Both Neutralization Resistance and High Infectivity Phenotypes Are Caused by Mutations of Interacting Residues in the Human Immunodeficiency Virus Type 1 gp41 Leucine Zipper and the gp120 Receptor- and Coreceptor-Binding Domains

Neutralization resistance of human immunodeficiency virus type 1 (HIV-1) is a major impediment to vaccine development. We have found that residues of HIV-1 MN strain in the C terminus of gp120 and the leucine zipper (LZ) region of gp41 viral envelope proteins interact cooperatively to determine neutralization resistance and modulate infectivity. Further, results demonstrate that this interaction, by which regions of gp120 are assembled onto the LZ, involves amino acid residues intimately related to those which participate in the binding of the envelope to its receptor and coreceptor. Variations in this critical assembly structure determine the concordant, interdependent evolution of increased infectivity efficiency and neutralization resistance phenotypes of the envelopes. The results elucidate important structure-function relationships among epitopes that are important targets of vaccine development.     

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.The entry process of the human immunodeficiency virus type 1 (HIV-1) has become a major target for new antiviral drugs. Viral entry is initiated by binding of the HIV-1 envelope glycoprotein subunit gp120 to the CD4 receptor and a chemokine coreceptor, generally CCR5 or CXCR4. Upon coreceptor binding, the viral transmembrane subunit gp41 undergoes conformational changes that eventually lead to the formation of the six-helix bundle (6HB) and membrane fusion. The 6HB is composed of a central trimeric coiled-coil structure formed by the N-terminal heptad repeat (NHR) domains of three gp41 molecules and the corresponding C-terminal heptad repeats (CHRs) that pack into the longitudinal grooves on the surface of the NHR coiled-coil in an antiparallel orientation (23). C-peptide fusion inhibitors (CFI) derived from the CHR of gp41 compete with the viral CHR for binding to the NHR trimer, thus blocking 6HB formation and viral entry (18).T-20 (enfuvirtide) is the first clinically approved CFI with high antiviral activity and a low-toxicity profile. However, as with many anti-HIV-1 drugs, resistance can emerge rapidly (13). The majority of the resistance mutations are found in the NHR of gp41 among the amino acids 544 to 553 (32, 35) (numbering refers to gp160 of the HIV-1 HXB2 strain throughout the article). Most of these mutations cause resistance by reducing the affinity of the NHR target region to inhibitory C peptides (13). Additionally, viral entry kinetics were found to correlate with the baseline susceptibility of different HIV strains to CFI. Determinants for viral entry kinetics are found in gp41 as well as in gp120 (1, 14, 35). Here, the influence of coreceptor affinity on virus entry kinetics and CFI susceptibility has been studied extensively (28, 30, 31). Recently, a statistical approach was used that highlighted positions in gp120 that underwent mutations in patients under enfuvirtide treatment (38). However, to our knowledge, selected CFI resistance mutations outside of gp41 have never been confirmed experimentally.Previously, we developed a retroviral vector expressing a membrane-anchored antiviral C peptide (maC46) that efficiently inhibits a broad range of different HIV-1 isolates. Enfuvirtide-resistant HIV-1 strains with mutations in the GIV motif of NHR were fully susceptible to maC46 (10). In the present study, we selected an HIV-1 variant with reduced sensitivity to maC46 by passaging an enfuvirtide-resistant BaL strain of HIV-1 on cells expressing increasing concentrations of maC46. Mutations in gp120 and gp41 were found to contribute to maC46 resistance.     

Determinants of Human Immunodeficiency Virus Type 1 Resistance to gp41-Derived Inhibitory Peptides

A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1_IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.     

