Role of HIV membrane in neutralization by two broadly neutralizing antibodies

Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.     

Mouse marginal zone B cells harbor specificities similar to human broadly neutralizing HIV antibodies

A series of potent, broadly neutralizing HIV antibodies have been isolated from B cells of HIV-infected individuals. VRC01 represents a subset of these antibodies that mediate neutralization with a restricted set of IGHV genes. The memory B cells expressing these antibodies were isolated years after infection; thus, the B-cell subpopulation from which they originated and the extent of participation in the initial HIV antibody response, if any, are unclear. Here we evaluated the frequency of anti-gp120 B cells in follicular (FO) and marginal zone (MZ) B-cell compartments of naïve WT mice and comparable human populations in uninfected individuals. We found that in non–HIV-exposed humans and mice, the majority of gp120-reactive B cells are of naïve and FO phenotype, respectively. Murine FO B cells express a diverse antibody repertoire to recognize gp120. In contrast, mouse MZ B cells recognize gp120 less frequently but preferentially use IGHV1-53 to encode gp120-specific antibodies. Notably, IGHV1-53 shows high identity to human IGHV1-2*02, which has been repeatedly found to encode broadly neutralizing mutated HIV antibodies, such as VRC01. Finally, we show that human MZ-like B cells express IGHV1-2*02, and that IGHV1-53 expression is enriched in mouse MZ B cells. These data suggest that efforts toward developing an HIV vaccine might consider eliciting protective HIV antibody responses selectively from alternative B-cell populations harboring IGHV gene segments capable of producing protective antibodies.     

Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Vaccines are the most important medical countermeasure for protecting entire populations against viruses, of which smallpox and measles vaccines are successful examples. In fact, a safe and effective HIV vaccine is considered to be the best way to end the global AIDS epidemic (1). However, how to produce a universal vaccine for highly antigenically variable viruses like HIV is a daunting and yet unsolved problem. The high variability of this virus allows it to elude the immune system, making the produced antibodies ineffective; that is, they are generally specific for a given strain of the virus but not for other strains resulting from mutation. In some cases, HIV-infected patients can elicit antibodies that can recognize and neutralize a broad range of different viral strains (2, 3). These broadly neutralizing antibodies (bnAbs) usually take a long time to appear naturally in infected patients and then only in a subset of such individuals.The reason that bnAbs can arise is that even highly variable pathogens have regions with a well-defined, relatively conserved structure, which is required for their function. In HIV, entry depends on the trimeric spike exposed on the external lipid membrane of the virion, a heterotrimer formed by the gp120 and gp41 glycoproteins produced by posttranslational cleavage of a gp160 precursor. This protein binds to the CD4 coreceptor on CD4 T lymphocytes during HIV infection, and it has some relatively conserved regions that can be used as a target for bnAbs. Indeed, many bnAbs target the CD4 binding site (CD4bs) (4–8). If naive B cells that can bind to one of these relatively conserved regions can be expanded upon exposure to different variants of the virus, antibodies could evolve to better recognize the conserved portions, while avoiding the variable ones. The resulting antibodies can acquire breadth in this way, thereby becoming bnAbs. A successful vaccine would contain immunogens that can guide the immune system to produce bnAbs, rather than strain-specific antibodies.In the past, numerous approaches for the development of an effective HIV vaccine have been tried. They include the use of cleverly chosen natural HIV proteins, the design of a consensus (9) or “center-of-tree” (10) antigens, and the creation of a mosaic protein from different HIV strains (11). All these methods used a single optimized antigen in the vaccine and were shown to be ineffective at eliciting bnAbs (12, 13). One possible reason for this is that, when exposed to a single antigen, the immune system will produce antibodies specific for that particular antigen, and neutralization escape variants can easily develop. A possible solution is to use more than one antigen in a vaccination protocol. This raises a number of questions: How many antigens are necessary? How different should they be from each other? And in what temporal order should they be administered? Answering such questions is far from trivial, in particular due to the limited mechanistic understanding of affinity maturation (AM) in vivo. Another problem is that bnAbs have an unusually high number of somatic mutations, not only in the complementarity-determining regions (CDRs) but also in the immunoglobulin framework regions (7, 14). Recent computational data on the flexibility of the antibody and the need for framework mutations in the simulated AM showed how important it is for a vaccination protocol to have a specific antigen that can prime a good antibody precursor B cell receptor (BCR) (15). Moreover, it has been shown that putative precursors of known classes of bnAbs are generally not able to neutralize HIV or recognize envelope (Env), often due to clashes of the antibody with the glycosylation shield that protects the HIV Env protein (8, 16–18). For example, VRC01-class bnAbs are known to introduce a deletion or a mutation to a flexible glycine in the CDRL1 loop to avoid the glycan at N276 (19, 20).The above discussion led to the proposal of a vaccination strategy consisting of three steps. First, a special purpose antigen is used to activate the correct naïve or precursor B cell (17, 21). Since this precursor will generally not bind to native HIV, as a second step, one or more antigens are used as intermediates to induce somatic mutations and to allow recognition of the native virus. In the third step, one or more antigens are used in a mixture or in sequence to increase the breadth of the antibody population (19, 22, 23). Implementations of the first and second steps have already been shown to be promising in experiments (16, 21, 24–27). However, much less is known about the third step. Some insights into this question can be obtained by in silico simulations of AM. Using coarse-grained models, it has been shown that, while administering a single mixture containing multiple antigens may induce too much frustration to lead to bnAbs formation, a sequential approach, in which antigens are administered one after another, seems to be more effective (23). It was also observed that the number of antigens required in a mixture is correlated with their sequence dissimilarity, and optimal breadth is obtained at an optimal number of antigens and dissimilarity (28). Given the coarse-grained nature of these studies, the actual antigen sequences to use in experiments cannot be obtained from them.In this work, we focus on the third step of the proposed vaccination protocol. In particular, we derive a set of empirical rules and protocols to select an optimal panel of antigens to maximize the breadth of the produced antibodies upon AM. To be able to do so, it is essential to understand, at an atomistic level of detail, the role of each antigen amino acid in the antibody/antigen interaction. This aspect will be presented in the next section based on an analysis of the available crystallographic structures of bnAbs bound to the gp160 Env glycoprotein. However, the structures do not provide information concerning HIV stability and function. For example, generating antigen sequences by introducing purely random mutations will likely lead to sequences that are lethal for the virus and/or are not representative of HIV in vivo. To overcome this problem, it is useful to consider the structural data together with a model of the gp160 fitness landscape (29), which is a measure of the ability of HIV to tolerate mutations in its gp160 sequence to escape immune pressure. Structural and fitness information together provide a classification of the antibody/antigen interface and indicate the residues to mutate and the amino acids that are more probable at those positions.While this analysis helps to reduce the number of antigen sequences to consider by highlighting the “hot spots” of antibody/antigen binding, it leaves open the question of how to select a combination of antigen sequences for use in a vaccine. Given rules of optimal sequence dissimilarity and optimal fitness according to the HIV landscape, a Pareto frontier approach will be described. It is able to select, from all possible panels of antigen sequences, the few that are predicted to best elicit antibodies with a broad activity spectrum. Experimental evidence of the viability of the designed antigens and of their immunogenic properties is presented in the final section.     

Difficulties in eliciting broadly neutralizing anti-HIV antibodies are not explained by cardiolipin autoreactivity

OBJECTIVE: In a recent report [Haynes et al. Science 2005; 308:1906-1908], difficulties in eliciting broadly neutralizing antibodies to HIV were linked to the binding of prototypic broadly neutralizing monoclonal antibodies to autoantigens and in particular, to the binding of two antigp41 antibodies, 2F5 and 4E10, to the autoantigen cardiolipin. We used a number of assays to understand whether 2F5 and 4E10 are autoreactive, polyreactive, or have a generalized affinity for lipids that may facilitate recognition of their membrane proximal epitopes. METHODS: 2F5 and 4E10 were evaluated for autoreactivity using diagnostic assays developed to detect serum antibodies associated with antiphospholipid syndrome (APS). As an indication of polyreactivity, we measured the binding of 2F5 and 4E10 to liposomal bilayers of differing composition using surface plasmon resonance (SPR) spectroscopy and to protein microarrays using biochip technology. RESULTS: 2F5 showed completely negative results in the APS and SPR studies, indicating that it is neither autoreactive nor absolutely requires phospholipid binding for epitope recognition. In contrast, 4E10 bound to more than one lipid and showed weak activity in the APS studies. The activity displayed by 4E10 more closely resembles that of antiphospholipid antibodies elicited during many infections than that of autoimmune APS antibodies, at variance with the notion that difficulites in eliciting 4E10-like antibodies can be attributed to tolerance mechanisms. The microarray studies further indicated that broadly neutralizing anti-HIV mAb are not exceptionally polyreactive. CONCLUSION: These results suggest that autoantigen mimicry cannot be reliably invoked as a general mechanism for HIV immune evasion.     

