The advantages of protein vaccine include many aspects, such as no MHC type restriction for human use, and inducing high-intensity integrated immune response containing CD4+ T, CD8+ T cells and B cells after uptake and presentation by professional antigen presenting cell(APC)in vivo . In this study, we found that HCA587 combined with CFA plus 50 µg CpG could induce potent cellular and humoral immune responses, and it did have specific antitumor effect in vivo and significantly delayed tumor growth.
It is important to understand the mechanism of action of adjuvants in order to establish an ideal immune response using existing and new adjuvants. The use of adjuvant at priming may presumably program the immune response to generate a stronger innate transcriptional response at enhancement, which can increase / enhance the adaptive immune response [10, 11]. A vaccine adjuvant is a necessary condition to activate dendritic cells (DCs) and inducing a favorable immunologic milieu . Freund’s adjuvant reagent is a classic adjuvant, which divides into the complete Freund’s adjuvant(CFA) and incomplete Freund’s adjuvant (IFA). We have learned that they were widely used in protein vaccines to enhance the body’s robust host immune response [12, 13]. Therefore, HCA587 protein combined with CFA/IFA could induce strong humoral immune response with or without the presence of CpG.
But some clinical researches report that Freund’s adjuvant reagent need to be associated with other adjuvants to enhance its activity, such as toll-like receptor (TLR) 3 agonist and TLR4 agonist [14, 15], and CpG ODN and so on . The research of Marit M. Melssen et al indicated that LPS and polyiclc combined with IFA are safe and effective vaccine adjuvants. When added to TLR agonists, IFA enhanced the response of T cells to peptide vaccines . The research of Daniel E. Speiser et al indicated that vaccination with peptide, IFA and CpG ODN 7909 could lead rapid and strong human CD8+ T cell responses . Moreover, previous reports showed that immunization with peptides in an emulsion with Montanide ISA 51 adjuvant (water-in-oil emulsion) might induce a Th2-dominant microenvironment, which suggested that the vaccine site microenvironment was not fully utilized for induction of strong Th1/Tc1 responses, with induction instead of FoxP3+ cells and eosinophils. It suggests that there is a Th2 dominant microenvironment . The above supports our conclusion that Freund’s adjuvant need other adjuvant to help inducing enough cellular immune response.
CpG oligodeoxynucleotides, which are TLR 9 ligands, are being widely tested as a potent adjuvant for peptide- and protein-based cancer vaccines that stimulate cancer-reactive CTLs . CpG is a synthetic oligodeoxynucleotides containing CpG motifs, which is a potent driver of B cells and Th1 immune responses, and promotes the production of IFN-γ secreting cytotoxic cells by cross-presentation [16, 19, 20]. The cornerstone of vaccine effectiveness is whether the host immune response against an antigen can cause a memory T cell response over time . CpG adjuvant can induce strong immune response in vaccination trials. The research of P. Baumgaertner et al indicated that vaccination with the long synthetic NY-ESO-1 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses and a significant enhancement of tumor-specific antibodies in melanoma patients [22, 23]. Type I cytokines such as IFN-γ secreted by T cells play an important role in T cell-mediated antitumor mechanism . In the experiment, several immune strategies were compared by IFN-γ ELISpot and Ab ELISA. HCA587 protein was combined with CFA/IFA and CpG. The results showed that HCA587 protein induced a large amount of IFN-γ-producing splenocytes when combined with CFA and CpG 50 µg. At the same time, in order to understand the type of immune response after vaccination and the ability of immune cells to secrete cytokines, we also carried out intracellular cytokine staining. CD4+ T cells were the main group of IFN-γ-secreting splenocytes induced by HCA587 and 50 µg CpG. This indicates that HCA587 protein vaccine can induce specific Th1 type cellular immune response in mice, and the presence of CpG is vital for the elicitation of Th1 type cellular immune response.
In order to evaluate the antitumor effect of HCA587 protein vaccine in vivo, we constructed the HCA587-positve B16 melanoma cells to inoculate C57BL/6 mice. HCA587 protein combined with CFA and 50 µg CpG could specifically retard HCA587 antigen-positive tumor cells in mice, but it could not prolong the survival time of tumor-bearing mice. The reason for this may be related to the complexity of tumor microenvironment. PD-1/PD-L1 signaling pathway can inhibit T cell immune response and promote tumor immune escape . The equilibrium state maintained by adaptive immunity is destroyed . It is also possible that regulatory T (Treg) cells inhibit T cell response and function through a variety of direct and indirect mechanisms in tumor microenvironment . In addition, we have learned that antigen-specific CD8+ T cells induced with IFA are disordered in their cytokine production and proliferative capability, and this may be a vital mechanism for the transient immune response and the low clinical response rate in Freund's adjuvant . It also reported that the adjuvant fosters the recruitment, exhaustion and eventual death of T cells within the vaccination site . Therefore we consider the possibility of better combinations of adjuvants, such as using the adjuvant combination of CpG and ISCOM [8, 29]. It can also be treated with the combined checkpoint inhibitor PD-1 antibody. PCSK9 can also be inhibited so as to avoid the degradation of tumor cells such as MHC-1 .