A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.
Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-γ ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD 50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP 366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains.Keywords: Consensus sequence r Heterosubtypic protection r Immunodominance hierarchy r T-cell immunity r Universal influenza vaccine Additional supporting information may be found in the online version of this article at the publisher's web-site , and the resemblance of the highly pathogenic avian influenza (HPAI) H5N1 to Spanish flu caused great concern regarding future pandemic preparedness. Therefore, H5N1 has been included as part of the regular updating of the candidate vaccine strain since 2004 and vaccine manufacturers have been developing prepandemic vaccines against H5N1, which currently appear to be the only option. Nevertheless, the potential for future emergence of a highly transmissible HPAI H5N1 through spontaneous acquisition of transmission capability and antigenic variation is of great potential risk [3]. Unfortunately, the current time-consuming vaccine manufacturing process cannot respond quickly to emerging variants. The ultimate solution would be a universal influenza vaccine providing broad heterosubtypic protection. The M2 protein and HA stem are attractive targets for broad neutralization [4,5]. Vaccination studies have shown promising results for heterosubtypic protection [6,7]; however, the suboptimal neutralizing activity of the antibodies, due to poor accessibility to those target molecules, resulted in limited effectiveness against only low-dose challenges.T-cell-based vaccines are considered promising vaccine candidates, particularly for protection against highly variable viruses such as human immunodeficiency viru...
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