Background: Influenza is a contagious respiratory illness caused by an acute infection of influenza viruses, among which influenza A virus causes seasonal epidemic infections nearly every year. Due to the unpredictability of the evolving influenza A virus and time-consuming vaccine development cycles, novel universal influenza vaccines designed to induce broadly cross-reactive immune responses against frequently mutant influenza A virus strains are urgently required.

Objective: The aim of this study was to synthesize a novel vaccine through the dual-site specific conjugation of the constant epitope of 23 amino acids (M2e) of the influenza A virus with a highly immunogenic carrier protein of Cross-Reacting Material (CRM₁₉₇) under denaturation and to evaluate its primary immunogenicity in mice.

Methods: The antigen (M2e) and the carrier protein (CRM₁₉₇) were linked with different types of hetero-functionalized linkers, α-Maleimide-ε-Hydrazide Polyethylene Glycol 2k (MAL-PEG-HZ) and N-β-Maleimidopropionic Acid Hydrazide (BMPH) separately. The immunogenicity of the M2e-CRM₁₉₇ conjugates with different types of linkers was evaluated in mice, and the M2especific total IgG and IgG-isotypes were determined by ELISA.

Results: Immunogenicity studies revealed that anti-M2e antibody could be induced by the conjugate products, M2e-PEG-CRM₁₉₇ and M2e-BMPH-CRM₁₉₇, by approximately 30 and 90-fold higher than that of the M2e group. In addition, the anti-M2e antibody level induced by M2e-PEG-CRM₁₉₇ conjugate was three times higher than that of M2e-BMPH-CRM₁₉₇ conjugate, and the former could simultaneously activate both cellular and humoral immune responses.

Conclusion: The M2e-CRM₁₉₇ conjugated vaccines we synthesized in this study are highly immunogenic compared with M2e alone. Besides, evidence presented here indicated that the hydrophilic, non-immunogenic and biocompatible chain of the cross-linker might be a better choice for the development of a conjugate vaccine.

背景：流感是由流感病毒急性感染引起的一种传染性呼吸道疾病，其中甲型流感病毒几乎每年都会引起季节性流行性感染。由于甲型流感病毒进化的不可预测性和耗时的疫苗开发周期，迫切需要新型通用流感疫苗，旨在诱导针对频繁突变的甲型流感病毒株的广泛交叉反应免疫反应。

目的：本研究的目的是通过甲型流感病毒 23 个氨基酸 (M2e) 恒定表位与交叉反应材料 (CRM ₁₉₇₎的高免疫原性载体蛋白的双位点特异性偶联来合成一种新型疫苗。) 变性并评估其在小鼠中的主要免疫原性。

方法：将抗原 (M2e) 和载体蛋白 (CRM ₁₉₇ ) 与不同类型的杂功能化接头连接，α-马来酰亚胺-ε-酰肼聚乙二醇 2k (MAL-PEG-HZ) 和 N-β-马来酰亚胺丙酸酰肼 (BMPH) 分开。在小鼠中评估了具有不同类型接头的 M2e-CRM ₁₉₇偶联物的免疫原性，并通过 ELISA 确定了 M2e 特异性总 IgG 和 IgG 同种型。

结果：免疫原性研究表明，结合产物 M2e-PEG-CRM ₁₉₇和 M2e-BMPH-CRM ₁₉₇可诱导抗 M2e 抗体，比 M2e 组高约 30 和 90 倍。此外，M2e-PEG-CRM ₁₉₇偶联物诱导的抗 M2e 抗体水平是 M2e-BMPH-CRM ₁₉₇偶联物的三倍，前者可以同时激活细胞和体液免疫反应。

结论：与单独的 M2e 相比，我们在本研究中合成的 M2e-CRM ₁₉₇偶联疫苗具有高度免疫原性。此外，这里提供的证据表明，交联剂的亲水性、非免疫原性和生物相容性链可能是开发结合疫苗的更好选择。