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Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNAsequencing analyses of samples frommouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

In this study, by characterizing several human monoclonal antibodies (mAbs) isolated from single B cells of the COVID-19–recovered individuals in India who experienced ancestral Wuhan strain (WA.1) of SARS-CoV-2 during early stages of the pandemic, we found a receptor binding domain (RBD)–specific mAb 002-S21F2 that has rare gene usage and potently neutralized live viral isolates of SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) with IC 50 ranging from 0.02 to 0.13 μg/ml. Structural studies of 002-S21F2 in complex with spike trimers of Omicron and WA.1 showed that it targets a conformationally conserved epitope on the outer face of RBD (class 3 surface) outside the ACE2-binding motif, thereby providing a mechanistic insights for its broad neutralization activity. The discovery of 002-S21F2 and the broadly neutralizing epitope it targets have timely implications for developing a broad range of therapeutic and vaccine interventions against SARS-CoV-2 variants including Omicron sublineages.

B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna's mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern.

While humoral immune responses to infection or vaccination with ancestral SARS-CoV-2 have been well-characterized, responses elicited by infection with variants are less understood. Here we characterized the repertoire, epitope specificity, and cross-reactivity of antibodies elicited by Beta and Gamma variant infection compared to ancestral virus. We developed a high-throughput approach to obtain single-cell immunoglobulin sequences and isolate monoclonal antibodies for functional assessment. Spike-, RBD- and NTD-specific antibodies elicited by Beta- or Gamma-infection exhibited a remarkably similar hierarchy of epitope immunodominance for RBD and convergent V gene usage when compared to ancestral virus infection. Additionally, similar public B cell clones were elicited regardless of infecting variant. These convergent responses may account for the broad cross-reactivity and continued efficacy of vaccines based on a single ancestral variant.WA1, Beta and Gamma variants of SARS-CoV-2 all elicit antibody responses targeting similar RBD epitopes; public and cross-reactive clones are common.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1–3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

Neutralizing Activity against SARS-CoV-2 Variants Among samples obtained from persons who had received the mRNA-1273 or BNT162b2 vaccines, neutralizing antibody titers against the B.1.617.1 variant...

SummarySARS-CoV-2 Omicron is highly transmissible and has substantial resistance to antibody neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-specific vaccines would enhance immunity and protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing antibody titers against D614G were 4760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (pre-boost), respectively, and 320 and 110 for Omicron. Two weeks after boost, titers against D614G and Omicron increased to 5360 and 2980, respectively, for mRNA-1273 and 2670 and 1930 for mRNA-Omicron. Following either boost, 70-80% of spike-specific B cells were cross-reactive against both WA1 and Omicron. Significant and equivalent control of virus replication in lower airways was observed following either boost. Therefore, an Omicron boost may not provide greater immunity or protection compared to a boost with the current mRNA-1273 vaccine.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant emerged in November 2021 and consists of several mutations within the spike. We use serum from mRNA-vaccinated individuals to measure neutralization activity against omicron in a live-virus assay. At 2–4 weeks after a primary series of vaccinations, we observe a 30-fold reduction in neutralizing activity against omicron. Six months after the initial two-vaccine doses, sera from naive vaccinated subjects show no neutralizing activity against omicron. In contrast, COVID-19-recovered individuals 6 months after receiving the primary series of vaccinations show a 22-fold reduction, with the majority of the subjects retaining neutralizing antibody responses. In naive individuals following a booster shot (third dose), we observe a 14-fold reduction in neutralizing activity against omicron, and over 90% of subjects show neutralizing activity. These findings show that a third dose is required to provide robust neutralizing antibody responses against the omicron variant., Graphical abstract, Here in this study, Edara et al. report that 6 months after the primary series (initial two doses) of mRNA vaccination, the majority of the subjects lost detectable neutralizing antibody titers against omicron. The data suggest that a third booster dose is necessary to sustain neutralizing activity against omicron.

We describe rapid detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant using targeted spike single-nucleotide polymorphism polymerase chain reaction and viral genome sequencing. This case occurred in a fully vaccinated and boosted returning traveler with mild symptoms who was identified through community surveillance rather than clinical care.