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.The development of an AIDS vaccine is an ongoing and urgent challenge. One of the major hurdles is that the specific correlates of protection against human immunodeficiency virus (HIV) are still largely unknown. Nonetheless, most agree that the full complement of cellular and humoral components of the immune system will be needed to combat this virus. This is especially true given that the virus resides permanently in its host, infects the very cells needed to direct effective immune responses, and evades the immune system, either by changing in appearance or hiding in subcellular compartments.A broadly reactive neutralizing antibody response is likely to be critical as a first line of defense upon initial HIV exposure by aiding in the clearance of cell-free virions, targeting infected cells for destruction, and preventing viral spread through cell-to-cell transmission. The presence of inhibitory antibodies in highly exposed persistently seronegative individuals testifies to the importance of the humoral response (9, 37). Additionally, broadly neutralizing serum has been associated with healthier prognoses for infected individuals (27, 65) and may be vital for protecting offspring from their infected mothers (7, 79) and preventing superinfection by heterologous HIV strains (23, 84). Even if complete protection cannot be achieved by vaccine-derived antibodies, an early, well-poised and effective neutralizing antibody repertoire may be able to lower the set point of the viral load following the initial burst of viremia, an outcome that has been reported to translate into improved disease outcomes and reduced transmission of HIV (66, 74). Further benefits of neutralizing antibodies have been seen with passive immunization studies in macaques, in which administration of broadly neutralizing monoclonal antibodies (MAbs) has demonstrated that it is possible to provide protection from—and even sterilizing immunity against—HIV infection (5, 51, 66). There is also evidence that such antibodies may provide therapeutic benefits for chronically infected individuals, analogous to benefits realized with anti-HIV drug treatment regimens (87).Despite the promising potential of broadly neutralizing MAbs, designing immunogens that can elicit such cross-reactive neutralizing responses against HIV has been a surprisingly difficult task. Since the majority of the host''s B-cell response is directed against the envelope (Env) glycoproteins, gp120 and gp41, vaccine efforts have concentrated on these proteins and derivatives thereof in approaches ranging from the use of Env-based peptide cocktails to recombinant proteins and DNAs made with varied or consensus sequences and diverse, heterologous prime/protein boost regimens (reviewed in references 36, 58, and 70). These iterative studies have shown notable improvements in the potency and breadth of neutralizing responses induced. However, concerns exist regarding immunogens containing extraneous epitopes, as is the case with intact subunits of Env, and the nature of the immune responses they may elicit. A polyclonal burst of antibodies against a multitude of nonfunctional epitopes may include a predominance of antibodies that are (i) low affinity and/or nonfunctional (reviewed in reference 72); (ii) isolate specific (25); (iii) able to interfere with the neutralizing capabilities of otherwise-effective antibodies (via steric hindrance or by inducing various forms of B-cell pathology) (67); or (iv) directed against irrelevant epitopes instead of more conserved (and sometimes concealed) epitopes that might be able to elicit more potent and cross-reactive neutralizing responses (28, 71, 91).We have developed a system that can be used to present essentially any chosen epitope in a stable, well-exposed manner on the surface of the cold-causing human rhinovirus (HRV). HRV is itself a powerful immunogen and is able to elicit T-cell as well as serum and mucosal B-cell responses (reviewed by Couch [22]) and has minimal immunologic similarity to HIV (data not shown). Chimeric viruses displaying optimal epitopes should be able to serve as valuable components in an effective vaccine cocktail or as part of a heterologous prime/boost protocol. We have shown previously that HRV chimeric viruses displaying HIV-1 gp120 V3 loop sequences are able to elicit neutralizing responses against HIV-1 (75, 82, 83).In this study, we focused our attention on presenting part of the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41, a region of approximately 30 amino acids adjacent to the transmembrane domain (reviewed in references 59 and 97). The MPER plays an important role in the process of HIV fusion to the host cell membrane (60, 78). This region is also involved in binding to galactosylceramide, an important component of cell membranes, thus permitting CD4-independent transcytosis of the virus across epithelial cells at mucosal surfaces (1, 2). These functions likely explain this region''s sequence conservation and the efficacy of antibodies directed against the MPER (97), particularly given that an estimated 80% of HIV-1 infections are sexually transmitted at mucosal membranes. In fact, potent responses against the MPER are associated with stronger and broader neutralizing capabilities in infected individuals (68). A conserved, contiguous sequence of the MPER, the ELDKWA epitope (HIV-1 HxB2 gp41 residues 662 to 668), is recognized by the particularly broadly neutralizing human MAb 2F5 (11, 62, 85) and is highly resistant to escape mutation in the presence of 2F5 (49). 2F5 was also used in the MAb cocktails reported to confer passive, protective immunity in macaques (5, 51). In addition, infected individuals producing neutralizing antibodies directed against the ELDKWA epitope have been seen to exhibit better health (16, 29), including persistent seronegativity (8), and reduced transmission of HIV to offspring (89). While none of the vaccine-induced immune responses generated against this region has been effective thus far (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), more appropriate presentations of MPER epitopes should produce valuable immunogens that can contribute to a successful vaccine.In this study, we have grafted the ELDKWA epitope onto a surface loop of HRV connected via linkers of variable lengths and sequences and selected for viruses well recognized and neutralized by MAb 2F5. In so doing, we have been able to create immunogens capable of eliciting antibodies whose activities mimic some of those of 2F5. The combinatorial libraries produced were designed to encode a large set of possible sequences and, hence, structures from which we could search for valuable conformations. This work illustrates that HRV chimeras have the potential to present selected HIV epitopes in a focused and immunogenic manner.     

