Generating cross-reactive vaccines aimed at targeting all human influenza A virus subtypes is among high priority tasks in contemporary vaccinology. Such vaccines will be primarily demanded during pre-pandemic period as well as used to prime some population cohorts prior to vaccination with standard vaccines containing area-relevant epidemic virus. Unlike routine approach universal vaccines do not induce a sterilizing immunity, but significantly ameliorate overt infection and probable complications. Our study was aimed at evaluating characteristics of immune response in experimental animals primed with a candidate universal vaccine challenged with sublethal influenza A virus infection. Mice were immunized intranasally with the recombinant protein FlgH2-2-4M2e containing conservative peptides derived from two influenza A virus proteins: M2 protein ectodomain and 76-130 amino acid sequence from the second hemagglutinin (HA2) subunit genetically linked to bacterial flagellin protein, which is a ligand for Toll-like receptor 5 (TLR5). Control mice received saline. Two weeks after immunization, mice from both groups were infected with a sublethal dose of A/Aichi/2/68 AN3N2 influenza virus strain. Level of immunoglobulins G and A in the blood sera and bronchoalveolar lavages (BAL) were determined two weeks after immunization and 1 month post infection. Percentage of lung CD4+ T and CD4+ Tem (CD44+CD62L-) cells secreting cytokines TNFα, IFNγ, IL-2 was determined. Immunized vs. control mice responded to sublethal infection with the influenza virus by insignificant weight loss and more pronounced production of vaccine peptide-specific (M2e and aa76-130 HA2) and pan-influenza A/Aichi/2/68 virus IgG and A in the blood sera and BAL. After challenge the number of CD4+ T cells secreting cytokines TNFα and/or IL-2 in immunized mice significantly exceeded counterpart T cells in unimmunized animals that was true for both CD4+T and CD4+ Tem cells. Memory CD4+ T cells were previously shown to play a key role in the prime-boost event and heterosubtypic immune response. Thus, we were able to demonstrate a priming effect for recombinant cross-protective vaccine used in our experiment. © 2019 Saint Petersburg Pasteur Institute. All rights reserved.