The development of the highly efficacious mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology. However, reports of waning vaccine efficacy, coupled with the emergence of variants of concern that are resistant to antibody neutralization, have raised concerns about the potential lack of durability of immunity to vaccination. We recently reported findings from a comprehensive analysis of innate and adaptive immune responses in 56 healthy volunteers who received two doses of the BNT162b2 vaccination. Here, we analyzed antibody responses to the homologous Wu strain as well as several variants of concern, including the emerging Mu (B.1.621) variant, and T cell responses in a subset of these volunteers at six months (day 210 post-primary vaccination) after the second dose. Our data demonstrate a substantial waning of antibody responses and T cell immunity to SARS-CoV-2 and its variants, at 6 months following the second immunization with the BNT162b2 vaccine. Notably, a significant proportion of vaccinees have neutralizing titers below the detection limit, and suggest a 3rd booster immunization might be warranted to enhance the antibody titers and T cell responses.

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID50pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID50GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations.One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

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

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

BackgroundVaccine efficacy against the B.1.351 variant following mRNA-1273 vaccination in humans has not been determined. Nonhuman primates (NHP) are a useful model for demonstrating whether mRNA-1273 mediates protection against B.1.351.MethodsNonhuman primates received 30 or 100 µg of mRNA-1273 as a prime-boost vaccine at 0 and 4 weeks, a single immunization of 30 µg at week 0, or no vaccine. Antibody and T cell responses were assessed in blood, bronchioalveolar lavages (BAL), and nasal washes. Viral replication in BAL and nasal swabs were determined by qRT-PCR for sgRNA, and histopathology and viral antigen quantification were performed on lung tissue post-challenge.ResultsEight weeks post-boost, 100 µg x2 of mRNA-1273 induced reciprocal ID50 neutralizing geometric mean titers against live SARS-CoV-2 D614G and B.1.351 of 3300 and 240, respectively, and 430 and 84 for the 30 µg x2 group. There were no detectable neutralizing antibodies against B.1351 after the single immunization of 30 µg. On day 2 following B.1.351 challenge, sgRNA in BAL was undetectable in 6 of 8 NHP that received 100 µg x2 of mRNA-1273, and there was a ∼2-log reduction in sgRNA in NHP that received two doses of 30 µg compared to controls. In nasal swabs, there was a 1-log10 reduction observed in the 100 µg x2 group. There was limited inflammation or viral antigen in lungs of vaccinated NHP post-challenge.ConclusionsImmunization with two doses of mRNA-1273 achieves effective immunity that rapidly controls lower and upper airway viral replication against the B.1.351 variant in NHP.

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

SummaryHuman antibody responses are established by the generation of combinatorial sequence diversity in antibody variable domains, followed by iterative rounds of mutation and selection via T cell recognition of antigen peptides presented on MHC-II. Here, we report that MHC-II peptide epitope deletion from B cell receptors (BCRs) correlates with antibody development in vivo. Large-scale antibody sequence analysis and experimental validation of peptide binding revealed that MHC-II epitope removal from BCRs is linked to genetic signatures of T cell help, and donor-specific antibody repertoire modeling demonstrated that somatic hypermutation selectively targets the personalized MHC-II epitopes in antibody variable regions. Mining of class-switched sequences and serum proteomic data revealed that MHC-II epitope deletion is associated with antibody class switching and long-term secretion into serum. These data suggest that the MHC-II peptide epitope content of a BCR is an important determinant of antibody maturation that shapes the composition and durability of humoral immunity.HighlightsAntibody somatic hypermutation selectively removes MHC-II peptide epitopes from B cell receptors.Antibodies with lower MHC-II epitope content show evidence of greater T cell help, including class-switching.MHC-II peptide epitope removal from a BCR is linked to long-term antibody secretion in serum.MHC-II genotype provides a personalized selection pressure on human antibody development.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

Summary A population at low census might go extinct or instead transition into exponential growth to become firmly established. Whether this pivotal event occurs for a within-host pathogen can be the difference between health and illness. Here, we define the principles governing whether HIV-1 spread among cells fails or becomes established by coupling stochastic modeling with laboratory experiments. Following ex vivo activation of latently infected CD4 T cells without de novo infection, stochastic cell division and death contributes to high variability in the magnitude of initial virus release. Transition to exponential HIV-1 spread often fails due to release of an insufficient amount of replication-competent virus. Establishment of exponential growth occurs when virus produced from multiple infected cells exceeds a critical population size. We quantitatively define the crucial transition to exponential viral spread. Thwarting this process would prevent HIV transmission or rebound from the latent reservoir.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2∗02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2∗02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.