为了解决抗原性漂移的不确定性和当前流感疫苗的低效性，迫切需要一种通用疫苗。在本研究中，一种称为计算优化广谱反应抗原（COBRA）的策略被用于生成1918年至2021年收集的流行性感冒病毒样本的血凝素球状头部（HA1）的共识序列，以追踪其渐进性变化，并将其纳入设计的构建体中。利用基于塞姆利基森林病毒RNA依赖性RNA聚合酶（RdRp）的真核表达系统pJHL204，通过沙门氏菌介导的细菌感染，将携带不同HA1区域的构建体注入真核细胞。通过蛋白质免疫印迹和免疫荧光法证实了重组蛋白的表达。用所设计的构建体免疫小鼠产生了体液反应，其免疫球蛋白G（IgG）水平显著增加，以及细胞介导的免疫反应也显著增加，其中CD4⁺和CD8⁺ T细胞增加了1.5倍。具体来说，构建体#1和#5促进了产生CD4⁺和CD8⁺ T细胞的干扰素-γ（IFN-γ）的生成，使反应偏向了辅助性T细胞（Th1）途径。此外，产生白细胞介素-4（IL-4）的T细胞表达上调4倍。其保护效果得到验证，对选定的病毒株产生高达4倍的中和抗体，且血凝抑制滴度高于40。设计的构建体具有广泛的保护性免疫反应，导致流感A/PR8/34、A/Brisbane/59/2007、A/California/07/2009、KBPV VR-92和NCCP 43021株小鼠的病毒滴度显著降低，肺部炎症最小。这一发现彻底改变了流感疫苗的设计和递送；沙门氏菌介导的COBRA-HA1是一种高效的体内抗原呈递方法。这种方法可以有效地抗击季节性H1N1流感毒株和潜在的疫情暴发。

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella -mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4 ⁺ and CD8 ⁺ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4 ⁺ and CD8⁺ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.