Conventional dendritic cells 2, the targeted antigen-presenting-cell, induces enhanced type 1 immune responses in mice immunized with CVC1302 in oil formulation.

Multifunctional CD4+ T helper 1 (Th1) cells, producing IFN-γ, TNF-α and IL-2, define a correlate of vaccine-mediated protection against intracellular infection. In our previous study, we found that CVC1302 in oil formulation promoted the differentiation of IFN-γ+/TNF-α+/IL-2+Th1 cells. In order to extend the application of CVC1302 in oil formulation, this study aimed to elucidate the mechanism of action in improving the Th1 immune response. Considering the signals required for the differentiation of CD4+ T cells to Th1 cells, we detected the distribution of innate immune cells and the model antigen OVA-FITC in lymph node (LN), as well as the quantity of cytokines produced by the innate immune cells. The results of these experiments show that, cDC2 and OVA-FITC localized to interfollicular region (IFR) of the draining lymph nodes, inflammatory monocytes localized to both IFR and T cell zone, which mainly infiltrate from the blood. In this inflammatory niche within LN, CD4+ T cells were attracted into IFR by CXCL10, secreted by inflammatory monocytes, then activated by cDC2, secreting IL-12. Above all, CVC1302 in oil formulation, on the one hand, targeted antigen and inflammatory monocytes into the LN IFR in order to attract CD4+ T cells, on the other hand, targeted cDC2 to produce IL-12 in order to promote optimal Th1 differentiation. The new finding will provide a blueprint for application of immunopotentiators in optimal formulations.

Analysis of CVC1302-Mediated Enhancement of Monocyte Recruitment in Inducing Immune Responses.

Monocytes (Mos) are believed to play important roles during the generation of immune response. In our previous study, CVC1302, a complex of PRRs agonists, was demonstrated to recruit Mo into lymph nodes (LNs) in order to present antigen and secret chemokines (CXCL9 and CXCL10), which attracted antigen-specific CD4+ T cells. As it is known that Mos in mice are divided into two main Mo subsets (Ly6C+ Mo and Ly6C- Mo), we aimed to clarify the CVC1302-recruiting Mo subset and functions in the establishment of immunity. In this study, we found that CVC1302 attracted both Ly6C+ Mo and Ly6C- Mo into draining LNs, which infiltrated from different origins, injection muscles and high endothelial venule (HEV), respectively. We also found that the numbers of OVA+ Ly6C+ Mo in the draining LNs were significantly higher compared with OVA+ Ly6C- Mo. However, the levels of CXCL9 and CXCL10 produced by Ly6C- Mo were significantly higher than Ly6C+ Mo, which plays important roles in attracting antigen-specific CD4+ T cells. Under the analysis of their functions in initiating immune responses, we found that the ability of the Ly6C+ monocyte was mainly capturing and presenting antigens, otherwise; the ability of the Ly6C- monocyte was mainly secreting CXCL9 and CXCL10, which attracted antigen-specific CD4+ T cells through CXCR3. These results will provide new insights into the development of new immunopotentiators and vaccines.

Pattern-Recognition Receptor Agonist-Containing Immunopotentiator CVC1302 Boosts High-Affinity Long-Lasting Humoral Immunity.

Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.

Evaluation of CVC1302 for Improved Efficacy of FMD-Inactivated Vaccine in Oxidative Stressed Mice Generated with PCV2 Infection.

This study aimed to explore the effect of the immunopotentiator CVC1302 on foot-and-mouth disease (FMD) vaccination in animals placed under oxidative stress. We established oxidative stress models using porcine circovirus type 2 (PCV2)-infected PK-15 cells and mice model both in vitro and in vivo, respectively. The efficacy of CVC1302 on PK-15 cells or in addition to the FMD vaccine was evaluated by quantitative real-time polymerase chain reaction, histopathological and enzyme-linked immunosorbent assay (ELISA) analysis. CVC1302 affected apoptosis of PCV2-infected PK-15 cells and significantly inhibited PCV2 replication, while it had no effect on the viability for blank PK-15 cell in vitro test with varying dilutions of CVC1302. Results showed that PCV2 induced a strong oxidative stress response in mice. CVC1302 reduced the viral load in spleen of PCV2-infected mice and ameliorated the pathological injury of spleen. Furthermore, CVC1302 significantly increased IgG antibody titer, cytokine expression, superoxide dismutase activity, catalase concentrations, and glutathione content in mice immunized with FMD vaccine. In conclusion, CVC1302 inhibits PCV2 replication and regulates oxidative stress in PCV2-infected mice, which can improve the immune efficacy of the FMD vaccine, providing a safe and effective immune enhancement.

