Biology subjects

Upadhyay, A. A.

Publications and source records attributed to Upadhyay, A. A..

13 recordsLinked to original sources

bioRxiv2026

Germinal centers can persist for up to one year with declining somatic hypermutation and ongoing plasma cell output

The total duration of primary germinal center (GC) responses, and how B cell evolution changes as these responses mature, remain poorly defined. Here, longitudinal sampling of rhesus macaques immunized with HIV Envelope protein revealed that a single slow-delivery prime sustained ongoing GCs for nearly a year without additional antigen. Somatic hypermutation (SHM) accumulated throughout these prolonged responses, but its rate declined substantially over time, independent of both priming strategy and boosting. Over the same period, subdominant non-base Env specificities became increasingly represented within the GC response. Delaying boosting until after primary GC contraction increased recruitment of recalled memory B cells into the GC response. Bone marrow plasma cells were generated throughout prolonged GC maturation, with the timing of their generation modulated by booster timing. Together, these findings reveal how GCs evolve across nearly a year and how this prolonged maturation shapes the memory and plasma cell compartments that underpin long-term humoral immunity.

immunology↗

bioRxiv2025

Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1

Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity. Author SummaryUnderstanding how effective antibodies arise after HIV vaccination is essential for developing a protective vaccine, yet studying these responses in non-human primates has been limited by low- throughput methods. In this study, we adapted a high-throughput single-cell sequencing approach to identify HIV envelope-specific antibodies from vaccinated rhesus macaques. This method allowed us to recover paired antibody sequences together with their antigen specificity from thousands of individual B cells. We successfully identified known neutralizing antibodies and discovered additional antibodies capable of neutralizing HIV across multiple animals. Our analysis revealed that vaccine-elicited antibody responses were dominated by a small number of expanded lineages, with shared genetic features among animals with stronger neutralization. These findings demonstrate that this approach can efficiently define the antibody repertoires generated by HIV vaccines and provide a powerful tool for evaluating and improving immunogens in preclinical vaccine studies.

immunology↗

bioRxiv2025

A Broad Survey and Functional Analysis of Immunoglobulin Loci Variation in Rhesus Macaques

Rhesus macaques (RMs) are a vital model for studying human disease and invaluable to pre-clinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed novel experimental and computational methodologies to generate the largest collection to date of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian origin RMs. We created a comprehensive resource of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs), including the curation of 1474 novel alleles, unveiling tremendous diversity, and expanding existing IG allele sets by 60%. This publicly available, continually updated resource (https://vdjbase.org/reference_book/Rhesus_Macaque) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

immunology↗

bioRxiv2024

Diverse priming outcomes under conditions of very rare precursor B cells

Rare B cells can have special pathogen-recognition features giving them the potential to make outsized contributions to protective immunity. However, rare naive B cells infrequently participate in immune responses. We investigated how germline-targeting vaccine antigen delivery and adjuvant selection affect priming of exceptionally rare BG18-like HIV broadly neutralizing antibody-precursor B cells (~1 in 50 million) in non-human primates. Only escalating dose (ED) priming immunization using the saponin adjuvant SMNP elicited detectable BG18-like cells in germinal centers (GCs). All groups had strong GC responses, but only ED+SMNP and bolus+SMNP induced BG18-like memory B cells in >50% of animals. One group had vaccine-specific GC responses equivalent to ED+SMNP, but BG18-like memory B cells were rarely detected. Following homologous boosting, BG18-like memory B cells were more frequent in a bolus priming group, but had lower somatic hypermutation and affinities. This outcome was inversely associated with post-prime antibody titers, suggesting antibody feedback can significantly influence rare precursor B cell responses.

immunology↗

bioRxiv2024

Passive infusion of an S2-Stem broadly neutralizing antibody protects against SARS-CoV-2 infection and lower airway inflammation in rhesus macaques

