This study demonstrates that delivery of the juvenile and immature F. hepatica antigen Cat B2 via DNA vaccine vectors induces humoral and cellular responses in BALB/c mice. Antigen availability for B cell priming is an essential factor in designing DNA vaccinations for the induction of humoral responses . In DNA vaccination, small amounts of secreted protein would aid to select B cells with high avidity . The COS-7 transfection analysis with the panel of DNA vaccine vectors indicated that in vivo expressed Cat B2 protein should be secreted from cells and be available for the priming of B cells.
There are a number of reports explaining the speed and magnitude of IgG antibody induction in mice vaccinated with secretory vaccine vectors [13, 35–38]. A previous report by Smooker et al. (2001) showed that after DNA vaccination with constructs encoding liver fluke antigens, IgG antibody responses peaked (1/2000) at week 8 and remained high for 20 weeks. In this study, constructs encoding liver fluke FABP only induce antibodies when delivered in a form that will secrete FABP from the host cell. Antigen availability for B cell priming is an essential factor in designing DNA vaccinations for the induction of humoral responses .
DNA vaccination with the VR1012 DNA vaccine encoding F. hepatica cathepsin L showed total IgG antibody titre increased by week 8 and attained a peak at week 13 (1/2000) . In a similar pattern, VR1012 encoding Cat B2 elicited IgG antibody responses that reached its peak antibody titre at week 10 in our study (1/1500).
The kinetics of antibody induction was different between the four constructs, with CTLA-4 Cat B2 inducing a strong response at the earliest time point measured (4 weeks) compared to the three other constructs. Therefore it appears that the CTLA-4 fusion construct generally induces titres faster than other constructs, and induced a high antibody titre. This confirms what has been seen in several studies [18, 19] as one of the major advantages of using CTLA-4 fusion constructs is the early induction of immune responses .
In our experiments, the MCP3 construct did not induce very high antibody responses and other studies [38, 39] have shown increased responses. The reason is unknown, and is presumably related to the specific combination of antigen and chemokine that is expressed. MCP3 is supposedly acting in a similar way to CTLA-4 in delivering antigen to antigen presenting cells, but not giving the expected increase. This confirms that CTLA-4 is the superior targeting system in these experiments.
In our study, Cat B2 DNA vaccines induced very modest IgG2a antibody response and dominant IgG1 and IgE antibody responses. The dominant IgE antibody response of cysteine proteases was observed in our previous study with F hepatica cathepsin L5 DNA vaccination in mice . The presence of Fasciola specific IgE antibody and eosinophil responses is a good indicator of acquired immunity which has been demonstrated elsewhere [40–42]. In a rat trial with recombinant cathepsin L, vaccination induced significantly higher specific IgG1 antibodies in vaccinated groups than in the control group .
One way of characterising antibody responses is to estimate the avidity of antibodies. Our results clearly show the sharp drop in binding in the control group, which is obviously reflective of non-specifically bound antibody. Rainczuk et al. tested the relative avidity of DNA vaccines where malarial antigen MSP4-5 was fused with MCP3 or CTLA-4 and found that the avidity of both these constructs were comparable. The relative avidity of MCP3 Cat B2 induced antibodies were higher than those induced by other vaccines, as inferred by a urea IgG ELISA. MCP3 has been shown to bind to chemokine receptors CCR1, CCR2, and CCR3 which are all expressed on immune cells.
Generally, Th1 and Th2-associated responses in the murine system are reflected by IgG2a and IgG1 isotypes respectively . Dendritic cells are crucial for processing and presenting antigens to stimulate naїve T lymphocytes, and also differentiate into a Th1 or Th2 T cell responses which provide T cells with costimulatory signals, CD80/CD86 . In a mouse study with experimental Fasciola infection, spleen cells from BALB/c exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. The induction of IL4 by fluke infection in mice has been well documented by many groups [46–49]. As stated above, the migratory juvenile and adult liver fluke ES material elicits Th2 responses [41, 50].