Conserved, N-Linked Carbohydrates of Human Immunodeficiency Virus Type 1 gp41 Are Largely Dispensable for Viral Replication

The transmembrane subunit (TM) of human immunodeficiency virus type 1 (HIV-1) envelope protein contains four well-conserved sites for the attachment of N-linked carbohydrates. To study the contribution of these N-glycans to the function of TM, we systematically mutated the sites individually and in all combinations and measured the effects of each on viral replication in culture. The mutants were derived from SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope sequence that originated from a primary HIV-1 isolate. The attachment site mutants were generated by replacing the asparagine codon of each N-X-S/T motif with a glutamine codon. The mobilities of the variant transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that all four sites are utilized for carbohydrate attachment. Transfection of various cell lines with the resulting panel of mutant viral constructs revealed that the N-glycan attachment sites are largely dispensable for viral replication. Fourteen of the 15 mutants were replication competent, although the kinetics of replication varied depending on the mutant and the cell type. The four single mutants (g1, g2, g3, and g4) and all six double mutants (g12, g13, g14, g23, g24, and g34) replicated in both human and rhesus monkey T-cell lines, as well as in primary rhesus peripheral blood mononuclear cells. Three of the four triple mutants (g124, g134, and g234) replicated in all cell types tested. The triple mutant g123 replicated poorly in immortalized rhesus monkey T cells (221 cells) and did not replicate detectably in CEMx174 cells. However, at 3 weeks posttransfection of 221 cells, a variant of g123 emerged with a new N-glycan attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental virus. The quadruple mutant (g1234) did not replicate in any cell line tested, and the g1234 envelope protein was nonfunctional in a quantitative cell-cell fusion assay. The synthesis and processing of the quadruple mutant envelope protein appeared similar in transient assays to those of the parental SHIV-KB9 envelope. Given their high degree of conservation, the four N-linked carbohydrate attachment sites on the external domain of gp41 are surprisingly dispensable for viral replication. The viral variants described in this report should prove useful for investigation of the contribution of carbohydrate moieties on gp41 to recognition by antibodies, shielding from antibody-mediated neutralization, and structure-function relationships.     

Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.     

Peptide Ligands to Human Immunodeficiency Virus Type 1 gp120 Identified from Phage Display Libraries

We have used phage-displayed peptide libraries to identify novel ligands to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120. Screening of libraries of random 12-mers, 7-mers, and cyclic 9-mers produced two families of gp120 binding peptides. Members of a family with the prototype sequence RINNIPWSEAMM (peptide 12p1) inhibit the interaction between gp120 and both four-domain soluble CD4 (4dCD4) and monoclonal antibody (MAb) 17b, a neutralizing antibody that covers the chemokine receptor binding surface on gp120. Peptide 12p1 inhibits the interaction of 4dCD4 with gp120 from three different HIV strains, implying that it binds to a conserved site on gp120. Members of a second family of peptides, with the prototype sequence TSPYEDWQTYLM (peptide 12p2), bind more weakly to gp120. They do not detectably affect its interaction with 4dCD4, but they enhance its binding to MAb 17b. A common sequence motif in the two peptide families and cross-competition for gp120 binding suggest that they have overlapping contacts. Their divergent effects on the affinity of gp120 for MAb 17b may indicate that their binding stabilizes distinct conformational states of gp120. The functional properties of 12p1 suggest that it might be a useful lead for the development of inhibitors of HIV entry.     

Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV_AD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.     

A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies

The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W⁶⁷⁰NWFDITN⁶⁷⁷) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.The continued spread of human immunodeficiency virus (HIV) worldwide and, in particular, in sub-Saharan Africa, where an estimated 22 million people currently are living with HIV/AIDS, underscores the urgent need for a preventative vaccine. However, despite nearly 25 years of intense international research, a vaccine is not yet available. Passive immunization with broadly neutralizing antibodies can confer sterilizing protection against infection in animal models (4, 12, 39-41, 51, 64), providing encouragement for the development of an antibody-inducing component of an HIV type 1 (HIV-1) vaccine. Such a vaccine should elicit neutralizing antibodies with activity against the broadest range of primary circulating isolates. However, a lack of understanding of how to raise potent, cross-reactive antibodies by immunization, the so-called neutralizing antibody problem, is a major hurdle in this effort (6, 24, 72). Thus, an understanding of the structure and presentation of neutralizing epitopes on the virus and the antibodies that recognize them is vital for vaccine development.The targets of antibody neutralization are the surface envelope (Env) glycoprotein trimers (gp120/gp41) that mediate the fusion of the viral membrane with that of the host. The majority of antibodies elicited during natural infection or immunization show limited or no cross-reactivity against diverse isolates. However, a few rare, broadly neutralizing, monoclonal antibodies have been isolated from HIV-1-infected individuals and exhibit activity against a wide range of isolates by binding to functionally conserved epitopes exposed on native gp120/gp41 trimers. These epitopes include the CD4 binding site, recognized by antibody b12, and a relatively well-conserved cluster of N-linked glycans, located on the outer domain of gp120, that is recognized by antibody 2G12 (12, 13, 71, 76). V3-directed antibodies, which are common in natural infection, also are able to sporadically neutralize across clades, as exemplified by 447-52D and F425-B4e8 (7, 16, 49, 66). The identification of three broadly neutralizing antibodies, 2F5, Z13, and 4E10, that target the conserved tryptophan-rich membrane-proximal external region (MPER) of gp41 has implicated this region as a highly promising vaccine target and has, therefore, spurred