Identification of mechanisms conferring an enhanced immune response in mice induced by CVC1302-adjuvanted killed serotype O foot-and-mouth virus vaccine.

The adjuvant CVC1302 was previously shown to efficiently enhance the immunogenicity of killed foot-and-mouth disease virus (FMDV) in mice and piglets. However, the underlining mechanism of action of CVC1302 remains unclear, especially at local injection sites and draining lymph nodes. Since the FMDV vaccine is administrated intramuscularly in field settings, we studied local immune responses to FMDV following intramuscular injection in mice, and found that CVC1302-adjuvanted killed FMDV (KV-CVC1302) induced secretion of several chemokines in murine muscle tissues, including MCP-1, MIP-1α, and MIP-1ß. The number of monocytes recruited to the site of injection was significantly higher in mice immunized with KV-CVC1302 compared with mice immunized with killed FMDV alone (KV). iTAQ-based quantitative proteomic assays were additionally employed to explore the molecular mechanisms of CVC1302 action in the draining lymph nodes. A total of 35 proteins were identified as being differentially expressed among the control group, KV-immunized group and KV-CVC1302-immunized group at 10 days post immunization (dpi). Proteins exhibiting differential expression were mainly involved in signal transduction, apoptosis, endocytosis and innate immune responses. Pathway analysis demonstrated that AMPK, phospholipase D, cAMP, Rap1, and MAPK signaling pathways were potentially induced by the immunopotentiator CVC1302. Understanding the local mechanism of CVC1302 action at injection sites and draining lymph nodes will provide new insights into the development of FMDV vaccines.

The immunopotentiator CVC1302 enhances immune efficacy and protective ability of foot-and-mouth disease virus vaccine in pigs.

The immunological enhancement characteristics of the immunopotentiator CVC1302 were evaluated for foot-and-mouth disease virus (FMDV) inactivated vaccine in pigs. Eight-week-old piglets were vaccinated with the foot-and-mouth disease (FMD) vaccine alone (FMD-vaccine group) or with the addition of CVC1302 (FMD-CVC1302 group), and the serum liquid phase blocking ELISA (LPB-ELISA) antibody titers, IgG1 and IgG2 levels, and the levels of four cytokines secreted by peripheral blood lymphocytes were measured at 28 days post vaccination (dpv). In the FMD-CVC1302 group, the LPB-ELISA antibody titers, IgG1, and IgG2 titers, and IL-2, IL-4, IL-6, and IFN-γ levels at 28 dpv were significantly higher than those in the FMD-vaccine group. The FMD-CVC1302 group had long-lasting antibody titers (>7.8 log2), lasting for at least 6 months. In addition, piglets were vaccinated with or without addition of CVC1302 to the FMD vaccine at three different doses (1, 1/3, and 1/9 of the standard vaccine dose) and the serum LPB-ELISA antibody and serum neutralizing (SN) antibody titers were detected at 28 dpv. Then all pigs were challenged with virulent FMDV for PD50 value, and the levels of FMDV-specific RNA copies for the two full-dose groups at 3 and 10 days post challenge (dpc) were measured. The LPB-ELISA and SN antibody titers for the three doses in the FMD-CVC1302 groups were significantly higher than those in the FMD-vaccine groups at the same doses (p < 0.05). Post-virus challenge, the FMDV-specific RNA copy number in the FMD-CVC1302 group was lower than that in the FMD-vaccine group at 3 and 10 dpc. The PD50 value was 15.85 for the FMD-CVC1302 group, which was obviously higher than that for the FMD-vaccine group (10.96), and in the 1/9-dose of FMD-vaccine group only 3/5 pigs were protected. These results indicate that CVC1302 can enhance the immune efficacy and protective ability of the FMD vaccine in pigs.

Long-term humoral immunity induced by CVC1302-adjuvanted serotype O foot-and-mouth disease inactivated vaccine correlates with promoted T follicular helper cells and thus germinal center responses in mice.

Long-lasting humoral immunity is one of the necessary criteria for a successful vaccine. In our previous study, it was demonstrated that the immunopotentiator CVC1302 could improve the humoral immunity induced by the foot and mouth disease virus (FMDV) killed vaccine (KV) with only one dose. Significantly higher FMDV-specific antibody titers and more persistent antibody responses were observed in pigs receiving CVC1302-adjuvanted KV (KV-CVC1302) than in those inoculated with KV alone. In this study, we show that CVC1302 enhances murine IgG responses to FMDV by promoting a potent T follicular helper cell (TFH) response, which directly controls the magnitude of the germinal center (GC) B cell response. These results indicate a need for studies to assess the capacity of CVC1302 to enhance the efficacy of FMDV KV immunization in pigs, and provide new insights into the development of FMDV vaccines.