The continued evolution of SARS-CoV-2 variants capable of subverting vaccine and infection-induced immunity suggests the advantage of a broadly protective vaccine against betacoronaviruses ({beta}-CoVs). Recent studies have isolated monoclonal antibodies (mAbs) from SARS-CoV-2 recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other {beta}-CoVs. Many of these mAbs target the conserved S2 stem region of the SARS-CoV-2 spike protein, rather the receptor binding domain contained within S1 primarily targeted by current SARS-CoV-2 vaccines. One of these S2-directed mAbs, CC40.8, has demonstrated protective efficacy in small animal models against SARS-CoV-2 challenge. As the next step in the pre-clinical testing of S2-directed antibodies as a strategy to protect from SARS-CoV-2 infection, we evaluated the in vivo efficacy of CC40.8 in a clinically relevant non-human primate model by conducting passive antibody transfer to rhesus macaques (RM) followed by SARS-CoV-2 challenge. CC40.8 mAb was intravenously infused at 10mg/kg, 1mg/kg, or 0.1 mg/kg into groups (n=6) of RM, alongside one group that received a control antibody (PGT121). Viral loads in the lower airway were significantly reduced in animals receiving higher doses of CC40.8. We observed a significant reduction in inflammatory cytokines and macrophages within the lower airway of animals infused with 10mg/kg and 1mg/kg doses of CC40.8. Viral genome sequencing demonstrated a lack of escape mutations in the CC40.8 epitope. Collectively, these data demonstrate the protective efficiency of broadly neutralizing S2-targeting antibodies against SARS-CoV-2 infection within the lower airway while providing critical preclinical work necessary for the development of pan-{beta}-CoV vaccines. AUTHOR SUMMARYIn this study, we explore the development of a broadly protective vaccine against betacoronaviruses ({beta}-CoVs), including SARS-CoV-2. We focused on monoclonal antibodies (mAbs) from individuals who recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other {beta}-CoVs. Unlike current vaccines that target the S1 region of the virus, these mAbs target a highly conserved S2 region of the spike protein. One antibody, CC40.8, showed promising results in small animal models. To further test its effectiveness, we infused CC40.8 into rhesus macaques at different doses and then challenged them with SARS-CoV-2. We found that higher doses of CC40.8 significantly reduced viral loads and inflammation in the lower airway. Additionally, there were no escape mutations in the targeted region, suggesting that the virus could not easily evade the antibody. Our findings highlight the potential of S2-targeting antibodies to protect against SARS-CoV-2 and support the development of vaccines that can broadly protect against various {beta}-CoVs. Conflicting InterestsRA, TFR, and DRB are listed as inventors on pending patent applications describing the SARS-CoV-2 and HCoV-HKU1 S cross-reactive antibodies. DRB and RA are listed as inventors on a pending patent application describing the S2 stem epitope immunogens identified in this study. DRB is a consultant for IAVI. All other authors declare that they have no competing interests. ONE SENTENCE SUMMARYPan-beta-coronavirus neutralizing mAb CC40.8 reduces SARS-CoV-2 viral loads and inflammation within the lower airway of infected rhesus macaques and provides pre-clinical support for S2-directed immunization strategies.

immunology↗

bioRxiv2022

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFN2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN- and IFN-{beta} pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

immunology↗

bioRxiv2021

TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant(R)), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. One sentence summaryMulti-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib

microbiology↗

bioRxiv2021

From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/439712v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@de0663org.highwire.dtl.DTLVardef@757c30org.highwire.dtl.DTLVardef@12c4709org.highwire.dtl.DTLVardef@1ebd6d7_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryCryoEM and next generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

immunology↗

bioRxiv2020

Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures

The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNF, CXCL8. We also identified NF-{kappa}B and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCEThe current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.

microbiology↗

bioRxiv2019

Microbiome Stability with Chronic SIV Infection in AIDS-resistant Sooty Mangabeys

Sooty mangabeys (SMs) are a natural host species of simian immunodeficiency virus (SIV) and avoid acquired immune deficiency syndrome (AIDS) despite persistently high viral loads, making them a pivotal research model for HIV pathogenesis. Unlike pathogenic SIV infection of macaque species, or HIV infection of humans, SIV-infected SMs maintain gastrointestinal barrier integrity. Here, we characterize the gastrointestinal bacterial microbiota of SIV-infected and uninfected SMs and perform a comparative analysis of diet-matched, rhesus macaques (RM). We assessed the fecal microbiome in fifty SM and thirty RM in total, and conducted analyses of the effect of SIV-status, species, and housing. When examining indoor-outdoor and indoor-only housing in our SM cohorts, biodiversity reduction and mild phylogenetic taxonomic perturbances were present. No statistically relevant differences were seen for biodiversity richness and evenness, or phylogenetic taxonomic communities between SIV negative and positive SM cohorts. In contrast, with pathogenic early chronic SIV infections in RM a trend of alpha diversity loss and increase of beta diversity and few phyla taxonomic communities differed. Lastly, we observed lower levels of pathobiont bacterial communities in SIV-uninfected SMs relative to RMs. These data suggest that the pre-existing bacterial community structure may contribute to the divergent phenotype between SIV natural hosts and pathogenic macaque species.\n\nImportanceHuman immunodeficiency virus remains a global concern. The sooty mangabey (SM) monkey is an important biomedical research model for understanding HIV pathogenesis due to its ability to avoid AIDS disease progression despite high viremia. In people living with HIV, gastrointestinal dysbiosis towards enrichment of pathobiont communities has been frequently reported. In this study we characterized the fecal microbiota of a primate non-pathogenic SIV host, the SM, and made direct comparisons to a pathogenic SIV host species, the rhesus macaque. We observed that SMs exhibit stability of the microbiota community into chronic SIV infection, which contrasts with SIV-infected rhesus macaques, in which we observed bacterial community divergence relative to uninfected animals. Collectively, our observation of stabilization of beneficent taxa in the mucosa of AIDS-resistant primates suggests that therapeutic strategies to enrich these communities may have potential for ameliorating the gastrointestinal inflammation in people living with HIV.

immunology↗

bioRxiv2018

West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-{beta} mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.\n\nIMPORTANCEWest Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

microbiology↗

bioRxiv2018

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance

The observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other difficult pathogens, due to the significant immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine needle aspirates, we found that two independent methods of slow delivery immunization of rhesus macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified differential IgV gene usage between slow delivery immunized and conventional bolus immunized animals. The most immunodominant IgV gene used by conventionally immunized animals was associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb development by altering GCs and modulating immunodominance of non-neutralizing epitopes.

immunology↗