Antigen-experienced memory B-cells (MBC) are endowed with enhanced functional properties compared to naïve B cells and play an important role in the humoral response. However, the epigenetic enzymes and programs that govern their rapid differentiation are incompletely understood. Here, the role of the histone H3 lysine 27 methyltransferase EZH2 in the formation of MBC in response to an influenza infection was determined in Mus musculus. EZH2 was expressed in all postactivated B-cell subsets, including MBC and antibody-secreting cells (ASC), with maximal expression in germinal center (GC) B cells. Deletion of EZH2 resulted in a skewing of the MBC pool towards a non-GC, IgM+ MBC subset that failed to fully express CCR6 and CD73 at both early and late infection time points. Intriguingly, although EZH2 protein levels were reduced in knockout MBC, deletion was not fully efficient, indicating a strong selective pressure to maintain EZH2 methyltransferase activity. Single-cell RNA-seq of antigen-specific MBC identified a core set of upregulated genes that are likely EZH2 targets across MBC subsets. Finally, defects in the ability to form secondary ASC and GC cells in response to a lethal challenge were observed in EZH2-deficient mice, indicating significant functional impairment in the absence of EZH2. These data show that EZH2 is a critical epigenetic modulator of MBC differentiation and functional potential during reactivation.

Systemically administered influenza vaccines provide strain-limited protection, while influenza infection of the respiratory epithelium supports development of lung resident memory B and T cells and more broadly reactive antibody responses. To test whether local antigen delivery is critical for establishing broad immunity, we directly compared respiratory tract and systemic delivery of influenza antigens to mice and hamsters using a replication-deficient adenovirus serotype 5 vector (Ad5[E1-,E2b-,E3-]). Both immunization routes elicited antibody responses in the lower respiratory tract and antigen-specific B and T cells in the draining lymph node. However, only intranasal immunization established lung-resident memory B and T cells, induced IgA responses in the upper respiratory tract directly at the site of viral entry and supported generation of IgA and IgG antibodies that bound antigenically drifted and distantly related influenza strains, including those of avian origin. Intranasal immunization accelerated viral clearance following heterologous virus challenge and was associated with limited pulmonary inflammation and fibrosis. Thus, intranasal immunization with the immunologically stealthy Ad5[E1-,E2b-,E3-] platform supports respiratory and systemic immunity to divergent influenza strains in the absence of overt lung immunopathology, suggesting that local antigen delivery may be key to development of more broadly protective “universal” flu vaccines.

Background/Objectives: The emergence of SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus-vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). Methods: K18-hACE2 mice and LVG Syrian hamsters were vaccinated with a single intranasal dose of MV-014-212 or MV-014-212-delta. Systemic and mucosal immunity were assessed following vaccination, and protection was evaluated following Delta SARS-CoV-2 challenge. In vaccinated hamsters, morbidity, viral shedding, and lung inflammation and injury were also assessed following natural exposure to infected cagemates. Results: A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8⁺ lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against the Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung, and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Conclusions: Intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.

Memory B cells (MBCs) are an integral part of the humoral immune response with the capacity to both reseed germinal center reactions and rapidly form antibody-secreting plasma cells (ASCs) upon secondary antigen encounter. MBCs arise via both T cell–dependent and –independent routes and while CD4+ T cells are not required for their formation, it is still not clear if or how initial T:B-cell interactions influence the molecular programming and diversity of T cell–independent MBCs. To address this, we characterized MBCs that form in response to influenza infection in major histocompatibility complex class II knockout (MHCII-KO) mice, which lack CD4+ T cells. Consistent with T cell–independent responses, the MBCs that formed in MHCII-KO mice were reduced in number and did not acquire surface expression of CCR6 and class-switched BCR. Transcriptional profiling identified cytokine- and activation-induced genes that were reduced in expression in MHCII-KO MBCs compared to wild type (WT). Adoptive transfer of MHCII-KO B cells into WT hosts, which cannot receive peptide/MHCII–TCR cognate interactions, revealed an ability of MHCII-KO cells to form MBCs and ASCs. Single-cell RNA sequencing revealed minimal transcriptional differences between MHCII-KO and WT MBCs, indicating that cognate CD4+ T-cell interactions provide limited early programming instruction to MBCs. MHCII-KO MBCs were able to clonally expand, class-switch, and form the same diverse transcriptional clusters as WT. These data indicate that early differentiating MBCs can seed a diverse pool of MBC populations in response to influenza infection independent of cognate T-cell help.

The emergence of new SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission to others. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from either the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8⁺ lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Therefore, intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.