Applications of high-throughput single B-cell sequencing to accelerate rational vaccine design

Understanding the antibody repertoire response to vaccination is critical for the rational design and evaluation of experimental vaccines. Immune receptors comprise two chains encoded by separate mRNA strands and thus conventional NextGen sequencing fails to identify the native pairings encoded by individual lymphocytes. To overcome this limitation, we are applying recent technical advances in high-throughput sequencing of complete antibodies (i.e., paired heavy and light chain sequencing) to generate a quantitative understanding of experimental vaccine performance and to accelerate vaccine design. We apply repertoire-based metrics of vaccine-elicited antibodies to evaluate and select promising candidate immunogens for inducing HIV-1 Envelope-specific VRC01-class antibodies. The VRC01 class of broadly neutralizing antibodies have been observed in multiple individuals and targets the HIV CD4 binding site via a common recognition motif that requires specific features in both heavy and light chains (e.g., VH1-2 heavy chain V-gene and a short, £5 amino acid light chain CDR3). We are using paired heavy and light chain sequencing to quantify the performance of various candidate HIV immunogens for inducing VRC01-class broadly neutralizing HIV antibodies in transgenic mouse models. We are also elucidating the ontogeny of antibodies in vaccinated and naturally infected human subjects and animal models via interrogation of paired heavy and light chain antibody sequences and antibody synthesis/testing of promising clones, including experimental influenza vaccine trials and a Phase I Ebola vaccine trial. These next-generation immunoanalytic approaches are providing detailed molecular feedback regarding experimental vaccine performance to accelerate vaccine design efforts against pathogens of major public health importance

Influenza A virus evolution and spatio-temporal dynamics in Eurasian wild birds: a phylogenetic and phylogeographical study of whole-genome sequence data.

Low pathogenic avian influenza A viruses (IAVs) have a natural host reservoir in wild waterbirds and the potential to spread to other host species. Here, we investigated the evolutionary, spatial and temporal dynamics of avian IAVs in Eurasian wild birds. We used whole-genome sequences collected as part of an intensive long-term Eurasian wild bird surveillance study, and combined this genetic data with temporal and spatial information to explore the virus evolutionary dynamics. Frequent reassortment and co-circulating lineages were observed for all eight genomic RNA segments over time. There was no apparent species-specific effect on the diversity of the avian IAVs. There was a spatial and temporal relationship between the Eurasian sequences and significant viral migration of avian IAVs from West Eurasia towards Central Eurasia. The observed viral migration patterns differed between segments. Furthermore, we discuss the challenges faced when analysing these surveillance and sequence data, and the caveats to be borne in mind when drawing conclusions from the apparent results of such analyses.We thank all ornithologists and other collaborators for their continuous support. We thank V. Munster, E. Skepner, O. Vuong, C. Baas, J. Guldemeester, M. Schutten, G. van der Water, D. Smith and E. Bortz for technical support and stimulating discussions. This manuscript was prepared while D.E. Wentworth was employed at the JCVI. The opinions expressed in this article are the author’s own and do not reflect the view of the Centers for Disease Control, the Department of Health and Human Services, or the United States government. This work was supported by NIAID/NIH contract HHSN266200700010C, HHSN272201400008C, HHSN272201400006C and HHSN272200900007C, a Wellcome Trust Fellowship Strategic Travel Award under contract WT089235MF, a DTRA FRCWMD Broad Agency Announcement under contract HDTRA1-09-14-FRCWMD GRANT11177182, by the EU Framework six program NewFluBird (044490) by contracts with the Dutch Ministry of Economic Affairs and a NIAID/NIH CEIRS travel grant under contract HHSN266200700010C. The Swedish sampling and analysis was supported by the Swedish Research Councils VR and FORMAS.This is the final version of the article. It first appeared from the Society for General Microbiology via http://dx.doi.org/10.1099/vir.0.00015

Elite control of HIV is associated with distinct functional and transcriptional signatures in lymphoid tissue CD8+ T cells

The functional properties of circulating CD8+ T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8+ T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8+ T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8+ T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4+ T cell–associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8+ T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV

Avian Influenza Viruses in Wild Birds: Virus Evolution in a Multihost Ecosystem.

