Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance

We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (V_HDJ_H) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 V_HDJ_H insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse Igμ chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 V_HDJ_H knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 V_HDJ_H knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery.     

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.     

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The tremendous circulating sequence diversity of HIV-1 and its capacity to evade host immunity pose unique challenges for vaccine design (reviewed in refs. 1, 2). Broadly neutralizing antibodies (bNAbs) identified from HIV-1 patients target conserved vulnerable epitopes on the HIV-1 envelope protein (Env) to prevent HIV-1 infection, and HIV-1 bNAb elicitation has become a major goal for HIV-1 vaccine design (3, 4). Several HIV-1 vulnerable epitopes have been described (5) including the following: the V1V2 apex (6, 7), the CD4-binding site (8–10), the membrane-proximal external region (11, 12), the glycan-V3 region (also known as N332 glycan supersite) (10, 13), the highly glycosylated region at the center of the silent face on the gp120 subunit (14), and the fusion peptide (FP), which is required for viral entry (15–17). Several complementary approaches seek to develop immunogens that elicit broadly neutralizing HIV-1 antibodies, with promising results (4, 18, 19). However, vaccine-elicited HIV-1 antibodies are often either less potent or less broad than many of the bNAbs identified from human patients. There is a pressing need to better understand bNAb developmental pathways and outline the genetic and structural antibody features that can provide enhanced neutralization breadth and potency for HIV-1 vaccines.Clinical data show that broadly neutralizing serum antibodies develop naturally in around 20% of individuals with chronic HIV-1 infection (20, 21) and that a smaller number of individuals show highly potent neutralization (22, 23). bNAbs develop via the accumulation of somatic hypermutations (SHM) and affinity maturation following their initial B-cell selection and expansion. While HIV-1–infected individuals may have high titers against HIV-1 antigens, the rarity of bNAbs suggests that the mutations acquired during bNAb development are correspondingly rare and/or that the mutational pathways to effective bNAb development are explored only in the context of chronic HIV-1 antigen exposure over years of viral infection (24). Evidence that high viral load is correlated with higher HIV-1 serum breadth and potency also suggests that larger numbers of sampled antibody maturation pathways are correlated with broad HIV-1 neutralization (20). A major question for HIV-1 vaccine design is thus how to quickly and effectively induce B-cell maturation to bNAbs from a small number of controlled immunizations.HIV-1 bNAbs are often highly somatically mutated (25–28), and reverting these mutations to antibody germline sequences results in drastic reductions of neutralization breadth and potency (9, 29–31). Complementarity determining regions (CDRs) are known to be mutational hotspots and are often in direct contact with antigen to enable recognition. Antibody framework region (FR) mutations can also modulate neutralization breadth and potency by altering the structural orientation, particularly at heavy:light interface alignments, and by altering the intrinsic flexibility of the paratope (16, 32–35). Not all observed bNAb somatic mutations are required for high neutralization breadth and potency (36), and a better understanding of the critical mutations and antibody structural features (both naturally elicited and vaccine-induced) to improve neutralization breadth and potency would enhance our understanding of bNAb development and guide efficient HIV-1 vaccine strategies (16, 17, 37–41).Among various immunization approaches reported for eliciting broadly neutralizing antibodies (16–19, 38–42), one strategy targets the HIV-1 FP epitope and has elicited 59 and 31% HIV-1 neutralization breadth in rhesus macaques and mice, respectively, against a broad panel of 208 HIV-1 strains (16, 17). Priming the immune response with soluble FP followed by three Env trimer boosts elicited antibodies with FP-targeted trimer recognition and broad HIV-1 neutralization; however, the structural mechanisms that enable effective anti-FP antibody maturation and pathway selection are not fully understood. Several key unanswered questions include the following: 1) What are the structural features of FP recognition vs. trimer recognition that may appropriately guide antibody development?; 2) What critical parameters control virus neutralization of FP-targeting bNAbs?; and 3) How do beneficial mutations fit into the entire landscape of possible mutational pathways, and what fraction of possible mutations provide increased affinity, neutralization breadth, and potency?To address these questions, we characterized the genetic and functional fitness landscape of an anti-FP bNAb and used biophysical and structural techniques to follow the mechanisms of antibody improvement. We implemented these screening strategies using the anti-FP bNAb vFP16.02, a vaccine-elicited antibody that neutralizes ∼30% of HIV-1 viral isolates (16). We applied yeast display and fluorescence-activated cell sorting (FACS) coupled with next-generation sequencing (NGS) to identify single mutations that enhanced binding or fitness to multiple HIV-1 BG505 Env trimer variants (Fig. 1). We mapped possible single mutations by their functional impacts and identified a panel of mutations with enhanced binding affinity and neutralization. Structural analyses of a subset of these mutations provided insights into the mechanisms of enhanced neutralization. These data confirm that several parallel mutational pathways exist for HIV-1 bNAb improvement and underscore the importance of improved Env trimer affinity for enhancing neutralization potency and breadth in HIV-1 vaccine designs.Open in a separate windowFig. 1.Experimental workflow for comprehensive functional analysis of all possible single mutations in an HIV-1 bNAb. SSM libraries were designed for VH and VL regions of the anti–HIV-1 FP bNAb vFP16.02. SSM libraries were cloned into yeast Fab surface display libraries and screened by FACS to determine the functional impact of each possible single mutation. Single-mutation display libraries were sorted for their affinity against a BG505 DS-SOSIP HIV-1 Env trimer with two different FP sequences. Sorted yeast libraries were prepped for NGS to enable quantitative variant tracking across three screening rounds; bioinformatic analyses of NGS data were used to interpret the functional impact of each possible amino acid mutation. Selected variants were characterized for neutralization activity and affinity against soluble FP and against HIV-1 Env. Structural analyses were performed to understand the mechanistic basis of anti–HIV-1 FP bNAb improvement.     

