Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity.

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membrane-proximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate. Here, we performed antibody-virus "washout experiments" using both lab-adapted and a panel of clade B primary isolates to analyze MPER accessibility. The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the "static" spike. However, for more neutralization-resistant viruses, the 2F5 and 4E10 antibodies could neutralize only under the "no antibody-virus wash" conditions, implying that the MPER epitopes were not accessible prior to receptor engagement. Accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format. These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.

Vaccines and the induction of functional antibodies: time to look beyond the molecules of natural infection?

Infection with some pathogens induces weak functional antibody responses that are non-protective, and there has been some skepticism about a role for antibodies in vaccine design. However, newer data show that antibodies can protect against infection with these pathogens, and new methods to elicit production of functional antibodies should be sought.

Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1.

How well antibodies can protect against disease due to HIV-1 infection remains a pivotal but unresolved issue with important implications for vaccine design and the use of prophylactic antibody to prevent infection after accidental exposure to the virus and to interrupt transmission of virus from mother to child. Strong doubts about the possible utility of antibodies in vivo have been raised because of the relative resistance of primary viruses to antibody neutralization in vitro. Primary viruses are likely to be close to the viruses transmitted during natural infection in humans. Vaccine studies have been of little value in assessing antibody efficacy in vivo because none of the strategies described to date have elicited significant neutralizing antibody responses to primary viruses. Passive immunization studies are similarly hindered by the paucity of reagents able to neutralize primary viruses effectively and a single study has suggested some benefit. Here we describe experiments to explore the ability of passive antibody to protect against primary virus challenge in hu-PBL-SCID mice. In this model, severe combined immunodeficient (SCID) mice are populated with human peripheral blood mononuclear cells (PBMCs) and infected with HIV-1. We find that the potent neutralizing human monoclonal antibody IgG1b12 at high dose is able to completely protect even when given several hours after viral challenge. The results are encouraging for antibody-based postexposure prophylaxis and support the notion that antibody induction could contribute to an effective vaccine.

Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization.

Antibody-mediated neutralization of human immunodeficiency virus type-1 (HIV-1) is thought to function by at least two distinct mechanisms: inhibition of virus-receptor binding, and interference with events after binding, such as virus-cell membrane fusion. Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells. A correlation between the inhibition of virus binding and virus neutralization was observed for soluble CD4 and all anti-gp120 antibodies, indicating that this is a major mechanism of HIV neutralization. By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction. However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody.

The ability of antibodies to neutralize diverse primary isolates of human immunodeficiency virus-type 1 in vitro has been questioned, with implications for the likely efficacy of vaccines. A recombinant human antibody to envelope glycoprotein gp120 was generated and used to show that primary isolates are not refractory to antibody neutralization. The recombinant antibody neutralized more than 75 percent of the primary isolates tested at concentrations that could be achieved by passive immunization, for example, to interrupt maternal-fetal transmission of virus. The broad specificity and efficacy of the antibody implies the conservation of a structural feature on gp120, which could be important in vaccine design.

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.

Antibody: the flexible adaptor molecule.

Antibodies are flexible adaptor molecules linking target and potential killer i.e. antigen and effector. With the discovery of effector binding sites on antibodies we can begin to visualize at the molecular level how this adaptor role is fulfilled.

Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage.

Phage display of antibody libraries has been widely used for over a decade to generate monoclonal antibodies. Yeast display has been developed more recently. Here the two approaches were directly compared using the same HIV-1 immune scFv cDNA library expressed in phage and yeast display vectors and using the same selecting antigen (HIV-1 gp120). Yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting all the scFv identified by phage display and twice as many novel antibodies. Positive phage display selection appeared to largely reflect those antibodies that as phage-scFv gave the highest signal in phage ELISAs assessing antigen binding. This signal is thought to reflect the efficiency of expression of folded scFv at the phage surface. Increased access to immune repertoires may increase the rescue of novel antibodies of therapeutic or analytical value that often form a minor part of a typical antibody response.

CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range.

We describe the investigation of methodologies for the creation of very high affinity human antibodies. The high affinity human antibody b4/12 was optimized for its affinity to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1). Five libraries of b4/12 were constructed by saturation mutagenesis of complementarity-determining regions (CDRs). Libraries of antibody Fab fragments were displayed on the surface of filamentous phage and selected in vitro for binding to immobilized gp120. Sequential and parallel optimization strategies of CDRs were examined. The sequential CDR walking strategy consistently yielded b4/12 variants of improved affinity in each of the four different optimization sequences examined. This resulted in a 96-fold improvement in affinity. Additivity effects in the antibody combining site were explored by combining independently optimized CDRs in the parallel optimization strategy. Six variants containing optimized CDRs were constructed. Improvement of affinity based on additivity effects proved to be unpredictable but did lead to a modest improvement in affinity. Indeed, only one of the six combinations demonstrated additivity. The highest affinity Fab prepared using this strategy was improved 420-fold in affinity. The affinity of this Fab was 15 pM as compared to 6.3 nM for b4/12. Examination of the kinetics of Fab binding to gp120 revealed that improvements in affinity were dominated by a slowing of the off-rate of the Fab. The methodology presented here provides a route for the improvement of the affinities of antibodies typical of tertiary immune responses into the picomolar range. Such improvements may have profound effects on the utility of antibodies as therapeutic and prophylactic agents.

