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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (

SUMMARYThe emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1–7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10–13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14–18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

Simian immunodeficiency virus (SIV) infection of Indian rhesus macaques (RMs) is one of the best-characterized animal models for human immunodeficiency virus (HIV) infection. Monoclonal antibodies (mAbs) have shown promise for prevention and treatment of HIV infection. However, it has been difficult to isolate mAbs that potently neutralize the highly pathogenic SIVmac239 strain. This has been largely due to the low frequency of circulating B cells encoding neutralizing Abs. Here we describe a novel technique to isolate mAbs directly from bone marrow-derived, Ab-secreting plasma cells. We employed an automated micromanipulator to isolate single SIVmac239 SOSIP.664-specific plasma cells from the bone marrow of a SIVmac239-infected RM with serum neutralization titers against SIVmac239. After picking plasma cells, we obtained 44 paired Ab sequences. Ten of these mAbs were SIV specific. Although none of these mAbs neutralized SIVmac239, three mAbs completely neutralized the related SIVmac316 strain. The majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced Ab-dependent cellular cytotoxicity. This method is a first step in successful isolation of antigen-specific bone marrow-derived plasma cells from RMs., Graphical Abstract, Pedreño-Lopez et al. adapted a previously described innovative fluorescence-based method to isolate antigen-specific plasma cells from total bone marrow of rhesus macaques using a micromanipulator. This method facilitated isolation and subsequent characterization of SIV-specific non-neutralizing antibodies with antibody-dependent cellular cytotoxicity activity that might be efficacious in vivo.

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8β-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo. Keywords: SIV, monoclonal antibodies, rhesus macaques, CD8b depletion, antiretroviral therapy interruption

Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use.ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naïve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

Summary Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. A chimpanzee simian immunodeficiency virus (SIV) envelope (Env) shares a single bnAb site, the variable loop 2 (V2)-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Here, we generate a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determine the structure of the MT145K trimer by cryo-EM and show that its architecture is remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knockin mouse with the chimpanzee MT145K trimer induces HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induces responses that exhibit some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs., Graphical Abstract, Highlights • A designed chimpanzee SIV Env trimer binds HIV V2-apex bnAbs specifically • The trimer (MT145K) is engineered to bind inferred unmutated versions of HIV V2-apex bnAbs • The cryo-EM structure of the SIV MT145K trimer closely resembles that of HIV trimers • The MT145K SIV trimer induces HIV-specific nAb responses in a favorable animal model, Design of immunogens and strategies that can induce protective broadly neutralizing antibodies (bnAbs) is a priority for HIV vaccine development. Andrabi et al. design a chimpanzee simian immunodeficiency virus (SIV) envelope trimer immunogen that binds specifically to HIV V2-apex bnAbs and their unmutated versions. The SIV trimer immunogen induces HIV-specific neutralizing antibodies (nAbs) in a favorable animal model.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (β-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in β-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on β-CoV spike proteins for protective antibodies that may facilitate the development of pan-β-CoV vaccines.SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-β-CoV vaccine design strategies

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

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.

HIV Env trimers elicit antibodies that lead to their own destruction., Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing (“off-target”) antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

Pre-existing immunity to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, induced from natural infection or vaccination. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. Here we compare serum antibody and memory B cell responses to coronavirus spike proteins from pre-pandemic and SARS-CoV-2 convalescent donors using binding and functional assays. We show weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we find evidence of pre-existing cross-reactive memory B cells that are activated during SARS-CoV-2 infection. Monoclonal antibodies show varying degrees of cross-reactivity with betacoronaviruses, including SARS-CoV-1 and endemic coronaviruses. We identify one cross-reactive neutralizing antibody specific to the S2 subunit of the S protein. Our results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2., Pre-existing immune responses between antigenically related viruses can influence responses in viral infections or vaccinations. Here the authors assess and characterize the presence of antibody and memory B cell populations specific to SARS-CoV2 and endemic human coronaviruses.