Humanized V(D)J-rearranging and TdT-expressing mouse vaccine models with physiological HIV-1 broadly neutralizing antibody precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions (“N-regions”) by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC V_H1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of V_H1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.

Diverse antibody variable region exons are assembled in developing B cells from Immunoglobulin (Ig) heavy chain (HC) V, D, and J gene segments and from Igκ or Igλ light chain (LC) V and J segments (1). In humans, there are 55 germline HC Vs (V_Hs) and 70 Igκ and Igλ LC Vs. Vs encode most of the HC and LC variable region, including the antigen contact CDR1 and CDR2 sequences that vary among different HC and LC Vs. Ig HC V(D)J recombination occurs at the progenitor (Pro) B cell developmental stage in the fetal liver and in the postnatal bone marrow (2, 3). Ig LC V to J recombination takes place in the subsequent precursor (Pre) B cell developmental stage in these same sites (1). T cell receptor (TCR) variable region exon assembly also occurs in the fetal liver and thymus and then in the postnatal thymus (4, 5). Mice also have similar sets of Ig HC and LC and TCR variable region gene segments as those found in humans and, in general, assemble them in the context of similar developmental processes (6, 7).Primary B cell receptor (BCR) diversity is achieved, in part, by assorting HC and LC Vs along with each of their distinct sets of CDR1 and CDR2 sequences. However, several V(D)J junctional diversification mechanisms play an even greater role in V(D)J diversity generation (8). In this regard, terminal deoxynucleotidyl transferase (TdT), a DNA polymerase that adds nucleotides to 3''DNA ends without a template (9), plays a key role. V(D)J junctional diversity is immensely augmented by TdT-based nontemplated nucleotide additions, referred to as N regions (10), that are added to V(D)J junctions. While N-region addition generates CDR3 length and sequence diversity, it also suppresses recurrent CDR3s resulting from microhomology (MH)-mediated V(D)J joining (10–13). TdT expression is absent during fetal B and T cell development, resulting in less diverse repertoires dominated by variable region exons promoted by recurrent MH-mediated joins (14–21). In contrast, TDT expression diversifies antigen receptor variable region repertoires generated in mouse and human developing B and T cells that develop postnatally, with the notable exception of LC variable region repertoires in mice (10, 22, 23). Thus, while TdT is expressed during LC V(D)J recombination in postnatal human Pre-B cells (24), it is not expressed in postnatal mouse pre-B cells (25, 26), leading to decreased junctional diversity and much more abundant MH-mediated joins in primary mouse LC repertoires compared to those of humans (22, 23). Lack of TdT expression in fetal repertoires also is known to promote recurrent MH-mediated V(D)J junctions, that are not dominant in postnatal repertoires due to TdT expression. Some such recurrent MH-mediated V(D)J joins in fetal T or B cell repertoires generate TCRs or BCRs critical for certain physiological responses (13, 14, 27, 28). However, the potential role of TdT and N regions in promoting specific responses has remained largely unaddressed.VRC01-class bnAb HCs employ human V_H1-2, which encodes residues that contact the HIV-1 envelope protein (Env) CD4 binding site (29–37). VRC01-class LC variable regions are known to be encoded by several Vs; but all are associated with an exceptionally short 5 amino acid (5-aa) CDR3, which avoids steric clash with Env and contributes to Env interaction (29–37). As both requirements can be achieved by V(D)J recombination, they are predicted attributes of primary VRC01-class precursor BCRs. However, inferred primary VRC01-class BCRs lack detectable affinity for naive Envs (38–41). In this regard, following BCR antigen-activation, primary B cells are driven into germinal center (GC) reactions where they undergo rounds of variable region exon somatic hyper-mutation (SHM) followed by selection of SHMs that increase BCR antigen-binding affinity. This process ultimately leads to high-affinity antibody production. Correspondingly, a third VRC01-class bnAb attribute is abundant variable region SHMs with only a subset contributing to broad Env-binding and potent VRC01-class bnAb activity (37, 42), consistent with VRC01-class bnAb evolution occurring over long HIV-1 infection times and many SHM/selection cycles.To elicit VRC01-class broadly neutralizing antibodies (bnAbs), sequential vaccine immunization approaches propose a priming immunogen to drive precursors into GCs followed by boost immunogens designed to lead them through rounds of SHM/affinity maturation. Based on a structurally designed eOD-GT8 immunogen that binds to the inferred VRC01 unmutated common ancestor (UCA) BCR, potential human VRC01-like precursor B cell frequency was estimated to be one in 400,000 or fewer (43, 44). To test the priming and sequential immunogens that could elicit VRC01-class bnAbs in humans, mouse models are needed that reflect as closely as possible the biology of human B cell responses. Early models expressed knock-in V_H1-2 HCs and, in some, VRC01-class LC Vs, both with mature CDR3s (45–47). These models were nonphysiologic as their BCR repertoire was dominated by a single human HC/LC combination or a single human HC with diverse mouse LCs. Mice with fully human HC and LC gene segment loci assembled by V(D)J recombination were also tested; but precursor frequencies were 150- to 900-fold lower than that of humans (48), likely due to inability to express immense human-like CDR3 repertoires in mice with orders of magnitude fewer B cells. A V_H1-2-rearranging mouse model generated diverse V_H1-2 HC CDR3s, but it employed a germline-reverted VRC01 precursor LC with a 5-aa CDR3 from mature VRC01 bnAb (49). While useful for HC maturation studies during sequential immunization, this model was limited by over-abundance of VRC01 lineage LC precursors. More recently, B cells from transgenic VRC01-class UCA or eOD-GT8-binding precursor knock-in mice were adoptively transferred into congenic recipient mice at human-like frequencies (50–53). While this elegant approach has been very useful, it still has certain limitations as it focused only on eOD-GT8-priming and tested just a small subset of potential VRC01 lineage precursors (50–53).     

