Stable Docking of Neutralizing Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Monoclonal Antibodies 2F5 and 4E10 Is Dependent on the Membrane Immersion Depth of Their Epitope Regions

The binding of neutralizing antibodies 2F5 and 4E10 to human immunodeficiency virus type 1 (HIV-1) gp41 involves both the viral membrane and gp41 membrane proximal external region (MPER) epitopes. In this study, we have used several biophysical tools to examine the secondary structure, orientation, and depth of immersion of gp41 MPER peptides in liposomes and to determine how the orientation of the MPER with lipids affects the binding kinetics of monoclonal antibodies (MAbs) 2F5 and 4E10. The binding of 2F5 and 4E10 both to their respective nominal epitopes and to a biepitope (includes 2F5 and 4E10 epitopes) MPER peptide-liposome conjugate was best described by a two-step encounter-docking model. Analysis of the binding kinetics and the effect of temperature on the binding stability of 2F5 and 4E10 to MPER peptide-liposome conjugates revealed that the docking of 4E10 was relatively slower and thermodynamically less favorable. The results of fluorescence-quenching and fluorescence resonance energy transfer experiments showed that the 2F5 epitope was more solvent exposed, whereas the 4E10 epitope was immersed in the polar-apolar interfacial region of the lipid bilayer. A circular dichroism spectroscopic study demonstrated that the nominal epitope and biepitope MPER peptides adopted ordered structures with differing helical contents when anchored to liposomes. Furthermore, anchoring of MPER peptides to the membrane via a hydrophobic anchor sequence was required for efficient MAb docking. These results support the model that the ability of 2F5 and 4E10 to bind to membrane lipid is required for stable docking to membrane-embedded MPER residues. These data have important implications for the design and use of peptide-liposome conjugates as immunogens for the induction of MPER-neutralizing antibodies.The two broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10 target conserved core amino acid residues that lie in the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 (6, 9, 18, 25, 29). Structural studies of 2F5 and 4E10 in complex with their nominal epitope peptides led to the proposition that the long hydrophobic heavy chain CDR3 (CDR H3) loop might be involved in binding to the virion membrane due to the lack of direct contact of the tip of the CDR H3 loop with their bound epitopes (6, 25). MAbs 2F5 and 4E10 indeed were found to have enhanced binding to gp41 MPER in the presence of membrane (12, 25). Subsequent studies have revealed the lipid reactivity of both the 2F5 and 4E10 MAbs (2, 14, 23, 27), emphasizing the need to understand how MAbs 2F5 and 4E10 recognize their epitopes in the context of a membrane-gp41 MPER interface.It has been hypothesized that the ability of MAbs 2F5 and 4E10 to interact with membrane lipids is required for binding to the membrane-bound gp41 MPER region and subsequent HIV-1 neutralization (2, 14, 15). The binding of both the 2F5 and 4E10 MAbs to their epitope peptides presented on synthetic liposomes was remarkably different from that of epitope peptides alone and was best described by a two-step “encounter-docking” model (2). In this model, neutralizing MPER MAbs make an initial encounter complex, and such an interaction is associated with faster association and dissociation rates. The formation of the encounter complex induces the formation of the final “docked” complex, which is associated with slower dissociation rates and provides the stability of the overall interaction. A more recent study has also observed the same mode of interaction for MAb 4E10 when it binds to MPER peptide in liposomal form (31). The studies of Sun et al. revealed that critical residues of the 4E10 epitope may be buried in the viral membrane and that interaction of 4E10 with lipids is important in extracting the immersed residues from the lipid bilayer. Although 2F5 binding was not described in the study, the model shows that the N-terminal helix of the “L”-shaped MPER structure projects away from the membrane and that residues K₆₆₅ and W₆₆₆ of the core 2F5 epitope (residues DKW) are placed on the surface and in the interfacial region, respectively, of the membrane lipid (31). Thus, as for MAb 4E10, stable docking of 2F5 would also require some level of conformational rearrangement of MPER to release critical residues within the core epitope. This is consistent with binding kinetics data that showed that the final docking of MAbs 2F5 and 4E10 to MPER peptide-lipid conjugates might require conformational rearrangements (2). It is also likely that the CD4 and coreceptor-mediated triggering of HIV-1 Env (10, 28) that leads to the formation of the fusion intermediate conformation might also expose critical residues for MPER MAb binding. Both the 4E10 and 2F5 MAbs bound strongly to a recombinant trimeric gp41 intermediate design and either bound weakly or failed to bind, respectively, to the trimeric gp140 (11) and a putative prefusion-state trimeric MPER (22). However, the orientation of the MPER sequence in a viral-lipid-bound form is not known and, thus, it is possible that in the early stages of the triggered intermediate state, MPER residues may be lying in the plane of the membrane head groups and interaction of MPER MAbs with lipids and extraction of critical residues may be essential for stable docking (31).In order to gain further understanding of the binding mechanism involved in the interaction of MAbs 2F5 and 4E10 with their epitopes presented in the membrane environment, we have constructed three different novel gp41 MPER peptide-liposome conjugates, including a 2F5 nominal epitope peptide, a 4E10 nominal epitope peptide, and a peptide having sequences of epitopes for both the 2F5 and 4E10 MAbs. Unlike our previously designed constructs (2), the MPER peptides used in the current study were anchored to the liposomes by a hydrophobic sequence (YKRWIILGLNKIVRMYS), named GTH1, placed at their carboxyl termini. Using these second-generation