Immunity to FMDV following combined DNA and inactivated virus antigen.

尽管临床上接种了口蹄疫疫苗，但反刍动物在上呼吸道复制病毒并成为持续感染的携带者，对控制措施产生了重大影响。然而，证据支持疫苗效力、反应强度和病毒在口咽部复制发生率之间的关系。结论表明，高抗原有效载荷疫苗能够抑制局部病毒的复制、持久性和载体状态。这背后的效应机制尚不清楚。抗体介导的保护是可能的，因为增加抗原有效载荷促进更快速和更大的全身反应。然而，这一幅度不可避免地达到了一个上限，即额外的有效载荷显示出很少的有益影响，并且在许多观察中，面对高抗体滴度时会出现持久性。局部抗体可能影响持久性，但几乎没有证据支持这一点，即使增加有效载荷，被促进甚至引起这种反应。因此，其他因素极有可能发挥主要作用，如细胞介导和先天反应。高效口蹄疫疫苗能够在猪体内引发持久的全身细胞因子反应，如IL-6、IL-8和IL-12，这表明了单核细胞的活性。这是先天防御的重要组成部分，有助于早期保护宿主和缺乏持久性。这在反刍动物身上引起了类似的关注。干扰素γ在体外持续感染的牛上皮细胞中是口蹄疫病毒的有效抑制剂，α和β干扰素也抑制口蹄疫病毒的复制。在口蹄疫疫苗接种的绵羊中，IFN γ可以通过增加有效载荷而得到越来越多的系统性刺激，从而在挑战后不存在持续性。因此，与传统口蹄疫疫苗相比，持续增强多种细胞和体液反应的疫苗制度，应该对抑制持久性和载体状态有直接的有益作用。这在地方性和紧急情况下都有很大的好处。该提案将使用DNA疫苗来检验这些假设。一种编码“空衣壳”以及非结构蛋白2A、3C和3D的口蹄疫DNA疫苗与表达粒细胞巨噬细胞集落刺激因子（GM-CSF）的佐剂质粒一起使用，在猪和羊中诱导了极强的体液和细胞反应，并提供了保护。进一步的优化进一步放大了特异性和重要的中和抗体反应，并导致细胞免疫反应，检测为T细胞增殖。当使用口蹄疫灭活的同源病毒抗原和3D重组蛋白作为最后的促进剂并产生IFN-？、IFN-a、IL-2、4、6、8和10证实先天和特异性免疫应答均被激活。事实上，DNA疫苗现在被认为是一种极好的工具，可以启动广泛的免疫库，作为主要促进战略的一部分，并且在牛、山羊和马等宿主中有许多成功的例子。使用这种策略对抗牛疱疹病毒-1导致增强和保护性细胞和体液免疫，并显著减少病毒脱落。最近，两剂量的DNA -蛋白增强或反之，间隔14天，足以刺激牛对口蹄疫病毒的增强免疫力。因此，有可能缩短启动和增强之间的间隔时间，并检查用于紧急目的的蛋白质抗原同时接种DNA。将利用DNA疫苗接种制度增强免疫反应的能力，对那些对保护重要的因素提供更全面的了解，这些因素可以被新疫苗利用或更好地促进，以防止持久性和载体状态。

Despite being clinically protected from FMD vaccinated ruminants replicate virus in the upper respiratory tract and become persistently infected carriers with consequential impact on control measures. However, evidence supports a relationship between vaccine potency, magnitude of response, and incidence of virus replication in the oropharynx. It has been conclusively shown that higher antigen payload vaccines are capable of inhibiting local virus replication, persistence and the carrier state. The effector mechanisms behind this are unclear. Antibody mediated protection is a possibility since increasing the antigen payload promotes a more rapid and greater systemic response. However, the magnitude of this inevitably reaches a ceiling where additional payload shows little beneficial affect, and there are many observations where persistence occurs in the face of high antibody titre. Local antibody may influence persistence, but there is little evidence to support this, even with increased payload, being promoted or even eliciting, such a response. It is therefore extremely likely that other factors play a major role, like cell mediated and innate responses. High potency FMD vaccines are capable of eliciting durable systemic cytokine responses in pigs, such as IL-6, IL-8 and IL-12 , which is indicative of monocytic cell activity. This is an important part of an innate defence that contributes to early protection of the host and absence of persistence. This has led to a similar focus in the ruminant. IFN gamma is a potent inhibitor of FMD virus in persistently infected bovine epithelial cells in-vitro and alpha and beta interferons also inhibit FMDV replication. IFN gamma can be increasingly stimulated systemically in FMD vaccinated sheep, by augmented payload, resulting in no persistence following challenge. Thus, a vaccination regime which consistently enhances a diverse repertoire of cellular and humoral responses, over and above those afforded by traditional FMD vaccines, should have direct beneficial attributes to the inhibition of persistence and the carrier state. This could have major benefit both endemically and in emergency use. The proposal will use DNA vaccine, to examine these hypotheses. An FMD DNA vaccine encoding the 'empty capsid' along with the non-structural proteins 2A, 3C and 3D has been used with an adjuvant plasmid expressing granulocyte macrophage colony stimulating factor (GM-CSF), which induced extremely strong humoral and cellular responses and conferred protection in pigs and sheep. Further optimisation amplified the specific and important neutralising antibody response further, and resulted in cellular immune responses, detected as T cell proliferation. This was more dramatically enhanced when FMD inactivated homologous virus antigen and 3D recombinant protein was used as a final boost and the production of IFN-?, IFN-a, IL-2, 4, 6, 8, and 10 confirmed both innate and specific immune responses are activated. Indeed DNA vaccines are now considered to be an excellent tool for priming an extensive immune repertoire as part of a prime boost strategy and there are many examples where this has been successful in hosts such as cattle, goats and horses. Use of this strategy against bovine herpesvirus-1 led to enhanced and protective cellular and humoral immunity and a significant reduction in virus shedding. Recently, a two dose regime of DNA prime- protein boost or vice versa, 14 day apart, was sufficient in stimulating enhanced immunity against FMDV in cattle. There is therefore potential for shorter intervals between prime and boost and in examining simultaneous vaccination of DNA with the protein antigen for emergency purpose. The ability of DNA vaccination regimes to augment immune responses will be exploited to provide a more comprehensive understanding of those important to protection which can be utilized or better promoted by newer vaccines to prevent persistence and the carrier state.