Isotype modulates epitope specificity, affinity, and antiviral activities of anti-HIV-1 human broadly neutralizing 2F5 antibody

The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.     

Reduced coxsackie antibody titres in Type 1 (insulin-dependent) diabetic patients presenting during an outbreak of coxsackie B 3 and B 4 infection

Summary The potential role of antecedent viral infection in the pathogenesis of Type 1 (insulin-dependent) diabetes was investigated by measuring antibody titres to several viruses in serum obtained at the time of diagnosis of diabetes. An outbreak of Coxsackie B 4 infection folio wed by a wave of Coxsackie B 3 and B 5 infections occurred in Seattle during the time viral serology was obtained in the diabetic patients. Antibody titres to Cocksackie B 5 and Influenza A and B viruses were comparable in diabetics and matched control subjects, but antibody titres to Cocksackie B 3 and B 4 were lower in the diabetics and a low antibody titre to Coxsackie B 3/B 4 was associated with a significantly increased relative risk of diabetes.     

A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model

Vascular endothelial growth factor (VEGF) is a primary stimulant of tumor angiogenesis. We previously raised a neutralizing anti-VEGF monoclonal antibody 2C3 that blocks the interaction of VEGF with VEGFR2 (KDR/Flk-1) but not with VEGFR1 (FLT-1/flt-1). Here, we describe the therapeutic effects of 2C3 on tumor growth in an orthotopic model of MDA-MB-231 human breast carcinoma implanted in the mammary fat pads (MFP) of nude mice. Administration of 2C3 to mice with 100–150 mm³ tumors inhibited tumor growth by 75%, as compared to recipients of the isotype-matched irrelevant control IgG, C44. Treatment with 2C3 also inhibited the establishment of tumor colonies and reduced tumor burden in the lungs of mice injected intravenously with MDA-MB-231 cells. No toxicity was observed in these studies. The mean microvascular density (MVD) of tumors in 2C3-treated mice was 55 ± 5 per mm², as compared to 188 ± 5 per mm² in the C44-treated control group. The decrease in MVD closely correlated with the degree of inhibition of tumor growth. Treated tumors mostly contained mid-size and large vessels. Microvessels were mainly confined to the peripheral layer of tumor that bordered on the normal MFP epithelium. Tumor vessels had decreased expression of VEGFR2, indicating that neutralization of tumor-derived VEGF by 2C3 down-regulates the expression of VEGFR2 on tumor vasculature. This, in turn, may limit re-initiation of angiogenesis by either tumor-derived or stromal VEGF. These findings suggest that 2C3 is a candidate for treating primary cancer and for preventing the outgrowth of tumor metastases in cancer patients.