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter, or stage. The Republic of Georgia is located at the intersection of three migratory flyways: the Central Asian flyway, the East Africa/West Asia flyway, and the Black Sea/Mediterranean flyway. For six complete study years (2010 to 2016), we collected AIV samples from various duck and gull species that breed, migrate, and overwinter in Georgia. We found a substantial subtype diversity of viruses that varied in prevalence from year to year. Low-pathogenic AIV (LPAIV) subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, and H16N3, and two highly pathogenic AIVs (HPAIVs) belonging to clade 2.3.4.4, H5N5 and H5N8, were found. Whole-genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide sequence diversity for LPAIVs among different host species. Hemagglutinin clade 2.3.4.4 H5N8 viruses, which circulated in Eurasia during 2014 and 2015, did not reassort, but analysis after their subsequent dissemination during 2016 and 2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions, with maintenance of local AIVs in Georgia, whereas other lineages showed considerable genetic interrelationships with viruses circulating in other parts of Eurasia and Africa, despite relative undersampling in the area.IMPORTANCE Waterbirds (e.g., gulls and ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate the dispersal of AIVs at intercontinental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or reassort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates AIV evolutionary dynamics within a multihost ecosystem at a stopover site where three major migratory flyways intersect. Our analysis of this ecosystem over a 6-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to mitigating both the risk of incursion into domestic poultry and the potential risk to mammalian hosts, including humans

Characterization of the Neutralizing Antibody Response in a Case of Genetically Linked HIV Superinfection.

This report describes the identification of a genetically confirmed linked heterosexual human immunodeficiency virus (HIV) superinfection (HIV-SI) in a woman with chronic HIV infection who acquired a second strain of the virus from her husband. Serum neutralizing antibody (NAb) responses against their homologous and heterologous viruses, including the superinfecting strain, in the woman and her husband were examined before and after onset of HIV-SI. The woman displayed a moderately potent and broad anti-HIV NAb response prior to superinfection but did not possess NAb activity against the superinfecting strain. This case highlights the unique potential of linked HIV-SI studies to examine natural protection from HIV infection

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host's antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency

T cell differentiation and lineage choice are determined by the TCR

Next-generation sequencing technologies have revealed that adaptive immunity is underpinned by a vast array of T cell receptors (TCRs). However, it has proven difficult to interpret these extensive datasets, in part because the field previously lacked a comprehensive and internally controlled reference atlas encompassing the full spectrum of phenotypically defined subsets in each lineage. To address this knowledge gap, we sequenced 74 million TCRs expressed by discrete CD4+ and CD8+ memory T cell populations across genetically unrelated individuals, providing a resource to inform basic and applied studies of repertoire compartmentalisation within the adaptive immune system. Using this resource, we found that T cell differentiation could not be explained solely by the self-renewing effector model and, unexpectedly, that T cell fate could be predicted by specific genetic and physicochemical features of the TCR

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies

Epigenetic silencing of CD4 expression in nonpathogenic SIV infection in African green monkeys

African green monkeys (AGMs) are natural hosts of SIV that postthymically downregulate CD4 to maintain a large population of CD4–CD8aa+ virus-resistant cells with Th functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process, we performed genome-wide transcriptional analysis on T cells induced to downregulate CD4 in vitro from AGMs and closely related patas monkeys and T cells that maintain CD4 expression from rhesus macaques. In T cells that downregulated CD4, pathway analysis revealed an atypical regulation of the DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2 deoxycytidine. This signature was driven largely by the dioxygenase TET3, which became downregulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4–CD8aa+ T cells sorted directly ex vivo. These results suggest that AGMs use epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and HIV-1 entry receptor expression in hosts that become progressively infected with SIV, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo