Rashid Md Haroon Or, Yasui Fumihiko, Sanada Takahiro, Kono Risa, Honda Tomoko, Kitab Bouchra, Akter Lipi, Utsunomiya Masashi, Sato Risa, Yoshida Osamu, Hiasa Yoichi, Oda Yasunori, Goh Yasumasa, Miyazaki Takashi, Kohara Michinori, Tsukiyama-Kohara Kyoko
Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan.
Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
Vaccines (Basel). 2025 Apr 25;13(5):464. doi: 10.3390/vaccines13050464.
Hepatitis B virus (HBV) is a major cause of morbidity and mortality globally, and chronic infections are associated with cirrhosis and hepatocellular carcinoma. Issues with conventional treatments and vaccines mean there is a need for new therapeutic vaccines, which must elicit a strong and sustainable immune response. Here, we evaluated the immunogenicity of dual-antigen vaccines containing hybrid surface (hy-LHBs) and core (HBc) antigens, combined with a carboxyl-vinyl polymer (CVP) as a mucoadhesive excipient, following intranasal administration in mice. Mice were intranasally administered a mixed vaccine (10 µg of hy-LHBs and 2.5 or 10 µg of HBc) with or without a CVP excipient, and they were assessed for their immune response (levels of IgGs or IgA antibodies in an ELISA, IFN-γ level in splenocytes in an ELISpot assay, and cytokine/chemokine levels in a BioPlex assay). A protein stability assay was also conducted for vaccine formulations with and without excipients. Significantly enhanced IgG production was noted targeting hy-LHBs and (less markedly) HBc at 10 µg/antigen, but only a non-significant elevation was noted with the vaccine containing 2.5 µg HBc. The BioPlex assay showed a significant increase in IL-2 (#00-07, 0B), IL-12(p40)(#00), eotaxin (#00), MIP1α (#00, #00-07, 0B), and MCP-1 (#00-07, 0B) in mice that received treatment compared to those of untreated mice. The endpoint titers of IgG1 and IgG2a were measured, which were higher with CVP excipients than without. From the IgG2a/IgG1 ratio, a higher IgG1 response was induced by CVPs to hy-LHBs and a higher IgG2a response was induced to HBc. Th2-dominant phenotype to hy-LHBs was induced with CVP#00 in an ELISpot assay. The highest anti-hy-LHBs antibody titer was noted with the conventional CVP#00 excipient. Consistent with these results, a higher amount of neutralizing antibodies of HBV was induced with CVP#00 treatment and followed by #00-03 and #14-00. We consider that the addition of CVP excipients to vaccine formulation enhances immunogenicity and HBV antigen stability for intranasal vaccines. This effect was seen for both humoral and cell-mediated immune responses, indicating the potential of CVPs as excipients in intranasal HBV vaccines.
乙型肝炎病毒（HBV）是全球发病和死亡的主要原因，慢性感染与肝硬化和肝细胞癌相关。传统治疗方法和疫苗存在的问题意味着需要新型治疗性疫苗，这种疫苗必须引发强烈且可持续的免疫反应。在此，我们评估了含有杂交表面（hy-LHBs）和核心（HBc）抗原的双抗原疫苗，并结合羧基乙烯基聚合物（CVP）作为粘膜粘附性辅料，经鼻内给药小鼠后的免疫原性。给小鼠经鼻内接种混合疫苗（10μg hy-LHBs和2.5μg或10μg HBc），添加或不添加CVP辅料，然后评估它们的免疫反应（ELISA法检测IgG或IgA抗体水平、ELISpot法检测脾细胞中IFN-γ水平以及BioPlex法检测细胞因子/趋化因子水平）。还对含和不含辅料的疫苗制剂进行了蛋白质稳定性测定。在10μg/抗原时，针对hy-LHBs和（不太明显的）HBc观察到IgG产生显著增强，但含2.5μg HBc的疫苗仅观察到非显著升高。BioPlex分析显示，与未治疗的小鼠相比，接受治疗的小鼠中IL-2（#00 - 07，0B）、IL-12（p40）（#00）、嗜酸性粒细胞趋化因子（#00）、MIP1α（#00，#00 - 07，0B）和MCP-1（#00 - 07，0B）显著增加。测量了IgG1和IgG2a的终点效价，含CVP辅料的效价高于不含的。从IgG / IgG1比率来看，CVP对hy-LHBs诱导了更高的IgG1反应，对HBc诱导了更高的IgG2a反应。在ELISpot分析中，CVP#00对hy-LHBs诱导了Th2主导的表型。使用传统的CVP#00辅料时观察到最高的抗hy-LHBs抗体效价。与这些结果一致，CVP#00处理后诱导产生了更高量的HBV中和抗体，其次是#00 - 03和#14 - 00。我们认为，在疫苗制剂中添加CVP辅料可增强鼻内疫苗的免疫原性和HBV抗原稳定性。这种作用在体液免疫和细胞介导的免疫反应中均可见，表明CVP作为鼻内HBV疫苗辅料的潜力。
