A DNA Vaccine Encoding the VAA Gene of Vibrio anguillarum Induces a Protective Immune Response in Flounder

Abstract

Vibrio anguillarum is a pathogenic bacterium that infects flounder resulting in significant losses in the aquaculture industry. The VAA protein previously identified in flounder is associated with a role in immune protection within these fish. In the present study, a recombinant DNA plasmid encoding the VAA gene of V. anguillarum was constructed and its potential as a DNA vaccine, to prevent the infection of V. anguillarum in flounder fish, investigated. We verified the expression of the VAA protein both in vitro in cell lines and in vivo in flounder fish. The protective effects of pcDNA3.1-VAA (pVAA) were analyzed by determination of the percentage of sIgM+, CD4-1+, CD4-2+, CD8β+ lymphocytes, and the production of VAA-specific antibodies in flounder following their immunization with the DNA vaccine. Histopathological changes in immune related tissues, bacterial load, and relative percentage survival rates of flounder post-challenge with V. anguillarum, were all investigated to assess the efficacy of the pVAA DNA vaccine candidate. Fish intramuscularly immunized with pVAA showed a significant increase in CD4-1+, CD4-2+, and CD8β+ T lymphocytes at days 9, 11, and 14 post-vaccination, reaching peak T-cell levels at days 11 or 14 post-immunization. The percentage of sIgM+ lymphocytes reached peak levels at weeks 4–5 post-immunization. Specific anti-V. anguillarum or anti-rVAA antibodies were induced in inoculated fish at days 28–35 post-immunization. The liver of vaccinated flounder exhibited only slight histopathological changes compared with a significant pathology observed in control immunized fish. Additionally, a lower bacterial burden in the liver, spleen, and kidney were observed in pVAA protected fish in response to bacterial challenge, compared with pcDNA3.1 vector control injected fish. Moreover, the pVAA vaccine confers a relative percentage survival of 50.00% following V. anguillarum infection. In summary, this is the first study indicating an initial induction of the T lymphocyte response, followed by B lymphocyte induction of specific antibodies as a result of DNA immunization of flounder. This signifies the important potential of pVAA as a DNA vaccine candidate for the control of V. anguillarum infection.

Interleukin 34 Serves as a Novel Molecular Adjuvant against Nocardia Seriolae Infection in Largemouth Bass (Micropterus Salmoides)

“…The results of PCR and RT-PCR demonstrated that the Hrp1 and IL-34 genes were only found in the muscle tissue of fish immunized with pcHrp1, pcIL-34 or pcHrp1+pcIL-34, indicating that the recombinant DNA plasmids could avoid local degradation and uptake by the muscle cells at the injection site, resulting in expression at transcription levels. Our results are in agreement with studies investigated in other fish species, including channel catfish [23], orange-spotted grouper [47], snakehead fish [16,66], and the Japanese flounder [11,67]. It is reported that cell membrane permeability and susceptibility of foreign DNA to host enzymes is one of the pivotal factors to evaluate the efficiency of the cellular uptake of DNA vaccines [68]; hence, further studies are required to estimate the efficiency of the cellular uptake of pcIL-34, pcHrp1 and pcHrp1+pcIL-34.…”

“…However, various factors, such as vaccine type, antigen dose, vaccination schedule (with or without booster), vaccine delivery route and the application of adjuvants can influence the vaccine efficacy [95]. Similarly, recent vaccine studies in Vibrio alginolyticus [47,96], Vibrio anguillarum [10,11,97,98], Vibrio harveyi [48], and Edwardsiella tarda [13] indicated that vaccination of recombinant proteins in combination with adjuvants could confer higher RPS levels than immunization with a DNA vaccine alone. Interestingly, pcHrp1, when co-administrated with pcIL-34, could enhance the protection by generating better RPS (82.14%) compared with rHRP1 emulsified with ISA 763 A VG; hence, this result might explain the superiority of IL-34 as an molecular adjuvant.…”

“…Recent studies have shown that DNA vaccines could be used to control viral pathogens; however, only a few have been tested against bacterial infections. Reportedly, DNA vaccines have been utilized in fish to control Streptococcus iniae [9], Vibrio anguillarum [10,11], Edwardsiella tarda [12][13][14], and Nocardia seriolae [15,16], indicating that DNA vaccines could be suitable for controlling bacterial diseases by provoking both non-specific and specific immune host responses. Despite the advantages of DNA vaccines, including low cost, safety, stability, and efficacy against bacterial and viral diseases, there are only two DNA vaccines licensed for use in commercial aquaculture.…”

Kinetics of T lymphocyte subsets and B lymphocytes in response to immunostimulants in flounder (Paralichthys olivaceus): implications for CD4+ T lymphocyte differentiation

“…In the present study, based on the bioinformatics analysis of amino acid sequences, two 14-aa epitope peptide candidates were identified and employed as immunogens to develop mAbs against flounder CD4-1 and CD4-2. Although polyclonal antibodies against recombinant CD4-1 and CD4-2 proteins of flounder were produced, respectively, the dynamic change in the percentages of CD4 + cells was used as an indicator of the fish health status and vaccine evaluation in our lab [19][20][21][22][23][24][25] . However, some characteristics of polyclonal antibodies make them incomparable to monoclonal antibodies, and mAbs against CD4 molecules are sustainable and powerful tools in the functional study of fish CD4 + T cells.…”

“…Three CD4 + T cell subsets, CD4-1 single-positive T cells, CD4-2 single-positive T cells and CD4-1/CD4-2 doublepositive T cells, were found in different proportions in flounder peripheral blood, spleen and head kidney, and CD4-1 + /CD4-2 + T cells were the majority of CD4 + T cells 18 . In addition, dynamic changes in the percentages of CD4 + cells were detected as indicators of fish health and vaccine evaluation [19][20][21][22][23][24][25] . In this paper, to further explore the function of CD4 + Th cells, monoclonal antibodies against the flounder CD4-1 and CD4-2 molecules were developed.…”