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.

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus.

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.