interest in its structural characterization (15, 35, 45, 47, 48, 50, 80).The MPER plays a critical, but not fully understood, role in membrane fusion and is situated between the C-terminal heptad repeat (CHR) and the transmembrane domain (TM) of gp41 (Fig. ​(Fig.1).1). Following the binding of gp120 to the cell surface receptors CD4 and CXCR4/CCR5, the gp41 glycoprotein undergoes a series of conformational changes that trigger the membrane fusion activity. Notably, a relatively long-lived prehairpin intermediate of gp41 is formed, in which the coiled-coil of the N-terminal heptad repeats (NHR) extends so as to enable the fusion peptides to embed into the target membrane. In the postfusion or fusogenic state, the CHR and NHR reassemble into an antiparallel 6-helix bundle in a process that drives membrane fusion (18). The MPER contains several functionally conserved tryptophan residues that are critical for membrane fusion and viral entry, although the structural basis for their specific role has not been firmly established (22, 44, 58). Their mutation to alanine leads to the attenuation of viral infectivity, which is most pronounced for Trp666 and Trp672 (numbered according to the HXB2 isolate) (46, 58, 78). In addition, peptides based on the MPER can induce membrane leakage (68). Such membrane-disrupting properties of the MPER have been suggested to be functionally important in the expansion of the fusion pore created after receptor engagement (42, 44, 58, 68, 77).Open in a separate windowFIG. 1.Major features of gp41 include the fusion peptide (FP), NHR, CHR, TM, and cytoplasmic domain (CD). The MPER is located between the CHR and TM regions of gp41. The core epitopes of 2F5 (green), Z13e1 (yellow), and 4E10 (orange) are indicated. The epitope of Z13e1 is located between those of 2F5 and 4E10, but it overlaps more closely with 4E10.From initial explorations using solution nuclear magnetic resonance, the structure of a 19-residue MPER peptide (residues 665 to 683) was found to be helical in dodecylphosphocholine micelles, with the hydrophobic and hydrophilic residues distributed evenly around the helix axis (62). Another study found that an MPER peptide comprising residues 659 to 671 adopts a 3₁₀-helix in water (10). More recently, the structure of an MPER peptide (residues 662 to 683) in liposomes was elucidated by a combination of nuclear magnetic resonance and spin-label electron paramagnetic resonance (69), and it was found to adopt a kinked, amphipathic structure composed of two helices connected by a short hinge (Phe673 and Asn674). Crystal structures of Fab 2F5 in complex with a 7-mer (E⁶⁶²LDKWAS⁶⁶⁸) and 17-mer encompassing residues 654 to 670 previously had revealed a mostly extended conformation characterized by a central β-turn involving Asp664, Lys665, and Trp666 (47, 48). This motif is the key recognition determinant for 2F5 and becomes deeply buried in the antibody combining site, suggesting that it is exposed at some stage in viral entry (45, 47, 78). The crystal structure of Fab 4E10 in complex with peptide-spanning residues W⁶⁷⁰NWFDITNW⁶⁷⁸ revealed an amphipathic α-helical structure with a narrow hydrophilic face (15). The N terminus of the 4E10 epitope forms a 3₁₀-helix that transitions into a regular α-helix at residue Asp674 and continues to Lys683, which constitutes the end of the gp41 ectodomain (14). Thus, while the structure of the MPER within functional, membrane-embedded Env trimers is not known, the observation that unconstrained peptides are able to adopt more than one defined structure suggests an inherent degree of flexibility.Like 4E10, Z13 was identified from an HIV-1-infected individual, the former being isolated from an immortalized B-cell line and the latter from a bone marrow RNA phage display library (80). The epitope of MAb Z13 spans residues S⁶⁶⁸LWNWFDITN⁶⁷⁷, as determined by peptide mapping, scanning mutagenesis, and antibody competition studies (46, 80). This region lies between the 2F5 and 4E10 epitopes but overlaps more closely with 4E10 (Fig. ​(Fig.1).1). 