Human Immunodeficiency Virus Type 2 (HIV-2)/HIV-1 Envelope Chimeras Detect High Titers of Broadly Reactive HIV-1 V3-Specific Antibodies in Human Plasma

Deciphering antibody specificities that constrain human immunodeficiency virus type 1 (HIV-1) envelope (Env) diversity, limit virus replication, and contribute to neutralization breadth and potency is an important goal of current HIV/AIDS vaccine research. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 scaffolds may provide a sensitive, biologically functional context by which to quantify specific antibody reactivities even in complex sera. Here, we describe a novel HIV-2 proviral scaffold (pHIV-2_KR.X7) into which we substituted the complete variable region 3 (V3) of the env gene of HIV-1_YU2 or HIV-1_Ccon to yield the chimeric proviruses pHIV-2_KR.X7 YU2 V3 and pHIV-2_KR.X7 Ccon V3. These HIV-2/HIV-1 chimeras were replication competent and sensitive to selective pharmacological inhibitors of virus entry. V3 chimeric viruses were resistant to neutralization by HIV-1 monoclonal antibodies directed against the CD4 binding site, coreceptor binding site, and gp41 membrane proximal external region but exhibited striking sensitivity to HIV-1 V3-specific monoclonal antibodies, 447-52D and F425 B4e8 (50% inhibitory concentration of [IC₅₀] <0.005 μg/ml for each). Plasma specimens from 11 HIV-1 clade B- and 10 HIV-1 clade C-infected subjects showed no neutralizing activity against HIV-2 but exhibited high-titer V3-specific neutralization against both HIV-2/HIV-1 V3 chimeras with IC₅₀ measurements ranging from 1:50 to greater than 1:40,000. Neutralization titers of B clade plasmas were as much as 1,000-fold lower when tested against the primary HIV-1_YU2 virus than with the HIV-2_KR.X7 YU2 V3 chimera, demonstrating highly effective shielding of V3 epitopes in the native Env trimer. This finding was replicated using a second primary HIV-1 strain (HIV-1_BORI) and the corresponding HIV-2_KR.X7 BORI V3 chimera. We conclude that V3 is highly immunogenic in vivo, eliciting antibodies with substantial breadth of reactivity and neutralizing potential. These antibodies constrain HIV-1 Env to a structure(s) in which V3 epitopes are concealed prior to CD4 engagement but do not otherwise contribute to neutralization breadth and potency against most primary virus strains. Triggering of the viral spike to reveal V3 epitopes may be required if V3 immunogens are to be components of an effective HIV-1 vaccine.     

Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens.     

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

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

Stabilized HIV-1 Envelope Glycoprotein Trimers Lacking the V1V2 Domain,Obtained by Virus Evolution

The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on the induction of humoral immunity, but the mechanisms the virus has evolved to limit the induction and binding of neutralizing antibodies (NAbs) constitute substantial obstacles. Conserved neutralization epitopes are shielded by variable regions and carbohydrates, so one strategy to increase their exposure and, it is hoped, their immunogenicity is to delete the overlying variable loops. However, deleting the variable regions from Env trimers can be problematic, because hydrophobic patches that are normally solvent-inaccessible now become exposed, causing protein misfolding or aggregation, for example. Here, we describe the construction and characterization of recombinant gp140 trimers lacking variable domains 1 and 2 (ΔV1V2). The design of the trimers was guided by HIV-1 evolution studies that identified compensatory changes in V1V2-deleted but functional Env proteins (Bontjer, I., Land, A., Eggink, D., Verkade, E., Tuin, K., Baldwin, C., Pollakis, G., Paxton, W. A., Braakman, I., Berkhout, B., and Sanders, R. W. (2009) J. Virol. 83, 368–383). We now show that specific compensatory changes improved the function of ΔV1V2 Env proteins and hence HIV-1 replication. The changes acted by reducing the exposure of a hydrophobic surface either by replacing a hydrophobic residue with a hydrophilic one or by covering the surface with a glycan. The compensatory changes allowed the efficient expression of well folded, soluble gp140 trimers derived from various HIV-1 isolates. The evolved ΔV1V2 Env viruses were extremely sensitive to NAbs, indicating that neutralization epitopes are well exposed, which was confirmed by studies of NAb binding to the soluble ΔV1V2 gp140 trimers. These evolved ΔV1V2 trimers could be useful reagents for immunogenicity and structural studies.     

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.     

Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody

The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 Å. In the complex, the peptide adopts a β-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alanine-scanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.     

A Single Residue within the V5 Region of HIV-1 Envelope Facilitates Viral Escape from the Broadly Neutralizing Monoclonal Antibody VRC01

VRC01, a broadly neutralizing monoclonal antibody, is capable of neutralizing a diverse array of HIV-1 isolates by mimicking CD4 binding with the envelope glycoprotein gp120. Nonetheless, resistant strains have been identified. Here, we examined two genetically related and two unrelated envelope clones, derived from CRF08_BC-infected patients, with distinct VRC01 neutralization profiles. A total of 22 chimeric envelope clones was generated by interchanging the loop D and/or V5 regions between the original envelopes or by single alanine substitutions within each region. Analysis of pseudoviruses built from these mutant envelopes showed that interchanging the V5 region between the genetically related or unrelated clones completely swapped their VRC01 sensitivity profiles. Mutagenesis analysis revealed that the asparagine residue at position 460 (Asn-460), a potential N-linked glycosylation site in the V5 region, is a key factor for observed resistance in these strains, which is further supported by our structural modeling. Moreover, changes in resistance were found to positively correlate with deviations in VRC01 binding affinity. Overall, our study indicates that Asn-460 in the V5 region is a critical determinant of sensitivity to VRC01 specifically in these viral strains. The long side chain of Asn-460, and potential glycosylation, may create steric hindrance that lowers binding affinity, thereby increasing resistance to VRC01 neutralization.     

Mutation at a Single Position in the V2 Domain of the HIV-1 Envelope Protein Confers Neutralization Sensitivity to a Highly Neutralization-Resistant Virus

Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralization-resistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described α4β7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to α4β7.A major goal in human immunodeficiency virus type 1 (HIV-1) vaccine research is the identification of immunogens able to elicit protective immunity from HIV-1 infection. Results from the recent RV144 clinical trial in Thailand (53) have provided evidence that immunization with vaccines containing the recombinant HIV-1 envelope glycoprotein gp120 (6, 7) can protect humans from HIV infection when incorporated in a prime/boost immunization regimen. Although the level of protection observed in the RV144 trial (31%) was modest, it represents a significant advance in HIV-1 vaccine research and has rekindled the efforts to identify improved subunit vaccine antigens that might achieve even higher levels of protection. In these studies, we have sought to understand the molecular determinants of neutralization sensitivity and resistance in HIV-1 envelope proteins for the purpose of developing improved vaccine antigens.In previous studies (47), we have described a novel method of mutational analysis of the HIV-1 envelope protein, termed swarm analysis, for identification of mutations that confer sensitivity and/or resistance to broadly neutralizing antibodies (bNAbs). This method makes use of the natural amino acid sequence virus variation that occurs in each HIV-infected individual to establish panels of closely related envelope proteins that differ from each other by a limited number of amino acid substitutions. We have previously used this method to identify a novel amino acid substitution in gp41 that conferred sensitivity to neutralization by monoclonal and polyclonal antibodies as well as virus entry inhibitors. In this paper, we describe a mutation in the V2 domain of gp120 that similarly induces a neutralization-sensitive phenotype in an otherwise neutralization-resistant envelope sequence.Previous studies (10, 14, 33, 40, 43, 52, 72, 74) have suggested that sequences in the V2 domain act as the “global regulator of neutralization sensitivity” and confer neutralization resistance by restricting access to epitopes located in the V3 domain, the CD4 binding site, and chemokine receptor binding sites through “conformational masking” of neutralizing epitopes. Deletion of the V2 domain markedly increases neutralization sensitivity (10, 57, 62, 74), and several envelope proteins with V2 domain deletions have been developed as candidate HIV-1 vaccines (5, 42, 61). In this paper, we show that a single substitution of asparagine (N) for aspartic acid (D) at position 179 in the C-terminal portion of the V2 domain (corresponding to position 180 in HXB2 numbering) converts a highly neutralization-resistant virus to a neutralization-sensitive virus with a phenotype similar to that described for V2 domain deletion mutants. Position 179 has recently attracted attention as a critical element of the α4β7 integrin binding site that affects virus tropism to the gut (2). Our results suggest that mutation at position 179 results in a conformational change that increases neutralization sensitivity by exposure of epitopes in both gp120 and gp41 that are normally masked in the trimeric structure of gp160 and thus are unavailable for antibody binding.     

Enhanced Fusion and Virion Incorporation for HIV-1 Subtype C Envelope Glycoproteins with Compact V1/V2 Domains

In infected people, the HIV-1 envelope glycoprotein (Env) constantly evolves to escape the immune response while retaining the essential elements needed to mediate viral entry into target cells. The extensive genetic variation of Env is particularly striking in the V1/V2 hypervariable domains. In this study, we investigated the trade-off, in terms of fusion efficiency, for encoding V1/V2 domains of different lengths. We found that natural variations in V1/V2 length exert a profound impact on HIV-1 entry. Variants encoding compact V1/V2 domains mediated fusion with higher efficiencies than related Envs encoding longer V1/V2 domains. By exchanging the V1/V2 domains between Envs of the same infected person or between two persons linked by a transmission event, we further demonstrated that V1/V2 domains critically influence both Env incorporation into viral particles and fusion to primary CD4 T cells and monocyte-derived dendritic cells. Shortening the V1/V2 domains consistently increased Env incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env incorporation and fusion. Given that in a new host transmitted founder viruses are distinguished by compact Envs with fewer glycosylation sites, our study points to fusion and possibly Env incorporation into virions as limiting steps for transmission of HIV-1 to a new host and suggests that the length and/or the N-glycosylation profile of the V1/V2 domain influences these early steps in the HIV life cycle.     

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.     

Soluble HIV-1 Envelope Immunogens Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low Potency Neutralizing Antibodies

We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses.     

Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

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Trimeric Glycosylphosphatidylinositol-Anchored HCDR3 of Broadly Neutralizing Antibody PG16 Is a Potent HIV-1 Entry Inhibitor

PG9 and PG16 are two quaternary-structure-specific broadly neutralizing antibodies with unique HCDR3 subdomains. Previously, we showed that glycosylphosphatidylinositol (GPI)-anchored HCDR3 subdomains (GPI-HCDR3) can be targeted to lipid rafts of the plasma membrane, bind to the epitope recognized by HCDR3 of PG16, and neutralize diverse HIV-1 isolates. In this study, we further developed trimeric GPI-HCDR3s and demonstrated that trimeric GPI-HCDR3 (PG16) dramatically improves anti-HIV-1 neutralization, suggesting that a stoichiometry of recognition of 3 or 2 HCDR3 molecules (PG16) to 1 viral spike is possible.     

Drift of the HIV-1 Envelope Glycoprotein gp120 toward Increased Neutralization Resistance over the Course of the Epidemic: a Comprehensive Study Using the Most Potent and Broadly Neutralizing Monoclonal Antibodies

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.