Enhanced Mucosal Immune Responses Induced by a Combined Candidate Mucosal Vaccine Based on Hepatitis A Virus and Hepatitis E Virus Structural Proteins Linked to Tuftsin

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368–607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1–198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection.

甲型肝炎病毒（Hepatitis A virus, HAV）与戊型肝炎病毒（Hepatitis E virus, HEV）是引发传染性肝炎的最常见病原体。这两类病毒主要经粪-口途径传播，在发展中国家可引发具有重要临床意义的肝炎疾病。为评估同时靶向甲型、戊型肝炎感染的潜力，研究人员开发了一种联合黏膜候选疫苗：该疫苗包含戊型肝炎病毒部分开放阅读框2（open reading frame 2, ORF2）序列（氨基酸368~607，即HE-ORF2），以及甲型肝炎病毒部分病毒蛋白1（virus protein 1, VP1）序列（氨基酸1~198，即HA-VP1），上述序列均包含病毒中和表位。促吞噬肽（Tuftsin）是一种免疫刺激性肽段，可通过将蛋白靶向递送至巨噬细胞与树突状细胞，从而增强其免疫原性。本研究通过将促吞噬肽与HE-ORF2、HA-VP1偶联，开发了一种新型联合蛋白疫苗，并以合成的CpG寡脱氧核苷酸（CpG oligodeoxynucleotides, ODNs）作为佐剂。后续在BALB/c小鼠中开展的实验结果显示，经鼻内给药时，促吞噬肽可增强小鼠血清中针对戊型肝炎病毒与甲型肝炎病毒的特异性免疫球蛋白G（Immunoglobulin G, IgG）及免疫球蛋白A（Immunoglobulin A, IgA）抗体水平，且该增强效应在肠道、阴道与肺部黏膜界面处均有体现。此外，经鼻内免疫的促吞噬肽组（HE-ORF2-促吞噬肽 + HA-VP1-促吞噬肽 + CpG）小鼠，其分泌干扰素-γ（Interferon-γ, IFN-γ）的脾细胞水平（即1型辅助性T细胞（Type 1 T helper cell, Th1）免疫应答）以及CD4+/CD8+ T细胞比值，均显著高于无促吞噬肽组（HE-ORF2 + HA-VP1 + CpG）。综上，与无促吞噬肽组相比，促吞噬肽组可诱导更强的体液免疫与细胞免疫应答。此外，与肌内注射免疫相比，经鼻内免疫可诱导联合蛋白疫苗产生更强的免疫应答。本研究将HE-ORF2、HA-VP1与促吞噬肽整合至疫苗中，验证了开发联合黏膜抗甲型、戊型肝炎感染疫苗的重要理念，为后续相关研发提供了关键支撑。