Author(s): Rogers, Thomas F; Zhao, Fangzhu; Huang, Deli; Beutler, Nathan; Burns, Alison; He, Wan-Ting; Limbo, Oliver; Smith, Chloe; Song, Ge; Woehl, Jordan; Yang, Linlin; Abbott, Robert K; Callaghan, Sean; Garcia, Elijah; Hurtado, Jonathan; Parren, Mara; Peng, Linghang; Ricketts, James; Ricciardi, Michael J; Rawlings, Stephen A; Smith, Davey M; Nemazee, David; Teijaro, John R; Voss, James E; Andrabi, Raiees; Briney, Bryan; Landais, Elise; Sok, Devin; Jardine, Joseph G; Burton, Dennis R | Abstract: The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult., Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP (gp120-gp41 disulfide [SOS] with an isoleucine-to-proline mutation [IP] in gp41) alone, as well as B41 and BG505 coimmunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single-particle cryo-electron microscopy studies confirmed that B41- and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but we recovered only partial binding. Our data demonstrate that the lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope, and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult. IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.

A critical property of a prophylactic HIV vaccine is likely to be its ability to elicit broadly neutralizing antibodies (bnAbs). BnAbs typically have multiple unusual features and are generated in a fraction of HIV-infected individuals through complex pathways. Current vaccine design approaches seek to trigger rare B cell precursors and then steer affinity maturation toward bnAbs in a multi-stage multi-component immunization approach. These vaccine design strategies have been facilitated by molecular descriptions of bnAb interactions with stabilized HIV trimers, the use of an array of sophisticated approaches for immunogen design, the development of novel animal models for immunogen evaluation and advanced technologies to interrogate antibody responses. In this review, we will discuss leading HIV bnAb vaccine immunogens, immunization strategies and future improvements.

Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) SOSIP immunogens have revealed that the dominant autologous neutralizing site in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. BG505 and B41 SOSIP immunogens were combined to test whether immunization in rabbits could induce broader Tier 2 neutralizing responses to the common glycan hole shared between BG505 and B41. Here we isolated autologous neutralizing antibodies (nAbs) that were induced by immunization with B41 SOSIP alone, as well as B41 and BG505 co-immunization, and describe their structure in complex with the B41 SOSIP trimer. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both immunogens were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the highly conserved N241 glycan (>97% conserved), which is present in B41. Single particle cryo-electron microscopy (cryoEM) studies confirmed that B41 and BG505-specific nAbs bind to overlapping glycan hole epitopes. In an attempt to broaden the reactivity of a B41-specific nAb, mutations in the BG505 glycan hole epitope guided by our high-resolution data only recovered partial binding. Overall, designing prime-boost immunogens to increase the breath of nAb responses directed at glycan holes epitopes remains challenging even when the typically immunodominant glycan holes despite overlap with different Envs.IMPORTANCEA glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer immunized rabbits. Our high-resolution cryoEM studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that eliciting cross-reactive responses to this region would likely require hybrid immunogens that bridge between BG505 and B41.

As a consequence of their independent evolutionary origins in apes and Old World monkeys, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses of the SIVsmm/mac lineage express phylogenetically and antigenically distinct envelope glycoproteins. Thus, HIV-1 Env-specific antibodies do not typically cross-react with the Env proteins of SIVsmm/mac isolates. Here we show that PGT145, a broadly neutralizing antibody to a quaternary epitope at the V2 apex of HIV-1 Env, directs the lysis of SIVsmm/mac-infected cells by antibody-dependent cellular cytotoxicity (ADCC) but does not neutralize SIVsmm/mac infectivity. Amino acid substitutions in the V2 loop of SIVmac239 corresponding to the epitope for PGT145 in HIV-1 Env modulate sensitivity to this antibody. Whereas a substitution in a conserved N-linked glycosylation site (N171Q) eliminates sensitivity to ADCC, a lysine-to-serine substitution in this region (K180S) increases ADCC and renders the virus susceptible to neutralization. These differences in function correlate with an increase in the affinity of PGT145 binding to Env on the surface of virus-infected cells and to soluble Env trimers. To our knowledge, this represents the first instance of an HIV-1 Env-specific antibody that cross-reacts with SIVsmm/mac Env and illustrates how differences in antibody binding affinity for Env can differentiate sensitivity to ADCC from neutralization. IMPORTANCE Here we show that PGT145, a potent broadly neutralizing antibody to HIV-1, directs the lysis of SIV-infected cells by antibody-dependent cellular cytotoxicity but does not neutralize SIV infectivity. This represents the first instance of cross-reactivity of an HIV-1 Env-specific antibody with SIVsmm/mac Env and reveals that antibody binding affinity can differentiate sensitivity to ADCC from neutralization.

