The H7N9 virus is still occasionally detected in chickens [3]. Considering its high pathogenicity and the enormous damage to the poultry industry [27], the recurrence of the H7N9 avian influenza epidemic is of wide concern and widespread worry in the public health sector [4, 5, 27]. In order to improve animal welfare, reduce economic losses, and decrease human infections, vaccination is necessary. The development of influenza vaccine adjuvants that can effectively induce protective antibodies is important for the research and application of influenza vaccines. In this study, we investigated the immunization effect of H7N9 WIV with different doses of OX40L/Fc in co-immunized mice.
Our antibody assay results showed that OX40L effectively stimulated the production of high titers of IgG and HI antibodies, with 1.8 µg OX40L/Fc being the optimal concentration. The results of the challenge test showed that 1.5 µg H7N9 WIV + 1.8 / 3.0 µg OX40L/Fc exhibited a desirable adjuvant effect on H7N9 WIV in co-immunized mice, suggesting that OX40L could provide a good protection against lethal influenza virus infection. OX40L is expressed on the surface of mature dendritic cells, B cells, activated T cells, and other cells, and it is involved in the body's immune response and regulation of multiple diseases [28, 29]. OX40/OX40L, a co-stimulatory molecule pair, is a key stimulator of T cell activation, and it may maintain late T cell proliferation and survival [30]. The role of the OX40/OX40L signaling pathway in Th cell differentiation is controversial. Some studies claim that OX40/OX40L interactions do not directly determine the Th1/Th2 differentiation phenotype, but contribute to amplification of the ongoing immune response [10, 31]. The pre-existing host immune response determines the OX40/OX40L interaction, thus mediating Th1/Th2 immune response, eventually resulting in T cell proliferation and clonal expansion [31]. These findings are consistent with our results that although OX40L did not lead to no significant Th1 or Th2 immune response bias, OX40L increased IgG1 and IgG2 antibody levels.
In our study, H7N9 WIV + OX40L/Fc co-immunized mice produced a large number of Tfh cells, and thus OX40/OX40L was considered as an important costimulatory signal promoting Tfh differentiation. Consistently, OX40L has been reported to specifically bind to OX40 on the surface of T lymphocytes, thus providing a costimulatory signal to regulate the activation and proliferation of CD4+ T cells and convert activated CD4+ T lymphocytes into Tfh cells [32]. In a mouse model, OX40-deficient mice have been found to have about 70% fewer Tfh cells than WT mice after cowpox virus (V acV) infection [17]. One study of the SARS-CoV-2 vaccine reveals that the level of Tfh cell activation may reflect vaccine efficacy, that detection of Tfh cell responses after vaccination may predict long-term high-affinity antibody production, and that factors regulating Tfh cell responses may be potential targets for improving vaccine efficacy [33]. OX40L could enhance the efficacy of the H7N9 WIV vaccine by promoting the proliferation of Tfh cells, further enhancing the T cell-dependent humoral immune responses.
This study showed the more GC B cells produced in H7N9 WIV + OX40L/Fc co-immunized mice than in other immunized mice, which may be directly related to the amplification of Tfh cells. Without the assistance of Tfh cells, most B-cell immune responses cannot proceed. It has been reported that there are fewer Tfh cells and poorer development of GC B cells and Ab cells in OX40-deficient mice [34]. The interactions between OX40L+ B cells and OX40+ Tfh cells in the T-B border and GCs of V acV-infected mice further suggest a role of the OX40/OX40L signaling pathway in Tfh-helping B cells [17, 34]. Tfh cells play a fundamental role in the humoral immune response after vaccination, and Tfh cells can support B cell activation, plasma cell production, and antibody responses in GC cells [33]. Disruption of Tfh cell production in mice inoculated with SARS-CoV-2 spike protein results in an approximately 50% reduction in GC B cells, indicating that Tfh cells are essential for a strong GC response [35]. The interactions between B cells and Tfh cells in germinal center have been reported to promote the Tfh cell differentiation, thus leading to maturation of GC B cells [17].
The hallmark of protective immunity of vaccination is the production of long-lived plasma cells that secrete high-affinity antibodies [36]. In our study, H7N9 WIV + OX40L/Fc co-immunized mice exhibited significantly larger number of plasma cells in the bone marrow. OX40L is essential for the development of high-affinity Ig-producing plasma cells [37]. The deletion of OX40L in the systemic lupus erythematosus (SLE) mouse model leads to the reduction in the numbers of GC-B cells and plasma cells as well as antibodies [38]. The study of Sjogren's syndrome has revealed that the number of Tfh cells is significantly increased in patients with the increasing B cells and plasma cells [39]. Cross-linking of OX40 and OX40L expressed on stimulated B cells has significantly enhanced the proliferation of B cells and promoted the secretion of immunoglobulins [32]. GC Tfh cells can promote the development of long-term humoral immunity by producing memory B cells and high concentrations of plasma cells [40, 41]. In summary, the OX40-OX40L interaction induces GC B cell proliferation, which might be related to the role of OX40L in PC development. This interaction also enhances GC B-cell differentiation toward abundant long-lived PCs [17, 19, 42, 43], therefore in turn generating a large number of PCs.