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High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope

ABSTRACT: A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages

ORGANISM(S): Macaca mulatta

PROVIDER: GSE244293 | GEO | 2023/10/11

REPOSITORIES: GEO

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Genome-wide characterization of a novel necrotic granuloma model of latent tuberculosis infection and reactivation in mice

Project description:We sought to develop and characterize a novel paucibacillary model in mice, which develop necrotic lung granulomas following infection with Mycobacterium tuberculosis. Paucibacillary infection was established, recapitulating the sterilizing activities of human LTBI regimens. TNF neutralization led to increased lung bacillary load, disrupted granuloma architecture with expanded necrotic foci and reduced tissue hypoxia, and accelerated animal mortality. TNF-neutralized mouse lungs and sera showed significant upregulation of IFN?, IL-1?, IL-6, IL-10, CCL2, CCL3, and matrix metalloproteinase genes Six weeks after aerosol-immunization with recombinant M. bovis BCG overexpressing the 30-kilodalton antigen, C3HeB/FeJ mice were aerosol-infected with M. tuberculosis H37Rv. Six weeks later, mice were treated with one of three standard regimens for latent TB infection (LTBI) or TNF-neutralizing antibody. Mouse lungs were analyzed by histology, positron emission tomography/computed tomography, whole-genome microarrays, and RT-PCR. Lungs and sera were studied by multiplex enzyme-linked immunosorbent assays

2014-02-20 | E-GEOD-55183 | biostudies-arrayexpress

VIA of EM CD8 T cells in vaccinated monkeys

Project description:The goal of an effective AIDS vaccine is to generate immunity that will prevent HIV-1 acquisition. Despite limited progress towards this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime/rAd5 boost vaccination with SIVmac239 Gag/Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8+ T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8+ T cells showed strong virus inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8+ T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8+ T cells. The ability to elicit such virus-specific EM CD8+ T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection. Gag CM9-specific EM CD8+ T cells (CD28 low CD95 high tetramer+) from SIV-negative macaques at 12 wks post-DNA/rAd5 immunization were sorted by flow cytometry for microarray studies. RNA samples from strong VIA animals with (n=3) or without (n=6) CM9 peptide stimulation, along with CM9 peptide stimulated samples from weak VIA animals (n=2) were prepared using the Illumina beads station assay and hybridized to the Illumina HumanHT-12 version 4 Expression BeadChip.

2012-06-14 | E-GEOD-35628 | biostudies-arrayexpress

Intradermal but not intramuscular MVA immunizations protect against intravaginal tier2 SHIV challenges in macaques

Project description:Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 SHIV challenges in female macaques. Both routes of vaccination induced comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlated positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlated only with serum neutralizing activity. MVA-ID immunizations induced greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlated negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

2023-07-27 | GSE219118 | GEO

Cytokine-independent detection of antigen-specific germinal center T follicular helper (Tfh) cells in immunized non-human primates using a live cell Activation Induced Marker (AIM) technique

Project description:A range of current candidate AIDS vaccine regimens are focused on generating protective HIV neutralizing antibody responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective antibody responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GC) are the engines of antibody affinity maturation. GC T follicular helper (GC Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as antigen-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining (ICS). Cytokine production by GC Tfh cells may be intrinsically limited in comparison to other T helper effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify antigen-specific GC Tfh cells. RNAseq was performed using TCR stimulated GC Tfh cells to identify candidate markers based on differentially expressed genes and other criteria. Validation experiments determined CD25 (IL2Ra) and OX40 to be highly upregulated activation induced markers on the surface of GC Tfh cells after stimulation. In comparison to ICS, the activation induced marker (AIM) assay identified > 10-fold more antigen-specific GC Tfh cells in HIV Env protein immunized macaques (BG505 SOSIP). Additionally, the AIM assay detected antigen-specific CXCR5+ and CXCR5- CD4 T cells in peripheral blood. Thus, AIM demonstrates that antigen-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function. Total RNA obtained from GC Tfh cells from 3 rhesus macaque lymph node cell samples stimulated with Staphylococcal Enterotoxin B compared with 2 unstimulated cell samples.

