, the action of the anti-tetherin factors may involve specific sequence interactions. Indeed, the TM domain has been recognized as a target for HIV-1 Vpu (10, 15, 16, 25, 34), while a single point mutation introduced into the extracellular domain of human tetherin can block its antagonism by the SIVtan Env (11).In the present study, we investigated the roles of the different domains of tetherin in both promoting virus restriction and conferring susceptibility to the anti-tetherin factors encoded by HIV-1, HIV-2, and Ebola virus. We confirmed that tetherin restriction can be conferred by proteins that retain the two distinct membrane anchors, while signals for the cellular localization of the protein reside in the CT/TM domains of the protein. We found that the Vpu protein targets the TM domain of tetherin, while the HIV-2 Env targets the ectodomain of the protein. In contrast, the Ebola virus GP appears to use a non-sequence-specific mechanism to counteract tetherin restriction, since even an artificial tetherin could be successfully overcome by GP expression. Interestingly, Ebola virus GP counteracted tetherin restriction without removing the protein from the cell surface, suggesting that it is possible to overcome this restriction by mechanisms other than blocking tetherin''s cell surface expression.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC₅₀) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC₅₀ titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.Since the identification of human immunodeficiency virus type 1 (HIV-1) as the cause of AIDS, one of the greatest challenges has been the development of a vaccine that will prevent infection and/or ameliorate disease progression (38, 43). Although over 100 phase I, II, and III vaccine clinical trials of different candidates have been conducted all over the world, only a few candidates have advanced to efficacy testing and none has yet to show any benefit in prevention or control of HIV-1 (HIV Vaccine Database; www.iavi.org). In other viral diseases (such as polio, influenza, and measles), neutralizing antibodies are generated as part of either the natural immune response to infection or the response to immunization, and their role in protective immunity is well established (10, 12, 15, 22, 37, 42, 45, 47, 49, 52). For HIV-1, studies in animal models indicate that both broadly neutralizing antibodies and cell-mediated responses may be required to provide vaccine protection (7, 14, 16, 20, 29, 31, 33, 34, 39, 53). Unlike many other viruses, HIV-1 is highly variable, with multiple subtypes and recombinant forms circulating in different regions of the world. This high level of HIV-1 genetic variability, particularly in the envelope glycoproteins (gp120 and gp41), has been one of the greatest obstacles in development of a safe and effective HIV-1 vaccine and in particular in the elicitation of broadly neutralizing antibodies. In addition, HIV-1 has other mechanisms of immune escape preventing elicitation of broadly neutralizing antibodies, including the heavy glycosylation of the envelope glycoproteins, instability of such glycoproteins, and conformational masking of receptor-binding sites (6, 25, 32).Despite the enormous diversity of HIV-1, a relatively small number of broadly neutralizing monoclonal antibodies (bnMAbs) have been isolated, providing evidence that broad neutralization by single antibody specificities can be achieved (3-5, 8, 9, 17, 21, 23, 24, 29, 35, 36, 40, 41, 44, 50, 51, 55). Structures for such bnMAbs have been determined in complex with HIV-1 Env (26, 54) and provide starting points for the design of immunogens capable of eliciting broadly neutralizing antibodies. However, since there are only a few such bnMAbs, we established a global program as part of International AIDS Vaccine Initiative''s (IAVI''s) Neutralizing Antibody Consortium (6), aimed at screening HIV-1⁺ subjects with the goal of identifying individuals with broad and potent neutralizing activities as a potential source of novel bnMAbs, with an emphasis placed on individuals infected with non-clade B viruses. This paper describes the screening algorithm implemented to successfully identify HIV-1⁺ subjects with broadly neutralizing antibodies, including a subset of individuals termed “elite neutralizers.” These volunteers will be studied further to characterize the specificities of serum antibodies and will provide source materials for isolation of bnMAbs.     

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.     

Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.Feline leukemia viruses (FeLVs) are pathogenic retroviruses of domestic cats that induce proliferative, degenerative, and immunosuppressive disorders (17, 18, 27). Infected cats often contain a mixture of FeLVs that have been categorized into subgroups A, B, and C based on their interference and in vitro host range properties (39). These subgroups have been shown to be tightly associated with distinct feline diseases (17, 27). A fourth subgroup (T) that is highly related to FeLV-A has also been reported (26) and has been shown to be associated with immune deficiency (30). FeLV-A is the primary strain that is transmitted between cats and is the progenitor virus from which other subgroups arise. FeLV-B arises by recombination between endogenous retroviral sequences present in the cat genome and the gene encoding the FeLV-A envelope (Env) protein (32, 40, 42) that is responsible for host cell surface receptor recognition. FeLV-C and FeLV-T are formed by mutations in the FeLV-A Env gene (10, 27, 38). Env recombination and mutations are a major determinant of the host receptors used by the different FeLV subgroups (3, 23, 34, 46, 47).The emergence of pathogenic FeLV-C in cats infected with FeLV-A provides a classic example of Env mutations that switch the host receptor used for infection, leading to a fatal pathogenic disease in the host. The emergence of FeLV-C is tightly associated with red blood cell aplasia, a fatal feline anemia characterized by a specific disruption in erythroid progenitor cell development (2, 12, 19, 28). Moreover, FeLV-C emergence coincides with a switch in the host receptor used for infection from the thiamine transporter THTR1 (FeLV-A receptor) (23) to the heme exporter FLVCR1 (FeLV-C receptor) (34, 35, 46). Previous studies have suggested that the feline anemia is caused by the FeLV-C Env protein binding to, and disrupting, the cellular function of FLVCR1 (1, 34, 46). Indeed, expression of FeLV-C Env in hematopoietic stem cells (36) or disruption of FLVCR1 (21, 35) specifically disrupts early erythropoiesis, which mimics the anemia observed in cats with FeLV-C. Interestingly, the emergence of FeLV-C from FeLV-A is analogous to the emergence of cytopathic X4 strains of human immunodeficiency virus type 1 (HIV-1) in individuals infected with the R5 strain of HIV-1. Analogous to the emergence of FeLV-C, X4 HIV-1 arises by Env mutations in the R5 HIV-1 strain, which leads to a switch in the coreceptor used for infection allowing an expansion in HIV-1 cell tropism and subsequently to accelerated AIDS (4, 8). However, whereas HIV-1 pathogenesis also involves emergence of variants or X4/R5 intermediates that can use multiple related coreceptors for infection (5, 8, 11), the mechanism of how FeLV-C emerges, in terms of the presence of FeLV-A/FeLV-C intermediates, has yet to be elucidated.To better understand the emergence of pathogenic FeLV-C in infected cats and potential FeLV variants/intermediates that may arise, we isolated and characterized FeLV Env sequences from a primary FeLV isolate derived from a cat with pure red cell aplasia. In this study, we report the isolation and characterization of the novel FY981 Env, a hybrid FeLV-A/FeLV-C Env that, when pseudotyped, can use FLVCR1, THTR1, and the FLVCR1-related FLVCR2 for infection. Our findings suggest that the FY981 Env could represent a potential FeLV intermediate that arises during the emergence of pathogenic FeLV-C.     

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.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.     

Fundamental Difference in the Content of High-Mannose Carbohydrate in the HIV-1 and HIV-2 Lineages