4E10 and Z13 are both able to neutralize primary as well as laboratory-adapted isolates; nevertheless, Z13 is not as broadly neutralizing as 4E10, which has the greatest breadth of any HIV-1 antibody described to date (9). Z13e1 is an affinity-enhanced variant of Z13 and was evolved by randomizing the complementarity determining region (CDR) L3 loop sequence to identify tighter-binding mutants using phage display (46). Z13e1 displays higher affinity for both peptide and recombinant gp41 substrates, as well as increased neutralization potency, suggesting that the L3 mutations optimize binding to the linear MPER epitope. The neutralization breadth of Z13e1 is limited by the requirement for Asn671 and Asp674 in the MPER, which are approximately 71 and 58% conserved, respectively, among sequences in the Los Alamos HIV sequence database (80). Based on the clear relationship between Env trimer binding and neutralization, the neutralizing activity of Z13e1 derives from binding to a functional trimer (8, 20, 25, 43, 52, 55, 60, 73, 74). While Z13e1 and 4E10 have identical affinities for optimized linear peptides, Z13e1 is still about an order of magnitude less potent than 4E10 against a variety of primary isolates. Although the occlusion of the Z13e1 epitope on virion-associated trimers is thought to be the major limitation (46), the structural basis for the lower potency of Z13e1 relative to those of 2F5 and 4E10 is unclear.Whereas neutralization by 4E10 depends critically on Trp672 and Phe673, Z13e1 instead requires the flanking Asn671 and Asp674 residues (46). Based on a helical model of the MPER, it was predicted that Z13e1 binds the narrow hydrophilic face that displays Asn671, Asp674, and Asn677 that is opposite that recognized by 4E10. As Z13e1 and 4E10 bind to functional trimers, both epitopes must be exposed at some stage before membrane fusion (20). To examine how Z13e1 recognizes its MPER epitope, we determined the crystal structure of Fab Z13e1 in complex with a 12-residue peptide corresponding to the core epitope with C-terminal flanking lysines to aid peptide solubility (W⁶⁷⁰NWFDITN⁶⁷⁷KKKK). The crystal structure at 1.8-Å resolution uncovers a conformation of the MPER that is distinct from that visualized in complex with 4E10. Our findings show that Z13e1 and 4E10 recognize different conformers of the MPER and reveal a novel conformational switch that is relevant for HIV-1 neutralization and membrane fusion.     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. ​(Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man₉GlcNAc₂ precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man₅GlcNAc₂ intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. ​(Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man₃GlcNAc₂ pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. ​(Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc₃Man₉GlcNAc₂; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man₅GlcNAc₂ (Fig. ​(Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man₅GlcNAc₂ substrate (19) (Fig. ​(Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. ​(Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. ​Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc₃Man₉GlcNAc₂ precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man₉GlcNAc₂ precursor into Man₅GlcNAc₂ or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. ​(Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man₅GlcNAc₂ substrate, preventing the formation of hybrid and complex carbohydrates (Fig. ​(Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man₅GlcNAc₂ rather than with the larger Man₉GlcNAc₂ precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man₅GlcNAc₂ we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.     

Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity

It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associated gag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.     

Broadly Neutralizing Monoclonal Antibodies 2F5 and 4E10 Directed against the Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Protect against Mucosal Challenge by Simian-Human Immunodeficiency Virus SHIVBa-L

The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIV_Ba-L. In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.Eliciting broadly neutralizing antibodies is an important goal of HIV vaccine design efforts, and the study of broadly neutralizing monoclonal antibodies (bnMAbs) can assist in that goal. Human bnMAbs against both gp120 and gp41 of the HIV-1 envelope spike have been described. Three bnMAbs to gp41, 2F5, 4E10, and Z13e1, have been identified and shown to recognize neighboring linear epitopes on the membrane proximal external (MPER) region of gp41 (3, 24, 25, 37, 47). In a comprehensive cross-clade neutralization study by Binley et al., 2F5 neutralized 67% and 4E10 neutralized 100% of a diverse panel of 90 primary isolates (2). Similar broad neutralization was seen against sexually transmitted isolates cloned from acutely infected patients (22). More recently, a comprehensive study showed that 2F5 neutralized 97 isolates from a 162-virus panel (60%) and that 4E10 neutralized 159 isolates (98%) (41). Although less potent, the monoclonal antibody Z13, isolated from an antibody phage display library derived from a bone marrow donor whose serum was broadly neutralizing (47), has cross-clade neutralizing activity. Z13e1 is an affinity-enhanced variant of the earlier-characterized MAb Z13 that is directed against an access-restricted epitope between and overlapping the epitopes of 2F5 and 4E10. Both MAbs 2F5 and 4E10 were originally obtained as IgG3 antibodies in hybridomas derived from peripheral blood mononuclear blood lymphocytes (PBMCs) of HIV-1-seropositive nonsymptomatic patients and were later class switched to IgG1 to enable large-scale manufacturing and to prolong in vivo half-life (3, 6, 32).Despite the interest in the MPER as a vaccine target, there is limited information on the ability of MPER antibodies to act antivirally in vivo either in established infection or prophylactically. A study using the huPBL-SCID mouse model showed limited impact from 2F5 when the antibody was administered in established infection (31). Passive administration of 2G12, 2F5, and 4E10 to a cohort of acutely and chronically infected HIV-1 patients provided little direct evidence of 2F5 or 4E10 antiviral activity, whereas the emergence of escape variants indicated unequivocally the ability of 2G12 to act antivirally (18, 39). Indirect evidence did, however, suggest that the MPER MAbs may have affected virus replication, as indicated by viral rebound suppression in a patient known to have a 2G12-resistant virus prior to passive immunization (39). Another study of 10 individuals passively administered 2G12, 2F5, and 4E10 before and after cessation of combination antiretroviral therapy (ART) showed similarly that 2G12 treatment could delay viral rebound, but antiviral activity by 2F5 and 4E10 was not clearly demonstrated (21). In prophylaxis, an early 2F5 passive transfer study with chimpanzees suggested that the antibody could delay or lower the magnitude of primary viremia following HIV-1 challenge (7). A study using gene transfer of 2F5 in a humanized SCID mouse model suggested that continuous plasma levels of approximately 1 μg/ml of 2F5 may significantly reduce viral loads in LAI- and MN-challenged mice (34). Protection studies of rhesus macaques using simian-human immunodeficiency virus SHIV_89.6PD challenge did not provide definitive direct evidence for MPER antibody-mediated protection. One of three animals was protected against intravenous (i.v.) challenge when 2F5 was administered in a cocktail with HIVIG and 2G12 (19), but all three animals treated with 2F5 alone at high concentration became infected. In a vaginal challenge study with SHIV_89.6PD (20), four of five animals were protected with a cocktail of HIVIG, 2F5, and 2G12, but a 2F5/2G12 combination protected only two of five animals. Further protection studies have used MPER MAbs in combination with other MAbs, leaving the individual contributions of these antibodies uncertain (1, 8).In our previous studies, we successfully used the SHIV/macaque model to demonstrate neutralizing antibody protection against mucosal challenge, and we have begun to explore how that protection is achieved (12, 30). Here, we conducted a protection study with the two broadly neutralizing MPER-directed antibodies 2F5 and 4E10. We show that the antibodies can prevent viral infection and thereby support the MPER as a vaccine target.     

Epithelial Uptake and Transport of Cell-Free Human Immunodeficiency Virus Type 1 and gp120-Coated Microparticles

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4⁺ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and α-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.     

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

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

Analysis of the Critical Domain in the V3 Loop of Human Immunodeficiency Virus Type 1 gp120 Involved in CCR5 Utilization

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) lymphocytes and macrophages involves interaction of the surface subunit of the envelope protein (gp120) with coreceptors. Isolates have been found with specific tropism for macrophages and/or T-cell lines, through the utilization of chemokine receptor CCR5 (R5) or CXCR4 (X4). The third hypervariable loop (V3 loop) of gp120 is the major determinant of tropism. Using chimeric envelopes between HXB2 (X4) and ADA (R5), we found that the C-terminal half of the V3 loop was sufficient to confer on HXB2 the ability to infect CCR5-expressing cells. A sequence motif was identified at positions 289 to 292 allowing 30% of wild-type levels of infection, whereas full activity was achieved with the conversion of Lys to Glu at position 287 in addition to the above motif. Moreover, V3 loops from either SF2 (X4R5) or SF162 (R5) also allowed infection of CCR5-expressing cells, supporting the importance of V3 loops in influencing CCR5 utilization. The effects of amino acid changes at position 287 on the level of infection via CCR5 showed that negatively charged residues (Glu and Asp) were optimal for efficient interaction whereas only bulky hydrophobic residues drastically reduced infection. In addition, sequences at the N terminus of the V3 loop independently modulated the level of infection via CCR5. This study also examined the susceptibility of chimeric envelopes to neutralization by anticoreceptor antibodies and suggested the presence of differential interaction between the chimeric envelopes and CCR5. These findings highlight the critical residues in the V3 loop that mediate HIV-1 infection.     

Functional Analysis of the Disulfide-Bonded Loop/Chain Reversal Region of Human Immunodeficiency Virus Type 1 gp41 Reveals a Critical Role in gp120-gp41 Association

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.