Novel DNA vaccine development against porcine circovirus type 2 and swine influenza virus H1N1 subtype

Huiling Wei, Purdue University

Abstract

Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important viral pathogens in pig populations. Current vaccines against these viruses are based on inactivated viruses or recombinant proteins. They are effective in inducing humoral immunity only, while cellular immunity is essential to clear intracellular pathogens. DNA vaccines are capable of stimulating both humoral and cellular immunity. Therefore, developing DNA vaccines against these two viruses is the main goal in these studies. To evaluate the vaccine efficacy in the natural host pig of these two viruses, a virus challenge model was established successfully to reproduce diseases caused by these viruses via experimentally infecting pigs. Also, the challenge model was utilized to investigate the pathogenesis of PCV2-SIV co-infection. SIV H1N1 did not influence PCV2 replication in dually infected pigs under the conditions in this study. To develop DNA vaccines, a commercial vector pcDNA3.1 was modified by inserting several components in the plasmid backbone. A chimeric intron and a Kozak sequence were inserted downstream of the CMV promoter and upstream of the starting codon to enhance the expression of the target gene insert. The C-terminal region of VP22 gene (VP22c), encoding one of the major tegument proteins of bovine herpesvirus-1, was fused to the target gene at the N- or C-terminus. VP22c can transport the protein fused with it from the transfected cells to surrounding untransfected cells, thus enhancing the transfection and presentation efficiency. ORF2, encoding the most immunogenic capsid protein of PCV2, was the target gene in the effort to develop a DNA vaccine against PCV2. The plasmid with VP22c fused to the N-terminal of the ORF2 gene (pVP22cORF2) reached the highest percentage of ORF2-positive cells in an in vitro transfection experiment. A plasmid pGM-CSF, encoding the secreted porcine GM-CSF protein was constructed and co-delivered with pVP22cORF2 to pigs intramuscularly. Vaccinated pigs showed increased growth performance, reduced duration of clinical signs, as well as reduced viral load in serum, lung, and lymph nodes. The DNA vaccine protected pigs against virus infection and disease development in this study. SIVs are fast-evolving viruses and highly variable due to frequent mutation and reassortment. A modified pcDNA3.1 plasmid incorporating conservative epitopes among SIVs was constructed with the goal to protect animals against influenza virus infection. Firstly, a platform for a DNA-epitope vaccine was established in mice. In this study, two B-cell epitopes (HA91-108 and M2e), and two T-cell epitopes (NP366-374 and NP380-393), were quadruplicated and fused separately to the C-terminal of the VP22c gene in the modified pcDNA3.1 plasmid. Linker -KK- was used to space between each copy of the two B-cell epitopes and -RVKR- was used for the two T-cell epitopes in order to enhance the presentation of the epitopes to the immune system. After the DNA plasmids were administered to mice intramuscularly in combination or separately, or boosted with recombinant proteins of quadruplicated epitopes fused to VP22c, the vaccine stimulated the desired epitope-specific humoral immunity to the two B-cell epitopes and cellular immunity to the epitope NP380-393. Our results indicate that the vaccine design is a successful platform for developing DNA-epitope vaccines. The efficacy of this platform was evaluated in the following mice vaccination/challenge study. One B-cell epitope (HA2.30-130), a quadruplicated Th-cell epitope (NP55-69), and a quadruplicated CTL epitope (NP147-158) were fused separately to the C-terminal of VP22c gene in the modified pcDNA3.1 plasmid. Chitosan was used as an adjuvant to deliver plasmids intranasally. DNA plasmids were also administered to mice intramuscularly. In the intramuscularly vaccinated group, the vaccine stimulated epitope-specific immunity that provided 100% protection upon a lethal dose of SIV H1N1 challenge. However, the chitosan/plasmid vaccinated group only had 20% survival upon virus infection, compared to 0% in the challenge control group. Our results indicate that DNA plasmids administered intramuscularly are effective in mice. To conclude, the developed DNA vaccines in these studies provided efficient protection to animals against virus infections and the modified DNA plasmid can be used as a general DNA vehicle to deliver proteins or peptides against various targets.

Degree

Ph.D.

Advisors

Pogranichniy, Purdue University.

Subject Area

Animal Diseases|Virology|Immunology

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