2016-09-07 | E-GEOD-81382 | biostudies-arrayexpress

Identification of SARS-COV-2 specific antibodies from patient serum during the First Wave of the COVID-19 pandemic

Project description:Proteomic Ig-SEQ identification of SARS-COV-2 specific antibodies from serum of two convalescent patients during the first wave of the COVID-19 pandemic. Total IgG was isolated from serum, digested with IdeS protease to generate F(ab)2 fragments, and subjected to antigen affinity chromatography using agarose beads coupled with SARS-COV-2 spike protein. Purified F(ab)2 fragments were reduced, alkylated, and digested with trypsin prior to LC-MSMS analysis on an Orbitrap Fusion Lumos mass spectrometer. Resulting peptide IDs were used to identify IgG clonotypes in antigen-specific and flow-through fractions.

2024-01-26 | PXD046829 | Pride

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates

Project description:Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to human immunodeficiency virus (HIV) and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. We found that immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in 8 of 8 rhesus macaques to substantial frequencies, and with diverse lineages, in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. The results demonstrate proof of principle for priming of HCDR3-dominant bnAb precursors in outbred animals, suggest that N332-GT5 has promise to induce similar responses in humans, and encourage application of HCDR3-dominant germline-targeting for other bnAbs to HIV and additional pathogens.

2024-02-26 | MSV000094174 | MassIVE

Proteomic analysis of antigen-specific serum and gut antibodies in celiac disease patients

Project description:Comparison of serum and gut antibodies against two major antigens in celiac disease patients, the autoantigen transglutaminase 2 (TG2) and a deamidated gluten peptide (DGP) from food. The antigen-specific antibodies were affinity-purified from isolated total IgA of serum or gut biopsy secretions or from whole serum samples. Individual clonotypes could be identified among the purified antibodies based on matching to a database of IgH sequences obtained from antigen-specific gut plasma cells of the same patients.

2017-07-14 | MSV000081328 | MassIVE

Transcriptomic profiles of whole blood from SARS-CoV-2 rhesus macaques

Project description:Total RNAseq profiling of whole blood collected longitudinally from 8 macaques infected with SARS-CoV-2

2020-07-30 | GSE155363 | GEO

VIA of EM CD8 T cells in vaccinated monkeys

2012-06-15 | GSE35628 | GEO

Tissue resident memory T cells trigger rapid exudation and local antibody accumulation

Project description:Adaptive immunity is didactically partitioned into humoral and cell-mediated effector mechanisms, which may imply that each arm is separate and does not function together. Here, we report that the activation of CD8+ resident memory T cells in nonlymphoid tissues triggers vascular permeability, which facilitates rapid distribution of serum antibodies into local tissues. TRM reactivation was associated with transcriptional upregulation of antiviral signaling pathways as well as Fc receptors and components of the complement cascade. Effects were local, but evidence is presented that TRM in brain and reproductive mucosa are both competent to induce rapid antibody exudation. TRM reactivation in the mouse female genital tract increased local concentrations of virus-specific neutralizing antibodies including anti-vesicular stomatitis virus and passively transferred anti-HIV antibodies. We showed that this response was sufficient to increase the efficacy of ex vivo vesicular stomatitis virus neutralization. These results indicate that CD8+ TRM antigen recognition can enhance local humoral immunity. Microarray data comparing RNA isolated from murine female reproductive tract tissue from control tissue (SIIN) versus tissues where resident memory T cells were reactivated 9 hours prior (GP33). Control mice (SIIN) received the irrelevant peptide SIINFEKL transcervically. Experimental mice (GP33) recieved the peptide gp33, which P14 resident memory T cells specifically recognize, transcervically.

2022-12-09 | GSE219002 | GEO

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