Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

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 ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

mRNA-1273 protects against SARS-CoV-2 beta infection in nonhuman primates.

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.

Multiple Origins of Virus Persistence during Natural Control of HIV Infection.

Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

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.

HIV silencing and cell survival signatures in infected T cell reservoirs.

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.

Identification of astrocyte regulators by nucleic acid cytometry.

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 RNA sequencing analyses of samples from mouse 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.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

HIV infected CD4+ T cell clones are more stable than uninfected clones during long-term antiretroviral therapy.

Although combination antiretroviral therapy (ART) blocks HIV replication, it is not curative because infected CD4+ T cells that carry intact, infectious proviruses persist. Understanding the behavior of clones of infected T cells is important for understanding the stability of the reservoir; however, the stabilities of clones of infected T cells in persons on long-term ART are not well defined. We determined the relative stabilities of clones of infected and uninfected CD4+ T cells over time intervals of one to four years in three individuals who had been on ART for 9-19 years. The largest clones of uninfected T cells were larger than the largest clones of infected T cells. Clones of infected CD4+ T cells were more stable than clones of uninfected CD4+ T cells of a similar size. Individual clones of CD4+ T cells carrying intact, infectious proviruses can expand, contract, or remain stable over time.

Rapid Detection and Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Variant in a Returning Traveler.

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.

A non-human primate model for human norovirus infection.

Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection. We show that rhesus macaques are susceptible to oral infection with human noroviruses from two different genogroups. Variation in duration of virus shedding (days to weeks) between animals, evolution of the virus over the time of infection, induction of virus-specific adaptive immune responses, susceptibility to reinfection and preferential replication of norovirus in the jejunum of rhesus macaques was similar to infection reported in humans. We found minor pathological signs and changes in epithelial cell surface glycosylation patterns in the small intestine during infection. Detection of viral protein and RNA in intestinal biopsies confirmed the presence of the virus in chromogranin A-expressing epithelial cells, as it does in humans. Thus, rhesus macaques are a promising non-human primate model to evaluate vaccines and therapeutics against norovirus disease.

Adjuvanted SARS-CoV-2 spike protein vaccination elicits long-lived plasma cells in nonhuman primates.

Durable humoral immunity is mediated by long-lived plasma cells (LLPCs) that reside in the bone marrow. It remains unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein vaccination is able to elicit and maintain LLPCs. Here, we describe a sensitive method to identify and isolate antigen-specific LLPCs by tethering antibodies secreted by these cells onto the cell surface. Using this method, we found that two doses of adjuvanted SARS-CoV-2 spike protein vaccination are able to induce spike protein-specific LLPC reservoirs enriched for receptor binding domain specificities in the bone marrow of nonhuman primates that are detectable for several months after vaccination. Immunoglobulin gene sequencing confirmed that several of these LLPCs were clones of memory B cells elicited 2 weeks after boost that had undergone further somatic hypermutation. Many of the antibodies secreted by these LLPCs also exhibited improved neutralization and cross-reactivity compared with earlier time points. These findings establish our method as a means to sensitively and reliably detect rare antigen-specific LLPCs and demonstrate that adjuvanted SARS-CoV-2 spike protein vaccination establishes spike protein-specific LLPC reservoirs.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates.

As SARS-CoV-2 variants continue evolving, testing updated vaccines in non-human primates remains important for guiding human clinical practice. To date, such studies have focused on antibody titers and antigen-specific B and T cell frequencies. Here, we extend our understanding by integrating innate and adaptive immune responses to mRNA-1273 vaccination in rhesus macaques. We sorted innate immune cells from a pre-vaccine time point, as well as innate immune cells and antigen-specific peripheral B and T cells two weeks after each of two vaccine doses and used single-cell sequencing to assess the transcriptomes and adaptive immune receptors of each cell. We show that a subset of S-specific T cells expresses cytokines critical for activating innate responses, with a concomitant increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more cytotoxic phenotype. The second vaccine dose, administered 4 weeks after the first, elicits an increase in circulating germinal center-like B cells 2 weeks later, which are more clonally expanded and enriched for epitopes in the receptor binding domain. Both doses stimulate inflammatory response genes associated with elevated antibody production. Overall, we provide a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggest potential mechanisms for the enhanced response to secondary exposure.

High frequency of HIV precursor-target-specific B cells in sub-Saharan populations.

HIV gp120 engineered outer domain germline-targeting version 8 (eOD-GT8) was designed specifically to engage naive B cell precursors of VRC01-class antibodies. However, the frequency and affinity of naive B cell precursors able to recognize eOD-GT8 have been evaluated only in U.S. populations. HIV infection is disproportionally concentrated in sub-Saharan Africa, so we seek to characterize naive B cells able to recognize eOD-GT8 in sub-Saharan cohorts. We demonstrate that people from sub-Saharan Africa have a higher or equivalent frequency of naive B cells able to engage eOD-GT8 compared with people from the U.S. Genetically, the higher frequency of eOD-GT8-positive cells is accompanied by a higher level of naive B cells with gene signatures characteristic of the VRC01 class, as well as other CD4bs-directed antibodies. Our study demonstrates that vaccination with eOD-GT8 in sub-Saharan Africa could be successful at expanding and establishing a pool of CD4bs-directed memory B cells from naive precursors.

HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide.

Elicitation of antibodies that neutralize the tier-2 neutralization-resistant isolates that typify HIV-1 transmission has been a long-sought goal. Success with prefusion-stabilized envelope trimers eliciting autologous neutralizing antibodies has been reported in multiple vaccine-test species, though not in humans. To investigate elicitation of HIV-1 neutralizing antibodies in humans, here, we analyze B cells from a phase I clinical trial of the "DS-SOSIP"-stabilized envelope trimer from strain BG505, identifying two antibodies, N751-2C06.01 and N751-2C09.01 (named for donor-lineage.clone), that neutralize the autologous tier-2 strain, BG505. Though derived from distinct lineages, these antibodies form a reproducible antibody class that targets the HIV-1 fusion peptide. Both antibodies are highly strain specific, which we attribute to their partial recognition of a BG505-specific glycan hole and to their binding requirements for a few BG505-specific residues. Prefusion-stabilized envelope trimers can thus elicit autologous tier-2 neutralizing antibodies in humans, with initially identified neutralizing antibodies recognizing the fusion-peptide site of vulnerability.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera.

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.