Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

mRNA-1273 vaccination protects against SARS-CoV-2-elicited lung inflammation in nonhuman primates.

Vaccine-elicited SARS-CoV-2 antibody responses are an established correlate of protection against viral infection in humans and nonhuman primates. However, it is less clear that vaccine-induced immunity is able to limit infection-elicited inflammation in the lower respiratory tract. To assess this, we collected bronchoalveolar lavage fluid samples after SARS-CoV-2 strain USA-WA1/2020 challenge from rhesus macaques vaccinated with mRNA-1273 in a dose-reduction study. Single-cell transcriptomic profiling revealed a broad cellular landscape 48 hours after challenge, with distinct inflammatory signatures that correlated with viral RNA burden in the lower respiratory tract. These inflammatory signatures included phagocyte-restricted expression of chemokines, such as CXCL10 and CCL3, and the broad expression of IFN-induced genes, such as MX1, ISG15, and IFIT1. Induction of these inflammatory profiles was suppressed by prior mRNA-1273 vaccination in a dose-dependent manner and negatively correlated with prechallenge serum and lung antibody titers against SARS-CoV-2 spike. These observations were replicated and validated in a second independent macaque challenge study using the B.1.351/Beta variant of SARS-CoV-2. These data support a model wherein vaccine-elicited antibody responses restrict viral replication following SARS-CoV-2 exposure, including limiting viral dissemination to the lower respiratory tract and infection-mediated inflammation and pathogenesis.

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques.

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq.

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5' capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5' to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5' end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5' capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5' capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection.

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.

Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and metabolic profiling.

Generating effective and durable T cell immunity is a critical prerequisite for vaccination against dengue virus (DENV) and other viral diseases. However, understanding the molecular mechanisms of vaccine-elicited T cell immunity remains a critical knowledge gap in vaccinology. In this study, we utilize single-cell RNA sequencing (scRNAseq) and longitudinal TCR clonotype analysis to identify a unique transcriptional signature present in acutely activated and clonally-expanded T cells that become committed to the memory repertoire. This effector/memory-associated transcriptional signature is dominated by a robust metabolic transcriptional program. Based on this transcriptional signature, we are able to define a set of markers that identify the most durable vaccine-reactive memory-precursor CD8+ T cells. This study illustrates the power of scRNAseq as an analytical tool to assess the molecular mechanisms of host control and vaccine modality in determining the magnitude, diversity and persistence of vaccine-elicited cell-mediated immunity.

NSD1 mitigates caspase-1 activation by listeriolysin O in macrophages.

Mammals and plants share pathogen-sensing systems named nod-like receptors (NLRs). Some NLRs form the inflammasome, a protein scaffold that regulates the secretion of interleukin (IL)-1ß and IL-18 by cleaving catalytically inactive substrates into mature cytokines. Here, we show an immune conservation between plant and mammalian NLRs and demonstrate that the murine nuclear receptor binding SET domain protein 1 (NSD1), a protein that bears similarity to the NLR regulator enhanced downy mildew 2 (EDM2) in Arabidopsis, diminishes caspase-1 activity during extracellular stimulation with Listeria monocytogenes listeriolysin O (LLO). EDM2 is known to regulate plant developmental processes, whereas NSD1 is associated with developmental disorders. We observed that NSD1 neither affects nuclear factor (NF)-κB signaling nor regulates NLRP3 inflammasome gene expression at the chromatin, transcriptional or translational level during LLO stimulation of macrophages. Silencing of Nsd1 followed by LLO stimulation led to increased caspase-1 activation, enhanced post-translational maturation of IL-1ß and IL-18 and elevated pyroptosis, a form of cell death associated with inflammation. Furthermore, treatment of macrophages with LLO(W492A), which lacks hemolytic activity due to a tryptophan to alanine substitution in the undecapeptide motif, indicates the importance of functional LLO for NSD1 regulation of the NLRP3 inflammasome. Taken together, our results indicate that NLR signaling in plants may be used for gene discovery in mammals.