A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

Most antibodies induced by HIV-1 are ineffective at preventing initiation or spread of infection because they are either nonneutralizing or narrowly isolate-specific. Rare, "broadly neutralizing" antibodies have been detected that recognize relatively conserved regions on the envelope glycoprotein. Using stringently characterized, homogeneous preparations of trimeric HIV-1 envelope protein in relevant conformations, we have analyzed the molecular mechanism of neutralization by two of these antibodies, 2F5 and 4E10. We find that their epitopes, in the membrane-proximal segment of the envelope protein ectodomain, are exposed only on a form designed to mimic an intermediate state during viral entry. These results help explain the rarity of 2F5- and 4E10-like antibody responses and suggest a strategy for eliciting them.     

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1–infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man₅GlcNAc₂ glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.It is widely believed that a key characteristic of an effective HIV-1 vaccine would be its ability to induce broadly neutralizing antibodies (BnAbs). Known BnAbs have been shown to target conserved HIV-1 envelope (Env) regions including glycans, the glycoprotein 41 (gp41) membrane-proximal region, the gp120 variable loop 1/2 (V1V2), and the CD4 binding site (CD4bs) (1–7). Most mature BnAbs have one or more unusual features such as long heavy-chain third complementarity-determining regions, polyreactivity for non–HIV-1 antigens, and high levels of somatic mutations (2, 7, 8). In particular, CD4bs BnAbs have extremely high levels of somatic mutations, suggesting complex or prolonged maturation pathways (2–5). Adding to the challenge has been the difficulty in achieving binding of proposed antigens to germ-line or unmutated common ancestors (UCAs). Binding to BnAb UCAs would be a desirable characteristic for putative immunogens intended to induce BnAbs (5, 9–13).Immunization of humans with Env proteins has not resulted in high plasma titers of BnAbs (14, 15). Rather, dominant strain-specific neutralizing epitopes have selectively been induced. This was most clearly seen in the ALVAC/AIDSVAX RV144 HIV-1 vaccine efficacy trial, in which Env immunogens 92TH023 and A244 CRFAE_01 gp120s both expressed a dominant linear V2 epitope and bound with high-nanomolar affinity to the glycan-dependent V1V2 BnAbs PG9 and CH01 (16). Although both linear and glycan-dependent V2 epitopes were expressed on the A244 immunogen, the dominant V2 plasma antibody responses in this trial were targeted to linear V2 epitopes and not to the glycan-dependent BnAb epitope (14–16). A series of mAbs, the prototype of which is the mAb CH58, has been isolated from RV144 vaccines and shown to bind to linear V2 epitopes that include lysine 169 (16). However, they are strain-specific and only neutralize laboratory-adapted but not primary isolate HIV-1 strains (16). Although PG9 and CH01 V1V2 BnAbs also bind to V2 K169 and surrounding amino acids, they also bind to high-mannose glycans at N¹⁵⁶ and N¹⁶⁰ (17). Crystal structures of the CH58 antibody bound to V2 peptides demonstrated the V2 structure around K169 to be helical (16), whereas the crystal structure of the PG9 antibody with a V1V2 scaffold showed the same polypeptide region in a β-strand conformation (17).The rationale that undergirded the studies described below envisioned that an optimal immunogen for the V1V2 BnAb peptide–glycan envelope region would be one that presented a chemically homogeneous entity that binds to V1V2 BnAbs with high affinity. In addition, an optimal immunogen for the V1V2 BnAb site would be one that binds with high affinity to V1V2 BnAb UCAs. Recently, in a preliminary disclosure, we described chemically synthesized glycopeptides of the HIV-1 Env V1V2 148–184 aa region with Man₃GlcNAc₂ or Man₅GlcNAc₂ glycan units at N¹⁵⁶ and N¹⁶⁰ (18). It was found that these homogeneous glycopeptide constructs with oligomannose units bound avidly to the V1V2 BnAb PG9. In this study, we report that the disulfide-linked dimeric forms of these glycopeptides bound preferentially to V1V2 BnAb mature antibodies (PG9 and CH01) over the V2 strain-specific mAb CH58, to which the binding was minimal. Importantly, the V1V2 peptide–glycans also bound to both PG9 and CH01 V1V2 BnAb UCAs, thus providing a strong rationale for their evaluation as experimental immunogens.     

