Abstract
To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.
Highlights
Porcine circoviruses (PCVs) are very small non‐enveloped animal viruses with circular single‐stranded DNA genomes of approximately 2 kb, which belong to the genus Circovirus in the family Circoviridae [1]
The PCR product was subcloned into the restriction sites of XhoI and PstI in the baculovirus surface display vector [21] and the resultant plasmid was named as pBacSCCap(d41) (Figure 1)
The PCV2 capsid protein (Cap) protein is the major structural viral protein encoded by ORF2 and induces host-specific neutralizing antibody [4,5,6], which makes it an important target in the design of a subunit vaccine against PCV2 infections
Summary
Porcine circoviruses (PCVs) are very small non‐enveloped animal viruses with circular single‐stranded DNA genomes of approximately 2 kb, which belong to the genus Circovirus in the family Circoviridae [1]. PCV1 is non-pathogenic and exists in contaminated pig kidney cell lines 15, PK-15) and various pig tissues [1], wherase PCV2 is pathogenic and can cause pigs to develop postweaning multisystemic wasting syndrome (PMWS) [3]. Severe cases can lead to death, causing economic losses for pig farmers in various countries in the world [10, 11]. The first case of PMWS was discovered in North America in 1991 [12, 13], and similar case reports were subsequently submitted in Spain, the United States, Denmark, Ireland, and other countries [14,15,16,17]. The development of vaccines for comprehensive epidemic prevention is still the most effective way to deal with this problem
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