AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans.

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Multi-omics analysis of mucosal and systemic immunity to SARS-CoV-2 after birth.

The dynamics of immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infants and young children by analyzing blood samples and weekly nasal swabs collected before, during, and after infection with Omicron and non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, showed no sign of decay for up to 300 days. Infants mounted a robust mucosal immune response characterized by inflammatory cytokines, interferon (IFN) α, and T helper (Th) 17 and neutrophil markers (interleukin [IL]-17, IL-8, and CXCL1). The immune response in blood was characterized by upregulation of activation markers on innate cells, no inflammatory cytokines, but several chemokines and IFNα. The latter correlated with viral load and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell multi-omics. Together, these data provide a snapshot of immunity to infection during the initial weeks and months of life.

Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life.

The dynamics of innate and adaptive immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to SARS-CoV-2 infection in infants and young children in the first weeks and months of life by analyzing blood samples collected before, during, and after infection with Omicron and Non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, were stably maintained for >300 days. Antigen-specific memory B cell (MCB) responses were durable for 150 days but waned thereafter. Somatic hypermutation of V-genes in MCB accumulated progressively over 9 months. The innate response was characterized by upregulation of activation markers on blood innate cells, and a plasma cytokine profile distinct from that seen in adults, with no inflammatory cytokines, but an early and transient accumulation of chemokines (CXCL10, IL8, IL-18R1, CSF-1, CX3CL1), and type I IFN. The latter was strongly correlated with viral load, and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell transcriptomics. Consistent with this, single-cell ATAC-seq revealed enhanced accessibility of chromatic loci targeted by interferon regulatory factors (IRFs) and reduced accessibility of AP-1 targeted loci, as well as traces of epigenetic imprinting in monocytes, during convalescence. Together, these data provide the first snapshot of immunity to infection during the initial weeks and months of life.

Systems vaccinology of the BNT162b2 mRNA vaccine in humans.

The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) a greater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.

Systems biological assessment of human immunity to BNT162b2 mRNA vaccination.

The emergency use authorization of two COVID-19 mRNA vaccines in less than a year since the emergence of SARS-CoV-2, represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems biological approach to comprehensively profile the innate and adaptive immune responses in 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent strain and the variant of concern, B.1.351, but no induction of autoantibodies, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. The innate response induced within the first 2 days of booster vaccination was profoundly increased, relative to the response at corresponding times after priming. Thus, there was a striking increase in the: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of IFN- y in the plasma, which correlated with enhanced pSTAT3 and pSTAT1 levels in monocytes and T cells; and (iii) transcriptional signatures of innate responses characteristic of antiviral vaccine responses against pandemic influenza, HIV and Ebola, within 2 days following booster vaccination compared to primary vaccination. Consistent with these observations, single-cell transcriptomics analysis of 242,479 leukocytes demonstrated a ~100-fold increase in the frequency of a myeloid cluster, enriched in a signature of interferon-response transcription factors (TFs) and reduced in AP-1 TFs, one day after secondary immunization, at day 21. Finally, we delineated distinct molecular pathways of innate activation that correlate with CD8 T cell and nAb responses and identified an early monocyte-related signature that was associated with the breadth of the nAb response against the B1.351 variant strain. Collectively, these data provide insights into the immune responses induced by mRNA vaccines and demonstrate their capacity to stimulate an enhanced innate response following booster immunization.

Baseline immune profile by CyTOF can predict response to an investigational adjuvanted vaccine in elderly adults.

BACKGROUND: Mass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry. METHODS: In this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort. RESULTS: Both CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders. CONCLUSIONS: High parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.

Human papillomavirus status and gene expression profiles of oropharyngeal and oral cancers from European American and African American patients.

BACKGROUND: Disparities in prevalence, human papillomavirus (HPV) status, and mortality rates for head and neck cancer have been described between African American and European American patients. METHODS: We studied the HPV status and gene expression profiles in 56 oropharyngeal/oral cavity tumors and 9 normal tissue samples from European American and African American patients treated in South Carolina between 2010 and 2012. RESULTS: Overall, 59% of tumors were HPV DNA-positive, but only 48% of those expressed E7 mRNA (HPV-active). The prevalence of HPV-active tumors was 10% in African American patients and 39% in European American patients. Tumors positive for HPV DNA but negative for HPV mRNA exhibited gene expression profiles distinct from those of both HPV-active and HPV-negative cancers, suggesting that HPV DNA-positive/RNA-negative tumors may constitute a unique group. CONCLUSION: This study provides a direct assessment of differential expression patterns in HPV-related oropharyngeal cancer arising from African American and European American patients, for which there is a paucity of data. © 2015 Wiley Periodicals, Inc. Head Neck 00: 000-000, 2015.

TGF-ß regulation of gene expression at early and late stages of HPV16-mediated transformation of human keratinocytes.

In our in vitro model for HPV16-mediated transformation, HPV16-immortalized human keratinocytes (HKc/HPV16) give rise to differentiation resistant, premalignant cells (HKc/DR). HKc/DR, but not HKc/HPV16, are resistant to growth inhibition by transforming growth factor beta (TGF-ß), due to a partial loss of TGF-ß receptor type I. We show that TGF-ß activates a Smad-responsive reporter construct in HKc/DR to about 50% of the maximum levels of activation observed in HKc/HPV16. To investigate the functional significance of residual TGF-ß signaling in HKc/DR, we compared gene expression profiles elicited by TGF-ß treatment of HKc/HPV16 and HKc/DR on Agilent 44k human whole genome microarrays. TGF-ß altered the expression of cell cycle and MAP kinase pathway genes in HKc/HPV16, but not in HKc/DR. However, epithelial-mesenchymal transition (EMT) responses to TGF-ß were comparable in HKc/HPV16 and HKc/DR, indicating that the signaling pathways through which TGF-ß elicits growth inhibition diverge from those that induce EMT in HPV16-transformed cells.

A gene expression classifier of node-positive colorectal cancer.

We used digital long serial analysis of gene expression to discover gene expression differences between node-negative and node-positive colorectal tumors and developed a multigene classifier able to discriminate between these two tumor types. We prepared and sequenced long serial analysis of gene expression libraries from one node-negative and one node-positive colorectal tumor, sequenced to a depth of 26,060 unique tags, and identified 262 tags significantly differentially expressed between these two tumors (P < 2 x 10(-6)). We confirmed the tag-to-gene assignments and differential expression of 31 genes by quantitative real-time polymerase chain reaction, 12 of which were elevated in the node-positive tumor. We analyzed the expression levels of these 12 upregulated genes in a validation panel of 23 additional tumors and developed an optimized seven-gene logistic regression classifier. The classifier discriminated between node-negative and node-positive tumors with 86% sensitivity and 80% specificity. Receiver operating characteristic analysis of the classifier revealed an area under the curve of 0.86. Experimental manipulation of the function of one classification gene, Fibronectin, caused profound effects on invasion and migration of colorectal cancer cells in vitro. These results suggest that the development of node-positive colorectal cancer occurs in part through elevated epithelial FN1 expression and suggest novel strategies for the diagnosis and treatment of advanced disease.