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4., This work was supported by the National Institutes of Health, R01-5R01DE025167-04, by The Bill and Melinda Gates Foundation OPP1183956, by CHAVI-ID grant UM1 AI100663, by the Ramon y Cajal Merit Award from Ministerio de Ciencia, Innovacion y Universidades, Spain (RYC-2016–21155 to AG-M), and by a Marie-Curie Fellowship (FP7-PEOPLE-2013-IOF to LEM).

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans., The analysis of site-specific glycosylation of HIV Envelope glycoprotein (Env) is challenging as it contains 25–30 glycosylation sites with multiple glycan forms at each site. Here the authors present a generally applicable mass spectrometry-based method for site-specific analysis of protein glycosylation that they apply to the analysis of the HIV-1 Env.

Little is known about the neutralizing antibodies induced in HIV-1 patients on antiretroviral treatment, which constitute an interesting group of individuals with improved B cell profile. Plasma samples from 34 HIV-1 seropositive antiretroviral drug treated (ART) patients were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94 %) were found to neutralize ≥50 % viruses tested. In terms of overall neutralization frequency, approximately 86 %, 68 % and 17 % of the virus/plasma combinations showed 50 % neutralizing activity at 1 > 60, 1 ≥ 200 and 1 ≥ 2000 dilutions respectively. The improvement in neutralizing activity was shown to be associated with ART in two follow up patients. The neutralization of viruses by two representative plasma samples, AIIMS221 and AIIMS265, was exclusively mediated by immunoglobulin G fractions independent of ART drugs and IgG retained cross-reactive binding to recombinant gp120 proteins. We observed a positive trend of neutralization with duration of ART (p = 0.06), however no such correlation was found with clinical and immunological variables like CD4 count (p = 0.35), viral load (p = 0.09) and plasma total IgG (p = 0.46). Our study suggests that the plasma antibodies from ART patients display high neutralizing activity most likely due to an improved B cell function induced by ART despite low antigenic stimulation.

The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

We assessed the anti-V3 antibody content and viral neutralization potential of the plasma of 63 HIV-1-infected patients (antiretroviral naïve=39, treated=24) against four primary isolates (PIs) of clade C and a tier 1 clade B isolate SF162. Depletion and inhibition of anti-V3 antibodies in the plasma of five patients with high titers of anti-V3 antibodies led to modest change in the neutralization percentage against two PIs (range 0-21%). The plasma of antiretroviral-treated patients exhibited higher neutralization potential than that of the drug-naïve plasmas against the four PIs tested which was further evidenced by a follow-up study.

Limited information exists on the antibody responses elicited against the viral envelope in HIV-1-infected children. In this cross-sectional study, we assessed the antibody responses against three different immunogenic regions of HIV-1 envelope, namely V3 region of gp120, membrane proximal external region (MPER), and immunodominant loop (IDL) of gp41 in HIV-1-infected children from north India. We recruited 75 HIV-1-infected (40 antiretroviral naive and 35 treated) children, with age ranging from 1.5 to 16 y. Antibodies to V3 and the IDL region were found in a majority of the infected children, whereas antibodies to MPER were found in approximately one-third of the children studied. Higher antibody titers to the immunogenic regions corresponded to the symptomatic stages of HIV-1 infection in both naive and antiretroviral therapy (ART)-treated children. High titers of anti-V3C and anti-IDL antibodies were observed in a subset of antiretroviral-naive patients with suppressed viremia (

The viral reverse transcriptase gene was amplified from the plasma of 27 drug-naïve and five drug-experienced HIV patients in Northern India. Follow-up samples of naïve patients after antiretroviral therapy initiation were collected in six patients at 3 months and three patients at 6 months. Phylogenetic analysis revealed two new recombinant forms, CRF_CH and CRF_CK, and an accessory non-nucleoside reverse transcriptase inhibitor mutation E138A, not previously reported from India. The major DRMs found in two 6-month follow-up samples were absent in their baseline blood samples. This is the first follow-up study to determine anti-retroviral drug resistance mutations in Indian HIV patients.