HIV type 1 Env precursor cleavage state affects recognition by both neutralizing and nonneutralizing gp41 antibodies

HIV-1 is relatively resistant to antibody-mediated neutralization; however, rare antibodies to the exterior envelope glycoprotein, gp120, and the transmembrane glycoprotein, gp41, can neutralize a broad array of isolates. Two antibodies, 2F5 and 4E10, are directed against the gp41 membrane proximal external region (MPER); however, the kinetic neutralization signature of these antibodies remains unresolved. Previously, we reported that the fully cleaved, cell surface envelope glycoproteins (Env) derived from the primary isolate, JR-FL, are well recognized exclusively by gp120-directed neutralizing ligands and not by nonneutralizing gp120 antibodies. However, the gp120 nonneutralizing antibodies can recognize HIV spikes that are rendered fully cleavage defective by site-directed mutagenesis. Here, we extended such analysis to gp41 neutralizing and nonneutralizing antibodies and, relative to the rules of gp120-specific antibody recognition, we observed marked contrasts. Similar to gp120 recognition, the nonneutralizing gp41 cluster 1 or cluster 2 antibodies bound much more efficiently to cleavage-defective spikes when compared to their recognition of cleaved spikes. In contrast to gp120 neutralizing antibody recognition, the broadly neutralizing gp41 antibodies 2F5 and 4E10, like the nonneutralizing gp41 antibodies, did not efficiently recognize the predominantly cleaved, primary isolate JR-FL spikes. However, if the spikes were rendered cleavage defective, recognition by both the neutralizing and nonneutralizing ligand markedly increased. CD4 interaction with the cleaved spikes markedly increased recognition by most nonneutralizing gp41 antibodies, whereas such treatment had a minimal increase of 2F5 and 4E10 recognition. These data indicate again the profound influence that cleavage imposes on the quaternary packing of primary isolate spikes and have important implications for soluble trimer candidate immunogens.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.     

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.     

Designing immunogens to elicit broadly neutralizing antibodies to the HIV-1 envelope glycoprotein

To date HIV-1 vaccines have not been able to elicit potent, long lasting, and broadly neutralizing antibodies to the virus. Our knowledge of HIV envelope glycoprotein (Env) structure/function and the existence of a handful of broadly neutralizing antibodies is guiding rational immunogen design. We review here the potential targets on the HIV Env (the glycan shield, the CD4 binding site, the coreceptor binding site, Env fusion intermediates, and the membrane proximal region) and their associated rational immunogen design strategies. Moreover, we discuss immune dampening and immune refocusing strategies designed to counter immunodominant, decoy responses generated by the virus. In this regard, an immunogen design strategy of "in vitro de-evolution" is presented, which begins to distill the HIV Env to its most critical, core functional domains. While we are beginning to have some understanding as to where we would like out immune system to go, we find that our immune repertoire may actually have limits that preclude successful completion of the task at hand. The repertoire limits appear to be a byproduct of autoantibody tolerance mechanisms and the complex structural requirements for effective, potent broadly neutralizing antibodies. Nevertheless, the hope is that through novel insights and creative solutions that we will be able to design immunogens capable of eliciting broadly neutralizing antibodies to the HIV envelope glycoprotein.     

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.     

Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains

Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus−antibody “arms race.” Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus−antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.Any candidate HIV vaccine will need to induce an immune response effective for a wide range of potential variants. Approximately 15–25% of chronically infected HIV patients develop such broadly neutralizing antibodies (bnAbs) (1–3). These target four distinct epitope regions on the envelope glycoprotein—the V1−V2 loop, the CD4 binding site, a set of residues near the base of the V3 loop on gp120, and the membrane proximal external segment of gp41 (4, 5).A protective B-cell immune response requires antibody affinity maturation—selective proliferation of cells with antibody variable domain mutations that enhance antigen affinity (6–8). It is possible to study this process by isolating individual B cells from infected or vaccinated donors and cloning the recombined heavy- and light-chain variable regions of the antibodies (9–11). Understanding those patterns of antibody affinity maturation that give rise to the development of bnAbs could inform design of immunogens with increased likelihood of maturation along otherwise subdominant pathways (12).Data now available from donors followed from the onset of HIV infection until the development of bnAbs clarify some of these issues. Recent findings from an African patient, CH505, who was followed from acute HIV-1 infection through bnAb development, illustrate the interplay, or arms race, between virus evolution and antibody maturation (13). The structure of a gp120 core, which excludes a number of variable loops and glycosylation sites, has been determined in complex with the antigen-binding fragment (Fab) of a mature, CD4-binding-site broadly neutralizing antibody, CH103, derived from the same individual (13). Fixation in this lineage of mutations in residues in both partners that lie outside their interface indicate that there may be factors driving affinity maturation that are not evident from the crystal structure.Analysis of this CH103 antibody lineage (Fig. 1) indicates that the transmitter founder (T/F) virus binds with high affinity to the progenitor, or unmutated common ancestor (UCA), of the lineage but that virus envelopes (Envs) from later time points do not (13). Furthermore, the UCA has no detectable affinity for heterologous Envs, whereas the CH103 lineage antibodies show increasing affinity for heterologous Envs as maturation proceeds, and the more mature members of the lineage are more potent neutralizers of heterologous viruses (13). For example, Env binding and neutralization of a clade B virus (B.63521) both increased substantially as the antibody matured from UCA to a later intermediate, I2 (13). Thus, we have extensive sequence and biochemical information about the antibody−virus arms race in the CH505 individual.Open in a separate windowFig. 1.CH103 B-cell clonal lineage tree. The UCA is shown on the left, with increasingly mature antibodies on the right. The top branch resulted in bnAbs (blue). Branches that did not lead to broadly neutralizing antibodies are excluded from the diagram.To investigate the structural correlates of viral escape and of antibody affinity maturation in the CH103 lineage, we determined crystal structures of Fabs derived from the UCA and intermediate antibodies, I3 and I2. The structures show that the relative orientation of the heavy- and light-chain variable domains shifted during affinity maturation, changing the way the antibodies encountered gp120. The mutations that produced the shift occurred after the appearance of insertions into the gp120 V5 loop in Envs of the autologous virus. Binding measurements show that these insertions block binding by the UCA and that the shift in relative variable-domain orientation restores affinity. The shift depends largely on mutations in the light chain, and the timing of the relevant mutations appears to correlate with development of broad neutralization. Our analysis illustrates the importance of considering mutations outside the antibody−antigen interface when interpreting the contribution of mutations accumulated during antibody affinity maturation.     

HIV neutralizing antibodies: development and association with HIV related disease

Serum neutralization was measured in 72 sera collected during a 5.5-year period from 10 HIV infected individuals. Neutralizing antibodies (NA) were present in all sera. NA titers ranging from greater than 40 to greater than 640 were detected in sera from 4 patients, who all remained healthy and further an increase with time of NA was observed in these 4 patients. Progression to disease was observed in 3 persons with NA titers less than or equal to 40 who also lacked or lost anti-gag antibodies. Two of these patients were HIV antigenaemic prior to development of disease, whereas antigen was not detected in the remaining 7 healthy persons. A weak positive correlation (R(S) = +0.643, p less than 0.001) was found between titers of NA and whole virus antibody (WVA), with the ratios between titers (NA titer/WVA titer) varying a 100-fold. The results suggest that the presence of NA in some cases might be related to a healthy carrier state and that a combination of low titer NA with decline of anti-gag antibodies and/or HIV antigenaemia is associated with progression to clinical disease.