Background: Influenza remains a persistent public health challenge due to antigenic drift and shift, necessitating vaccines capable of eliciting broad and durable immunity. Hemagglutinin (HA) antigen serves as the critical target for eliciting protective immune responses against influenza. DNA vaccines offer distinct advantages over conventional platforms, including accelerated development and induction of both humoral and cellular immune responses. Methods: To optimize HA antigen presentation, we designed and systematically compared the immunogenicity and protective efficacy of HA antigen display strategies—bacteriophage T4 fibritin (HA-Foldon) and ferritin-based virus-like particles (HA-Ferritin)—versus monomeric HA DNA vaccines against seasonal influenza viruses. Results: HA-Ferritin showed superior structural stability. All vaccines induced similar HA-specific antibody levels, but HA-Ferritin elicited higher neutralizing antibodies and stronger T cell responses. Upon challenge, HA-Ferritin and HA-Foldon protected mice from weight loss and reduced lung virus loads by 3.27 and 0.76 times, respectively. Monomeric HA provided limited protection, with only 40% survival and minimal viral or pathological reduction. Conclusions: The HA-Ferritin DNA vaccine demonstrated enhanced immunogenicity and protection, supporting structured antigen display as a promising strategy for influenza DNA vaccine development.

Background: The current assessment method of the protective efficacy of adjuvanted vaccines remains slow and labor-intensive, hindered by prolonged immunization protocols and complex assays. Methods: To overcome this bottleneck, we demonstrate that early segregated cellular biomarkers enable rapid prediction of protection, using a respiratory syncytial virus (RSV) pre-fusion F (pre-F) protein model with diverse adjuvants in mice. Results: We identified that germinal center (GC) B cell responses (Days 7 and 9 post-immunization) strongly aligned with protective efficacy, except for Alum, which achieved MF59-level protection despite lower GC responses. Crucially, follicular dendritic cell (FDC) abundance at day 7 universally predicted protection across all adjuvants, including Alum, drastically shortening discovery time and effort from at least 4–6 weeks to within 1 week. Conclusions: FDCs and GC B cells serve as complementary early biomarkers that accurately forecast vaccine efficacy. This approach could potentially reduce the need for prolonged immunization regimens by cellular profiling on days 7–9, offering a modest step toward streamlining adjuvant selection and informing vaccine design.

Background/Objectives: Given the overlapping seasonality of influenza (Flu) and respiratory syncytial virus (RSV) infections in human populations, Flu–RSV combination vaccines are urgently needed. However, development of combo-vaccines is often faced with intra-vaccine interference which could compromise vaccination outcomes. Here we present an approach to overcoming this problem using a microneedle array patch (MAP)-based combo-vaccine with minimum intra-vaccine interference. Methods: Vaccine-laden dissolving MAPs were fabricated using a two-step micro-molding process with polyvinyl alcohol as major excipient. A partition-loading strategy was adopted to ensure spatially segregated distribution of a split-virus Flu vaccine and recombinant prefusion protein of RSV in separate MAP sectors. Serum samples from BALB/c mice post-vaccination were assessed for titers of binding and neutralizing antibodies against the viruses. Live virus challenge studies were carried out to assess the protection efficacy of the MAP-based vaccines. Results: Although i.m. administered standalone Flu and RSV vaccines were able to induce strong IgG responses in BALB/c mice, bidirectional intra-vaccine interference was observed when the two vaccines were co-administered in premixed form. However, when the two vaccines were loaded onto nonoverlapping sectors of D-MAPs for intradermal vaccination, the intra-vaccine interference effect was effectively overcome. The partition-loaded MAP-Flu/RSV combo-vaccine elicited antigen-specific IgG with robust virus-neutralizing activity and was strongly efficacious against either virus in challenge studies. Conclusions: Our data provide proof-of-concept evidence for the potential usefulness of partition-loaded MAPs in overcoming a critical barrier in vaccinology and offer a promising platform for future clinical translation.

Background: DNA vaccines are widely used due to their low production cost, ease of large-scale manufacturing, and rapid responsiveness to emerging epidemics. However, their relatively modest immunogenicity in larger species often requires potent adjuvants to elicit robust protective responses. GM-CSF and Flt3L are two cytokine adjuvants targeting dendritic cells (DCs). Although their combination has been reported to augment immune responses, the mechanism underlying the complementary effects of a genetically fused GM-CSF-Flt3L adjuvant plasmid remains incompletely understood. Methods: We constructed a GM-CSF-Flt3L fusion adjuvant plasmid and compared it with single-adjuvant plasmids for their effects on innate, humoral, and cellular immunity, as well as protective efficacy against lethal homologous virus challenge. Results: Compared with single-adjuvant plasmids, GM-CSF-Flt3L synergistically promoted the maturation of bone marrow-derived dendritic cells (BMDCs) and activated draining lymph node DCs, differentially expanded DC subsets, and enhanced the recruitment of migratory DCs. This adjuvant significantly elevated hemagglutinin (HA)-specific serum IgG titers, hemagglutination inhibition (HI) titers, and the responses of T follicular helper (TFH) cells and germinal center B (GCB) cells, while also enhancing the capacity of HA-specific CD4⁺ and CD8⁺ T cells to secrete IFN-γ and TNF-α. Following lethal homologous virus challenge, the GM-CSF-Flt3L group exhibited markedly reduced lung viral loads and no significant pathological damage in lung tissues. Conclusions: These findings demonstrate that the GM-CSF-Flt3L fusion adjuvant complementarily enhances humoral and cellular immune responses induced by the influenza DNA vaccine and improves protective efficacy, highlighting its potential as an effective DNA vaccine adjuvant.