International audience; Broadly neutralizing antibodies (bnAbs) directed to the V2 apex of the HIV envelope (Env) trimer isolated from individual HIV-infected donors potently neutralize diverse HIV strains, but strategies for designing immunogens to elicit bnAbs have not been identified. Here, we compared four prototypes (PG9, CH01, PGT145, and CAP256.VRC26.09) of V2 apex bnAbs and showed that all recognized a core epitope of basic V2 residues and the glycan-N160. Two prototype bnAbs were derived from VH-germlines that were 99% identical and used a common germline D-gene encoded YYD-motif to interact with the V2-epitope. We identified isolates that were neutralized by inferred germline (iGL) versions of three of the prototype bnAbs. Soluble Env derived from one of these isolates was shown to form a well-ordered Env trimer that could serve as an immunogen to initiate a V2-apex bnAb response. These studies illustrate a strategy to transition from panels of bnAbs to vaccine candidates.

Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

Summary Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an "anchor" for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses.

Objectives: A region in the conserved 5 ′ long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells. Methods: Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA. Results: One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for ≥3 weeks after a single transfection, even of a strain that had a mismatch in the target region. Conclusions: We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.

Methods We tested 30 HIV-1 drug naive plasma samples for neutralization against a broad panel of subtype-A, B and C tier 1 and tier 2 viruses in a TZM-bl assay. Three broadly neutralizing plasma (bNP) samples (AIIMS206, AIIMS239 and AIIMS249) were tested for ELISA binding with a set of 211 consensus-C gp160 overlapping peptides (NIH AIDS Research and Reference Reagent Program). The competition and depletion experiments (for neutralization) were carried out with peptides corresponding to consensus-C V3 (35mer), IDR (19mer) and MPER (24mer).

Background Little is known about the neutralizing activity in patients on antiretroviral therapy (ART), as most recent studies have focused on drug naive individuals. ART may lead to a significant increase in B cell numbers and normalization of B cell subpopulations, providing a possible explanation for improved B cell responses after ART. Methods Thirty-four HIV-1 seropositive patients on ART (25 males and 9 females) within the age range of 20-55 years were recruited in this study. The patients had a median CD4 count and viral load of 283 cells and 178 RNA copies respectively, and were on treatment for a few days up to two years. Heat inactivated plasma samples were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Results Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94%) samples were found to neutralize ≥50% viruses tested. Clustering analysis revealed that AIIMS253 (a clade-C virus) was the most sensitive while RHPA4259.7 (a clade-B isolate) was most resistant to antibody neutralization. The Immunoglobulin-G fractions from two representative samples AIIMS221 and AIIMS265 were shown to mediate neutralization exclusively. The IgG fractions retained binding to subtype-A, B and C recombinant gp120 proteins. We did not find any association of mean reciprocal ID50 neutralization titers with the plasma levels of ART drugs and clinical and immunological variables like CD4 count (p=0.35), viral load (p=0.37) and plasma total IgG (p=0.46). However we observed a positive association of neutralization with duration of ART (p=0.02) with a similar trend in two follow up patient samples. Conclusion

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19-27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

Methods A phage library of 7000 clones was constructed from a drug naive HIV-1 clade C infected Indian patient whose plasma exhibited high potential neutralizing potential against a panel of viruses and also displayed cross-reactive anti-V3 antibodies. PBMCs were isolated and EBV transformed. Cells (wells) producing anti-V3 antibodies were preselected with V3-CTB fusion protein and expanded. Total RNA was isolated and cDNA was constructed followed by VH and VL amplification. scFvs were constructed, cloned into phagemid vector and expressed in Escherichia coli. We assessed the expression of the scFvs by SDSPAGE and Western blotting. Specificity was examined by ELISA.

Neutralizing antibodies are an important component of the humoral immune response directed against viral infections So far the few available anti HIV-1 broadly neutralizing antibodies have a limited breadth and potency against clade C viruses. More than 50% of the HIV-1 infections worldwide belong to clade C. Clade C is the most prevalent subtype in India.

