Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial.

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2.

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host-receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface-expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.

A diverse collection of B cells responded to HIV infection in infant BG505.

Increasing evidence suggests infants develop unique neutralizing antibody (nAb) responses to HIV compared to adults. Here, we dissected the nAb response of an infant whose virus is in clinical trials as a vaccine immunogen, with a goal of characterizing the broad responses in the infant to this antigen. We isolated 73 nAbs from infant BG505 and identified a large number of clonal families. Twenty-six antibodies neutralized tier 2 viruses-in some cases, viruses from the same clade as BG505, and in others, a different clade, although none showed notable breadth. Several nAbs demonstrated antibody-dependent cellular cytotoxicity activity and targeted the V3 loop. These findings suggest an impressive polyclonal response to HIV infection in infant BG505, adding to the growing evidence that the nAb response to HIV in infants is polyclonal-a desirable vaccine response to a rapidly evolving virus like HIV.

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 µ g/mL; IC80 < 0.0006 to 0.0251 µ g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.

Neutralizing antibody VRC01 failed to select for HIV-1 mutations upon viral rebound.

Infusion of the broadly neutralizing antibody VRC01 has been evaluated in individuals chronically infected with HIV-1. Here, we studied how VRC01 infusions affected viral rebound after cessation of antiretroviral therapy (ART) in 18 acutely treated and durably suppressed individuals. Viral rebound occurred in all individuals, yet VRC01 infusions modestly delayed rebound and participants who showed a faster decay of VRC01 in serum rebounded more rapidly. Participants with strains most sensitive to VRC01 or with VRC01 epitope motifs similar to known VRC01-susceptible strains rebounded later. Upon rebound, HIV-1 sequences were indistinguishable from those sampled at diagnosis. Across the cohort, participant-derived Env showed different sensitivity to VRC01 neutralization (including 2 resistant viruses), yet neutralization sensitivity was similar at diagnosis and after rebound, indicating the lack of selection for VRC01 resistance during treatment interruption. Our results showed that viremia rebounded despite the absence of HIV-1 adaptation to VRC01 and an average VRC01 trough of 221 µg/mL. Although VRC01 levels were insufficient to prevent a resurgent infection, knowledge that they did not mediate Env mutations in acute-like viruses is relevant for antibody-based strategies in acute infection.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide.

A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man9-V3). V3-glycan bnAbs bound to Man9-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man9-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1-infected individual. In rhesus macaques, immunization with Man9-V3 induced V3-glycan-targeted antibodies. Thus, the Man9-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody.

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

New Member of the V1V2-Directed CAP256-VRC26 Lineage That Shows Increased Breadth and Exceptional Potency.

UNLABELLED: The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 µg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 µg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding.

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.

Vaccine-induced CD8+ T lymphocytes of rhesus monkeys recognize variant forms of an HIV epitope but do not mediate optimal functional activity.

The sequence diversity of HIV-1 presents a challenge for the development of an effective HIV-1 vaccine, because such a vaccine must confer protection against diverse forms of the virus. The present studies were initiated to explore how vaccine-induced clonal populations of CD8(+) T lymphocytes of rhesus monkeys recognize variants of an HIV-1 envelope epitope sequence. Evaluating a subset of variants of a selected epitope peptide that retain their binding to the MHC class I molecule of rhesus monkeys that presents this epitope peptide, we show that vaccine-elicited CD8(+) T lymphocytes comparably recognize the wild-type and a number of variant epitope peptides as determined by tetramer binding assays. In fact, the same clonal populations of CD8(+) T lymphocytes recognize the wild-type and variant epitope peptides. However, functional assays show that many of these variant epitope peptides stimulate suboptimal cytokine production by the vaccine-elicited CD8(+) T lymphocytes. These findings suggest that vaccine-induced CD8(+) T lymphocyte populations may recognize diverse forms of a viral epitope, but may not function optimally to confer protection against viruses expressing many of those variant sequences.

Epitope-specific CD8+ T lymphocytes cross-recognize mutant simian immunodeficiency virus (SIV) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during SIV infection.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) evade containment by CD8(+) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true representation of the plasticity of these viruses or the intensity of CD8(+) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8(+) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8(+) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8(+) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-specific CD8(+) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-specific CD8(+) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and fixation of the mutant epitope sequences in the viral quasispecies.

Compensatory substitutions restore normal core assembly in simian immunodeficiency virus isolates with Gag epitope cytotoxic T-lymphocyte escape mutations.

The evolution of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) as they replicate in infected individuals reflects a balance between the pressure on the virus to mutate away from recognition by dominant epitope-specific cytotoxic T lymphocytes (CTL) and the structural constraints on the virus' ability to mutate. To gain a further understanding of the strategies employed by these viruses to maintain replication competency in the face of the intense selection pressure exerted by CTL, we have examined the replication fitness and morphological ramifications of a dominant epitope mutation and associated flanking amino acid substitutions on the capsid protein (CA) of SIV/simian-human immunodeficiency virus (SHIV). We show that a residue 2 mutation in the immunodominant p11C, C-M epitope (T47I) of SIV/SHIV not only decreased CA protein expression and viral replication, but it also blocked CA assembly in vitro and virion core condensation in vivo. However, these defects were restored by the introduction of upstream I26V and/or downstream I71V substitutions in CA. These findings demonstrate how flanking compensatory amino acid substitutions can facilitate viral escape from a dominant epitope-specific CTL response through the effects of these associated mutations on the structural integrity of SIV/SHIV.

Hepatitis C virus core selectively suppresses interleukin-12 synthesis in human macrophages by interfering with AP-1 activation.

Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection, suggesting that it has evolved one or more strategies aimed at evading the host immune response. T cell responses, including interferon-gamma production, are severely suppressed in chronic HCV patients. The HCV core protein has been previously shown to circulate in the bloodstream of HCV-infected patients and inhibit host immunity through an interaction with gC1qR. To determine the role of the HCV core-gC1qR interaction in modulation of inflammatory cytokine production, we examined interleukin (IL)-12 production, which is critical for the induction of interferon-gamma synthesis, in lipopolysaccharide-stimulated human monocyte/macrophages. We found that core protein binds the gC1qR displayed on the cell surface of monocyte/macrophages and inhibits the production of IL-12p70 upon lipopolysaccharide stimulation. This inhibition was found to be selective in that HCV core failed to affect the production of IL-6, IL-8, IL-1beta, and tumor necrosis factor alpha. In addition, suppression of IL-12 production by core protein occurred at the transcriptional level by inhibition of IL-12p40 mRNA synthesis. Importantly, core-induced inhibition of IL-12p40 mRNA synthesis resulted from impaired activation of AP-1 rather than enhanced IL-10 production. These results suggest that the HCV core-gC1qR interaction may play a pivotal role in establishing persistent infection by dampening TH1 responses.

Direct binding of hepatitis C virus core to gC1qR on CD4+ and CD8+ T cells leads to impaired activation of Lck and Akt.

Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 x 10(-7) M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8(+) than on CD4(+) T cells, resulting in more severe core-induced suppression of the CD8(+)-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation.