Adenovirus-vectored SARS-CoV-2 vaccine expressing S1-N fusion protein.

Additional COVID-19 vaccines that are safe and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2 S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N). A single subcutaneous immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime-boost vaccination, and further improved through intramuscular heterologous prime-boost vaccination using subunit recombinant S1 protein. Priming with low dose (1 × 1010 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wild-type recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, which was sustained against Beta and Gamma SARS-CoV-2 variants. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19-based vaccines incorporating the nucleoprotein as a target antigen.

High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status.

Clinical definitions of asthma fail to capture the heterogeneity of immune dysfunction in severe, treatment-refractory disease. Applying mass cytometry and machine learning to bronchoalveolar lavage (BAL) cells, we find that corticosteroid-resistant asthma patients cluster largely into two groups: one enriched in interleukin (IL)-4+ innate immune cells and another dominated by interferon (IFN)-γ+ T cells, including tissue-resident memory cells. In contrast, BAL cells of a healthier population are enriched in IL-10+ macrophages. To better understand cellular mediators of severe asthma, we developed the Immune Cell Linkage through Exploratory Matrices (ICLite) algorithm to perform deconvolution of bulk RNA sequencing of mixed-cell populations. Signatures of mitosis and IL-7 signaling in CD206-FcεRI+CD127+IL-4+ innate cells in one patient group, contrasting with adaptive immune response in T cells in the other, are preserved across technologies. Transcriptional signatures uncovered by ICLite identify T-cell-high and T-cell-poor severe asthma patients in an independent cohort, suggesting broad applicability of our findings.

A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice.

Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.

Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype.

Although human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

ZBTB32 Restricts the Duration of Memory B Cell Recall Responses.

Memory B cell responses are more rapid and of greater magnitude than are primary Ab responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of Ab recall responses. ZBTB32 is highly expressed by mouse and human memory B cells but not by their naive counterparts. Zbtb32(-/-) mice mount normal primary Ab responses to T-dependent Ags. However, Zbtb32(-/-) memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32(-/-) secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32(-/-) secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of Ab recall responses.

A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells.

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies, we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system, which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor, yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures, revealing that the reaction engenders both MBC subsets with different immune effector function and, ultimately, LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity, for vaccine development, and for understanding long-term pathogen resistance.

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype.

Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.