Methods Plasma of eighty naive HIV-1 seropositive Indian patients were tested for neutralization against a panel of 3 subtype-B and 5 subtype-C tier 1 and tier 2 viruses. Three plasma, found to be broadly neutralizing (bNP), were mapped with a set of 212 consensus-C gp160 overlapping peptides (15mer each). 50% binding titers were determined by ELISA using consensus-C V3 (35mer), IDR loop (19mer) and MPER (24mer) peptides.

In this cross-sectional study, we evaluated the efficiency of the plasma of 38 antiretroviral-naïve HIV-1-infected children from northern India against a standard panel of pseudoviruses (3 clade C and 3 clade B) by TZM-bl assay. Neutralization potential was observed to a variable extent, with a potency ranging up to reciprocal ID(50) titers of 1967. Cross-neutralization was observed in 28.9 % (11/38) of the children. There was a significant positive correlation between viremia and neutralization efficiency against two of the viruses studied (Du172 r = 0.49; p = 0.007 and RHPA r = 0.47; p = 0.01), suggesting that persistent antigenic stimulation is necessary for the generation of broadly neutralizing antibody responses in these children. Further mapping of the epitope specificities of the neutralization determinants in the polyclonal plasma would provide important information for immunogen design.

Antibodies to two crucial regions, the third variable loop (V3) of gp120 and the membrane-proximal external region (MPER) of gp41 are important for HIV-1 neutralization. We here evaluated the relative binding of polyclonal plasma antibodies from 99 HIV-1-infected individuals from India to the consensus–C V3 and MPER peptides and observed immunodominance of V3 over MPER (p

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale.

Background Production of human monoclonal antibodies that shows broadly neutralising activity is needed for the prevention of HIV-1. Here we devised a novel approach and produced 11 different human scFvs against the V3 region of HIV-1 envelope. Method: The peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-1 clade C infected drug naive Indian patient whose plasma exhibited neutralising antibodies against a panel of viruses. PBMCs were EBV transformed in 96 well plate and then expanded into 24 well stage followed by 6 well and finally to flask stage. The transformed cells (wells) were screened against HIV-1 consensus C V3 peptide at each stage and only the positive cells (wells) were carried onto the next stage. Total RNA from these enriched positive antibody secreting cells at the flask stage was isolated and cDNA was synthesised. Results: By EBV transformation and preselection of V3 specific clones we successfully constructed a small phage library. One round of bio panning was done against HIV-1 consensus C and B V3 peptides. Randomly, 40 clones were selected and checked for their binding. Out of 40 clones, 13 showed positivity in phage ELISA. DNA fingerprinting analysis using BstN1 followed by sequencing showed that 11 out of 13 clones were distinct. Expression of positive clones was tested by SDS-PAGE and Western blot. All the 11 anti -V3 scFvs showed cross-reactivity against both the V3 peptides and did not show any reactivity against other unrelated peptides. The scFvs showed varying degrees of neutralisation against tier 1 and tier 2 viruses and cross neutralising activity against clade A, B and C viruses. Conclusion This is the first study to generate anti-V3 scFvs against HIV-1 Clade C. Further assessment of the neutralisation efficiency of these scFvs would reveal their potential for passive immunotherapy.

Methods A V3 specific phage library was constructed from EBV transformed B cells of a clade C HIV-1 infected Indian patient, whose plasma exhibited good neutralization potential against a panel of viruses. Diversity of the constructed phage library was analysed by DNA fingerprinting of 10 randomly selected clones from the unselected library using BstN1 enzyme. One round of biopanning was done against HIV-1 consensus V3C and V3B peptides. scFvs were than characterised for their binding, specifity and expression profile. VH and VL genes of anti-V3 scFvs were sequenced for their preferential gene usage.

One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

Background Subtype-C alone accounts for approximately 50% of global and more than 95% human immunodeficiency virus1(HIV-1) infection in India. Identification of antigenic epitopes that induce antibodies with cross-clade activity will be crucial to address the HIV-1viral diversity.

Background Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. We have isolated 3 anti-V3 mAbs 277, 903 and 904 by hybridoma technology from EBV transformed B cells of HIV-1 seropositive drug naive patients. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively.

Background Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.