peptide-liposome conjugates, we addressed the following questions. (i) How do MAbs 2F5 and 4E10 bind to the different peptide-liposome conjugates? (ii) How do the kinetics of MAb binding vary with temperature? (iii) How are the peptides oriented in the liposomal membrane in each construct? (iv) How does antibody binding correlate with differences in the membrane orientation of peptides? (v) Is there any difference in the secondary structures adopted by the peptides in the peptide-liposome complex?Our study of antibody interactions with their membrane-anchored epitope peptides indicates that both the 2F5 and 4E10 MAbs bind to their nominal epitope peptide-liposome conjugates with high affinity. The results of tryptophan fluorescence-quenching and fluorescence resonance energy transfer (FRET) experiments showed that the nominal 2F5 peptide is exposed on the surface of the membrane close to the polar head group, whereas the nominal 4E10 peptide is immersed in the interfacial region of the lipid bilayer. Circular dichroism (CD) spectroscopic studies revealed that the nominal epitope and biepitope peptides adopted ordered structures when anchored to the liposomal membrane. The membrane orientation data and secondary structural features of MPER peptides correlated well with antibody binding characteristics, thus suggesting that membrane-anchored MPER peptide conformations are a physiologic component of the native 2F5 and 4E10 binding epitopes in HIV-1 virions.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.Hepatitis C virus (HCV) is a major cause of liver failure and infects more than 170 million people worldwide. HCV is a member of the Flaviviridae family and contains a 9.6-kb positive-strand RNA genome. The genome is translated into a single polypeptide that is cleaved by viral and cellular proteases into at least nine different proteins. The major HCV surface glycoproteins, E1 and E2, form a noncovalent heterodimer on the virion surface (23) and are believed to mediate viral entry via a complex set of poorly understood interactions with cellular coreceptors, including CD81 (28), claudin-1 (8), occludin (29), scavenger receptor class B type I (30), and others (38). The E2 glycoprotein has been shown to interact directly with receptors (38); currently, no function has been assigned to E1, although it is known to be required for viral infection. These viral glycoproteins provide an obvious target for neutralizing monoclonal antibodies (MAbs).Isolation of potently neutralizing HCV-specific MAbs has been complicated by the lack of an in vitro cell culture system to study the full infection cycle of the virus. Recently, systems have been developed that allow for the generation of infectious viral particles, highlighting the importance of E1 and E2 in viral binding and entry. A novel in vitro infection system employs HCV pseudotyped viral particles (HCVpp) generated from a lentivirus that are devoid of native glycoproteins and engineered to contain HCV glycoproteins E1 and E2 (4, 15). HCVpp specifically infect cell lines derived from human liver cells and can be neutralized by polyclonal and MAbs directed against the HCV envelope glycoproteins.HCVpp have allowed the identification of antibodies that can neutralize HCV infection in cell culture. E1 has proven to be a difficult target for MAb-mediated neutralization, possibly because it appears to have low immunogenicity (32), has no identified binding proteins on the cell surface, and has an undefined role in cell entry. Despite this challenge, two groups have identified HCV neutralizing MAbs directed to E1: these MAbs are H-111, which has moderate neutralizing activity (17), and the recently isolated IGH505 and IGH526, which neutralize numerous HCV genotypes (1a, 1b, 2a, 4a, 5a, and 6a but not 2b and 3a) (22). Although they are predicted to inhibit viral binding or fusion, the mechanism by which these E1-directed MAbs neutralize HCV infection is unclear.A diverse group of mouse anti-E2 antibodies, recognizing both linear and discontinuous epitopes, has been generated. Many of these MAbs showed broad neutralization of multiple HCV genotypes, but not surprisingly, several HCV isolates were refractory to neutralization. In contrast, AP33, a mouse MAb that largely recognizes a highly conserved linear epitope in the N terminus of E2 (amino acids 412 to 423), was identified as a broadly cross-reactive antibody that neutralized strains from all genotypes tested (1a, 1b, 2a, 2b, 3a, 4, 5, and 6), with the exception of one genotype 5 virus (UKN5.14.4; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY894682","term_id":"58220837","term_text":"AY894682"}}AY894682) (24). Recently, several cross-reactive neutralizing MAbs have been identified that are of human origin and have the capacity to neutralize a significant fraction of the genotypes tested (1, 5, 12, 13, 27, 31) or to neutralize all genotypes tested (16, 20, 25). As with the vast majority of previously described human MAbs (HuMAbs), these MAbs recognize conformation-dependent epitopes of E2. One broadly neutralizing human antibody, AR3B, was tested in a mouse model of infection and showed significant protection from viremia (20). Given the known function of the E2 envelope glycoprotein, the high level of immunogenicity, the surface vulnerability, and the abundance of data pertaining to E2 and HCV neutralization, E2 provides a promising target for the development of fully human neutralizing antibodies.Liver deterioration due to HCV infection is the leading reason for liver transplantation in the United States. Unfortunately, it is highly likely that the transplanted liver will also become infected with HCV, and 10 to 25% of these patients develop cirrhosis within 5 years of transplant (9, 40). Here we describe the characterization of HuMAbs directed against the HCV E2 envelope glycoprotein, generated using transgenic mice. Based on epitope conservation and broad neutralization capacity, HuMAbs HCV1 and 95-2 provide excellent candidates for prevention of graft reinfection of HCV-infected individuals undergoing liver transplantation.     

Postnatal Passive Immunization of Neonatal Macaques with a Triple Combination of Human Monoclonal Antibodies against Oral Simian-Human Immunodeficiency Virus Challenge

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.     

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.     

Induction and Characterization of Neutralizing Antibodies against a Human Immunodeficiency Virus Type 1 Primary Isolate

Chimpanzees infected with the primary isolate DH012 mount potent neutralizing antibodies. This DH012 neutralizing activity is highly strain specific. Immune sera from guinea pigs immunized with recombinant DH012 gp120 could also neutralize this primary isolate. The neutralizing activity in chimpanzee and guinea pig sera against wild-type DH012 appears to be independent of a linear epitope in the V3 region of gp120. Interestingly, the neutralization escape mutant derived from growing DH012 in the presence of the potent neutralizing chimpanzee serum is at least 50-fold more sensitive than wild-type DH012 to neutralization by guinea pig immune sera. The unusually potent neutralizing activity against the DH012 neutralization-resistant virus is due to the presence of anti-V3 antibodies in guinea pig sera. These results suggested that recombinant gp120 could induce neutralizing antibodies against primary isolate DH012. The V3 of wild-type DH012 is poorly immunogenic in infected chimpanzees and is not accessible to neutralizing V3 antibodies. It is likely that this cryptic V3 region became exposed when the virus escaped the neutralizing activity of the chimpanzee serum.     

A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.     

Neutralizing Capacity of Monoclonal Antibodies That Recognize Peptide Sequences Underlying the Carbohydrates on gp41 of Simian Immunodeficiency Virus

Extensive glycosylation of the envelope spikes of human and simian immunodeficiency virus (HIV and SIV) is an important factor for the resistance of these viruses to neutralization by antibodies. SIVmac239 gp41 has three closely spaced sites for N-linked carbohydrate attachment. Rhesus macaques experimentally infected with mutant versions of SIVmac239 lacking two or three of these carbohydrate sites developed strong serum reactivity against mutated peptide sequences at the site of these glycosylations, as well as high titers of neutralizing activity to the mutant viruses (E. Yuste et al., J. Virol. 82:12472–12486, 2008). However, whether antibodies that recognize these underlying peptides have neutralizing activity has not been directly demonstrated. Here we describe the isolation and characterization of three gp41-specific monoclonal antibodies (4G8, 6G8, and 7D6) from one of these mutant-infected monkeys. All three antibodies reacted with mutant gp41 from viral particles and also with peptides corresponding to mutated sequences. Slight differences in peptide specificities were observed among the three antibodies. Sequence analysis revealed that the heavy chains of all three antibodies were derived from the same germ line heavy-chain segment (IGHV4-59*01), but they all had very different sequences in complementarity-determining region 3. The light chains of all three antibodies were very closely related to one another. All three antibodies had neutralizing activity to mutant viruses deficient in gp41 carbohydrate attachment, but they did not neutralize the parental SIVmac239. These results demonstrate unambiguously that antibodies with specificity for peptide sequences underlying gp41 carbohydrates can effectively neutralize SIV when these carbohydrates are absent. However, the presence of these gp41 carbohydrates effectively shields the virus from antibodies that would otherwise neutralize viral infectivity.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

Effector Function Activities of a Panel of Mutants of a Broadly Neutralizing Antibody against Human Immunodeficiency Virus Type 1

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcgammaR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcgammaR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.     

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.     

Synergistic Neutralization of Simian-Human Immunodeficiency Virus SHIV-vpu+ by Triple and Quadruple Combinations of Human Monoclonal Antibodies and High-Titer Anti-Human Immunodeficiency Virus Type 1 Immunoglobulins

We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu⁺). This virus encodes the HIV-1 strain IIIB env, tat, rev, and vpu genes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27–55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC₉₀], 2.0 μg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of <1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC₉₀, 1.8 μg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1.     

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.