Highlights d Slow delivery immunization enhances HIV neutralizing antibody development in monkeys d Slow delivery immunization alters immunodominance of the responding B cells d Weekly longitudinal germinal center (GC) B and T FH analyses provides new GC insights d High-resolution rhesus immunoglobulin locus genomic reference sequence

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B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR.Electronic supplementary materialThe online version of this article (10.1186/s13073-018-0528-3) contains supplementary material, which is available to authorized users.

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-β 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. IMPORTANCE West 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.

232 words; Importance: 122 words; and Text: 4,850 words. 22 ABSTRACT 23West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. 24Neuroinvasive WNV infection results in encephalitis and can lead to prolonged 25 neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting 26 antiviral defenses through the induction of antiviral effectors, which function to restrict 27 viral replication and spread. However, our understanding of the antiviral response to 28 WNV infection is mostly derived from analysis of bulk cell populations. It is becoming 29 increasingly apparent that substantial heterogeneity in cellular processes exists among 30 individual cells, even within a seemingly homogenous cell population. Here, we present 31 WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the 32 transcriptional variation and viral RNA burden across single cells. We observed that 33 only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, 34 and this did not appear to depend on viral RNA abundance within the same cell. 35 Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I 36 response, with genes displaying high unimodal and bimodal expression patterns. 37 Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, 38 corresponding with a precipitous decline in expression in cells with high viral RNA levels. 39 Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a 40 high-throughput technique for single-cell transcriptomics and WNV RNA detection. This 41 approach can be implemented in other models to provide insights into the cellular 42 features of protective immunity and identify novel therapeutic targets. 43 44 45 IMPORTANCE 46 West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent 47 epidemics of neuroinvasive disease. Type I interferon is essential for promoting an 48 antiviral response against WNV infection; however, it is unclear how heterogeneity in 49 the antiviral response at the single-cell level impacts viral control. Specifically, 50 conventional approaches lack the ability to distinguish differences across cells with 51 varying viral abundance. The significance of our research is to demonstrate a new 52 technique for studying WNV infection at the single-cell level. We discovered extensive 53 variation in antiviral gene expression and viral abundance across cells. This protocol 54 can be applied to primary cells or in vivo models to better understand the underlying 55 cellular heterogeneity following WNV infection for the development of targeted 56 therapeutic strategies. 57 58 59 60 61