Evaluation of a yeast-based BVDV vaccine approach

牛病毒性腹泻病毒（BVDV）是养牛业的主要经济疾病风险，导致各种相关问题。控制BVDV感染对养牛业具有重要的经济意义，因为该病毒可引起多种临床疾病和生殖障碍，以及潜在的继发性感染，如乳腺炎，对生产周期的所有阶段产生典型的不良影响，并且根据不同的国家，实施了各种BVDV根除计划（Ridpath，2013）。生产损失主要源于繁殖失败和急性BVDV感染期间的免疫抑制，这使小牛易患呼吸道或肠道疾病。事实上，BVDV感染对先天性和获得性免疫系统的影响是独特的，并导致显著的免疫功能障碍，单独接种疫苗不足以保护胎儿免受BVDV感染和损失。这是因为没有单一的BVDV疫苗显示出完全的胎儿保护。尽管对生物安全性测量和PI根除计划的认识不断提高，但在许多国家，疫苗接种仍然是控制BVDV感染的最重要控制策略（Ridpath，2013）。由于BVDV毒株之间存在异质性，因此提供针对BVDV感染的获得性免疫保护具有挑战性。改良的活疫苗和灭活疫苗都被证明是有效的。体液免疫和细胞免疫反应都具有保护作用。在自然感染或用改良活疫苗接种后，血清抗体主要针对病毒蛋白E2和NS 2/3，而针对Erns和E1蛋白的反应较少。目前的BVDV疫苗需要注射和“冷链”，到目前为止，所有商业上可用的疫苗都是在细胞培养中生产的。在这里，新的口服疫苗可能提供更方便的给药途径，因此具有很大的优势，如果显示出与目前的注射疫苗一样的保护性。酿酒酵母（Saccharomyces cerevisiae，S.酿酒酵母），一种非侵入性、非致病性的生物体，通常被认为是“一般认为是安全的”（GRAS）物种，因此，它是一种有吸引力的递送抗原和治疗分子的工具（Chen et al.，2005; Garrait等人，2007）。事实上，少至5个重组酵母颗粒的S.表达卵清蛋白的肽能诱导树突状细胞为基础的T细胞增殖，其程度与用饱和量的卵清蛋白肽致敏的T细胞相同。这伴随着CD 80、CD 86、CD 40、CD 54和MHCII的上调以及树突细胞产生的IL-12的增加（Stubbset等人，2001年）。与在小鼠中产生的这些数据相似，Lu埃塔尔和Franzusoff等人在人类中的工作已经证明了用灭活的S.与单独的抗原相比，表达肿瘤抗原的酿酒酵母产生了强得多的应答，并且与通过使用它们的活对应物免疫产生的抗肿瘤应答相等（Ardiani等，2010;Franzusoff等人，2005; Lu等人，2004年）。事实上，S。阿萨疫苗载体使用的酿酒酵母可以通过施用到粘膜部位，包括口腔、呼吸道和生殖道，有效地引发粘膜和全身免疫（Allnutt等，2007; Sasagawa等人，2005; Shin等人，2005年）。特别是在哺乳动物中，S.已经证明，酿酒酵母具有上级安全性和功效，其中由于其细胞壁中TLR配体的存在，其显示出引发先天性和适应性免疫应答（Ogra埃塔尔，2001）以及由于其大小而具有的抗原性特征。因此，我们建议评估使用在其表面以非分泌形式表达BVDV E2和/或NS 2/3蛋白的重组酿酒酵母作为口服施用的候选疫苗的价值。

Bovine Viral Diarrhoea Virus (BVDV) has been implicated as a majoreconomic disease risk for the cattle industry, resulting in a variety ofassociated problems. Control of BVDV infection is economicallyimportant to the cattle industry because the virus causes a varietyof clinical diseases and reproductive disorders, as well as potentialsecondary infections such as mastitis that adversely affectessentially all stages of the production cycle, and depending on thecountry, various BVDV eradication programs are in place (Ridpath,2013). Production losses primarily stem from reproductive failureand from immunosuppression during acute BVDV infection, whichpredisposes calves to respiratory or enteric diseases. Indeed, theeffects of a BVDV infection on the innate and acquired immunesystem are unique and result in dramatic immune dysfunction.Vaccination alone is not sufficient to protect against foetal infectionand losses due to BVDV. This is because no single BVDV vaccine hasbeen shown to give complete foetal protection. Despite increasedawareness regarding bio-security measurements and PI eradicationprograms, vaccination is still the most important control strategy forcontrolling BVDV infections in many countries (Ridpath, 2013).Providing acquired immune protection against infection with BVDVis challenging due to the heterogeneity that exists among BVDVstrains. Both modified live and killed vaccines have been shown tobe efficacious. Both humoral and cellular immune responses areprotective. Following natural infection or vaccination with amodified live vaccine, the majority of the serum antibodies aredirected against the viral proteins E2 and NS2/3, with minorresponses against the Erns and E1 proteins. Current BVDV vaccinesrequire injection and a 'cold chain', and to date all commerciallyavailable vaccines are produced in cell culture.Here, new oral vaccines may provide a more convenient route ofadministration, and thus being of great advantage, if shown to be asprotective as the current injectable ones. Saccharomyces cerevisiae(S. cerevisiae), a non-invasive, non-pathogenic organism, is generallyconsidered to be a "generally regarded as safe" (GRAS) species andfor this reason, it is an attractive tool for delivering antigens andtherapeutic molecules (Chen et al., 2005; Garrait et al., 2007).Indeed, as little as 5 recombinant yeast particles of S. cerevisiaeexpressing ovalbumin have been shown to induce dendriticcell-based T cell proliferation to the same degree as those primedwith saturating amounts of ovalbumin peptide. This wasaccompanied by up-regulation of CD80, CD86, CD40, CD54 and MHCII as well as an increase in IL-12 production by dendritic cells (Stubbset al., 2001). Similar to these data generated in mice, work by Lu etal and by Franzusoff et al in humans has demonstrated thatimmunisation with inactivated S. cerevisiae expressing tumourantigens elicits a far stronger response compared to antigen alone,and was equal to the anti-tumour response generated byimmunisation using their live counter-parts (Ardiani et al., 2010;Franzusoff et al., 2005; Lu et al., 2004). Indeed, S. cerevisiae used asa vaccine vehicle can effectively elicit mucosal and systemicimmunization by administration to mucosal sites, including the oral,respiratory and genital tracts (Allnutt et al., 2007; Sasagawa et al.,2005; Shin et al., 2005). In mammals in particular, S. cerevisiae hasbeen demonstrated to have superior safety and efficacy, where ithas been shown to elicit both innate and adaptative immuneresponses due to the presence of TLR ligands in its cell wall (Ogra etal., 2001) as well as its antigenic feature due to its size.Thus, we propose to assess the value of using recombinant S.cerevisiae yeast expressing BVDV E2 and/or NS2/3 proteins on itssurface in a non-secreted form as a vaccine candidate for oraladministration.