Mapping the protein surface of human immunodeficiency virus type 1 gp120 using human monoclonal antibodies from phage display libraries.

Panels of hybridoma-derived monoclonal antibodies against diverse epitopes are widely used in defining protein surface topography, particularly in the absence of crystal or NMR structural information. Here we show that recombinant monoclonal antibodies from phage display libraries provide a rapid alternative for surface epitope mapping. Diverse epitopes are accessed by presenting antigen to the library in different forms, such as sequential masking of epitopes with existing antibodies or ligands prior to selection and selection on peptides. The approach is illustrated for a recombinant form of the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 which has been extensively mapped by rodent and human monoclonal antibodies derived by cellular methods. Human recombinant Fab fragments to most of the principal epitopes on gp120 are selected including Fabs to the C1 region, a C1/C5 epitope, a C1/C2 epitope, the V2 loop, the V3 loop and the CD4 binding domain. In addition an epitope linked to residues in the V2 loop and CD4 binding domain is identified. Most of these specificities are associated with epitopes presented poorly on native multimeric envelope, consistent with the notion that these antibodies are associated with immunization by forms of gp120 differing in conformation from that found on whole virus or infected cells.

Neutron scattering studies of subcomponent C1q of first component C1 of human complement and its association with subunit C1r2C1s2 within C1.

Neutron scattering studies are reported on subcomponent C1q of component C1 of human complement, and on C1, the complex of C1q with subunit C1r2C1s2. For C1q, the molecular weight was determined as 460,000. The radius of gyration at infinite contrast RC is 12.8 nm. The RC values for the proteolytically cleaved forms of C1q, namely the heads and the stalks, are 1.5 to 2 nm and 11 nm, respectively, and thus the axis-to-arm angle of C1q is estimated at 45 degrees. Neutron data for subunit C1r2C1s2 are published elsewhere. The neutron data on C1 lead to an RC value of 12.6 nm for proenzymic C1 and a molecular weight of 820,000. The wide-angle scattering curve of C1q exhibits a minimum at Q = 0.28 nm-1 and a maximum at 0.39 nm-1; on the addition of C1r2C1s2, this minimum disappears. The neutron data on C1 indicate that C1q and C1r2C1s2 have complexed with a large conformational change in one or both parts. No conformational changes can be detected on the activation of C1 by this method.

Antibody binding defines a structure for an epitope that participates in the PrPC-->PrPSc conformational change.

The X-ray crystallographic structures of the anti-Syrian hamster prion protein (SHaPrP) monoclonal Fab 3F4 alone, as well as the complex with its cognate peptide epitope (SHaPrP 104-113), have been determined to atomic resolution. The conformation of the decapeptide is an Omega-loop. There are substantial alterations in the antibody combining region upon epitope binding. The peptide binds in a U-shaped groove on the Fab surface, with the two specificity determinants, Met109 and Met112, penetrating deeply into separate hydrophobic cavities formed by the heavy and light chain complementarity-determining regions. In addition to the numerous contacts between the Fab and the peptide, two intrapeptide hydrogen bonds are observed, perhaps indicating the structure bound to the Fab exists transiently in solution. This provides the first structural information on a portion of the PrP N-terminal region observed to be flexible in the NMR studies of SHPrP 90-231, SHaPrP 29-231 and mouse PrP 23-231. Antibody characterization of the antigenic surfaces of PrPC and PrPSc identifies this flexible region as a component of the conformational rearrangement that is an essential feature of prion disease.

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

Site directed mutagenesis of the complement C5a receptor--examination of a model for its interaction with the ligand C5a.

C5a is a 74 amino acid peptide cleaved from the fifth component of the complement system after activation of either the alternative or classical pathways. It is a potent chemoattractant for neutrophils and monocytes binding to identical receptors on the cell surface. Following the cloning of the cDNA encoding for the human complement C5a receptor, revealing it to be a member of the rhodopsin superfamily of G-protein coupled receptors, a model for the interaction of the C5a receptor with its ligand was proposed, the structure for the receptor being modelled on that of the well defined receptor bacteriorhodopsin. In this model two key residues of the receptor, aspartate82 and either glutamate179 or glutamate 180 were proposed to make up part of the binding site for C5a, acting as counter ions for arginine74 and arginine40, respectively of the C5a molecule. Replacement of aspartate82, glutamate179 and glutamate180 of the C5a receptor with asparagine and glutamine, respectively was shown to have little effect on the dissociation constant of the receptor as detected by Scatchard analysis and competitive binding assays. Hence this modus operandi for the interaction of C5a with its receptor can be rejected.

Localisation of the monocyte-binding region on human immunoglobulin G.

Earlier studies, which provided indirect evidence for the involvement of the C gamma 2 domain of human immunoglobulin G (IgG) in human immunoglobulin G (IgG) in human monocyte binding, have been extended to further localise the site of interaction on human IgG. A number of IgGs from several different species and fragments of human IgGs were assayed for ability to inhibit the interaction of radio-labelled human IgG and the human monocyte. By comparison of the amino-acid sequences of those IgGs found to exhibit relatively tight, intermediate or weak binding to human monocyte Fc receptors we are able to postulate a possible monocyte-binding site on human IgG. In addition, the results have implications for the applicability of monoclonal antibodies and antisera when used in the presence of human monocytes and possibly macrophages.