The virus-encoded envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) typically contain 26 to 30 sites for N-linked carbohydrate attachment. N-linked carbohydrate can be of three major types: high mannose, complex, or hybrid. The lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA), which specifically bind high-mannose carbohydrate, were found to potently inhibit the replication of a pathogenic cloned SIV from rhesus macaques, SIVmac239. Passage of SIVmac239 in the presence of escalating concentrations of GNA and HHA yielded a lectin-resistant virus population that uniformly eliminated three sites (of 26 total) for N-linked carbohydrate attachment (Asn-X-Ser or Asn-X-Thr) in the envelope protein. Two of these sites were in the gp120 surface subunit of the envelope protein (Asn244 and Asn460), and one site was in the envelope gp41 transmembrane protein (Asn625). Maximal resistance to GNA and HHA in a spreading infection was conferred to cloned variants that lacked all three sites in combination. Variant SIV gp120s exhibited dramatically decreased capacity for binding GNA compared to SIVmac239 gp120 in an enzyme-linked immunosorbent assay (ELISA). Purified gp120s from six independent HIV type 1 (HIV-1) isolates and two SIV isolates from chimpanzees (SIVcpz) consistently bound GNA in ELISA at 3- to 10-fold-higher levels than gp120s from five SIV isolates from rhesus macaques or sooty mangabeys (SIVmac/sm) and four HIV-2 isolates. Thus, our data indicate that characteristic high-mannose carbohydrate contents have been retained in the cross-species transmission lineages for SIVcpz-HIV-1 (high), SIVsm-SIVmac (low), and SIVsm-HIV-2 (low).The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are heavily glycosylated. N-linked carbohydrate is attached to the nascent protein at the asparagine of the consensus sequence N-X-S or N-X-T, where X is any amino acid except a proline (31, 52, 53). The number of potential N-linked carbohydrate attachment sites in the surface subunit of Env (gp120) ranges from 18 to 33, with a median of 25 (34, 65). There are typically 3 or 4 potential N-linked sites in the ectodomain of the Env transmembrane protein (gp41) (34).N-linked glycosylation of a protein consists of the en bloc transfer of the carbohydrate core oligosaccharide (two N-acetylglucosamines, nine mannoses, and three glucoses) from dolichol to the asparagine of the N-linked attachment site (8, 60). Initially the attached carbohydrate is processed into the high-mannose type (8). In the Golgi complex, high-mannose carbohydrate may be further processed into complex or hybrid oligosaccharides (58). Incomplete processing of N-linked carbohydrate results in the production of high-mannose carbohydrate chains, which terminate in mannose (58). Fully processed complex carbohydrate chains terminate in galactose, N-acetylglucosamine, sialic acid, or glucose (33, 57). Hybrid carbohydrate chains have two branches from the core, one that terminates in mannose and one that terminates in a sugar of the complex type (63).Glycoproteins exist as a heterogeneous population, exhibiting heterogeneity with respect to the proportion of potential glycosylation sites that are occupied and to the oligosaccharide structure observed at each site. Factors that influence the type of carbohydrate chain that is attached at any one N-linked site are the accessibility of the carbohydrate chain to processing enzymes (49), protein sequences surrounding the site (5, 40), and the type of cell from which the protein is produced (19).The N-linked carbohydrate chains of HIV and SIV Env are critical for the proper folding and cleavage of the fusion-competent envelope spike (20, 59, 61). After Env is assembled, enzymatic removal of N-linked carbohydrate does not dramatically affect the functional conformation (2, 6, 7, 13, 24, 38). It is generally accepted that the carbohydrate attached to Env limits the ability of the underlying protein to be recognized by B cells (11, 48, 62). This carbohydrate also shields protein epitopes that would otherwise be the direct targets of antibodies that neutralize viral infection (41, 48, 62, 64). Furthermore, the high-mannose carbohydrates of HIV and SIV Env bind dynamically to an array of lectin proteins that are part of the host lymphoreticular system. The interaction of viral high-mannose carbohydrate with host lectin proteins has been associated with the enhancement (9, 16, 17, 43-45) or suppression (42, 56) of viral infection of CD4-positive T cells. The high-mannose carbohydrate of Env is also known to activate the release of immune-modulatory proteins from a subset of host antigen-presenting cells (12, 54).The plant lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) specifically bind terminal α-1,3- and/or α-1,6-mannose of high-mannose oligosaccharides but not hybrid oligosaccharides (28, 55). GNA and HHA inhibit the replication of HIV-1 and SIVmac251, and uncloned, resistant populations of virus have been selected (3, 14). In this report, we define two N-linked sites in the external surface glycoprotein gp120 and one in the transmembrane glycoprotein gp41 whose mutation imparts high-level resistance to the inhibitory effects of GNA and HHA to cloned SIVmac239. Furthermore, using a GNA-binding enzyme-linked immunosorbent assay (ELISA), we show that assorted HIV-1 and SIVcpz gp120s consistently are considerably higher in mannose content than assorted gp120s from SIVmac, SIVsm, and HIV-2. These results shed new light on the impact of virus-host evolutionary dynamics on viral carbohydrate composition, and they may have important implications for the mechanisms by which long-standing natural hosts such as sooty mangabeys can resist generalized lymphoid activation and disease despite high levels of SIV replication.