Immunity to FMDV following combined DNA and inactivated virus antigen.

尽管在临床上可以保护接种疫苗的反刍动物免受口蹄疫的感染，但它们在上呼吸道中复制病毒，并成为持续感染的携带者，从而影响控制措施。然而，有证据支持疫苗效力、应答幅度和口咽部病毒复制发生率之间的关系。已经明确表明，较高抗原有效载荷的疫苗能够抑制局部病毒复制、持久性和载体状态。这背后的效应机制尚不清楚。抗体介导的保护是一种可能性，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，其中额外的有效载荷几乎没有显示出有益的影响，并且有许多观察结果表明，在面对高抗体滴度时会出现持久性。局部抗体可能会影响持久性，但几乎没有证据支持这一点，即使增加了有效载荷，被促进或甚至引发这样的反应。因此，其他因素极有可能发挥主要作用，如细胞介导的和先天的反应。高效FMD疫苗能够在猪中引发持久的全身性细胞因子应答，例如IL-6、IL-8和IL-12，其指示单核细胞活性。这是先天防御的重要组成部分，有助于宿主的早期保护和持久性的缺乏。这导致了反刍动物中的类似关注。IFN γ是体外持续感染的牛上皮细胞中FMD病毒的有效抑制剂，α和β干扰素也抑制FMDV复制。IFN γ可以通过增加有效载荷在口蹄疫疫苗接种的绵羊中越来越多地全身刺激，导致在攻击后没有持续性。因此，一致地增强多种多样的细胞和体液应答的疫苗接种方案，超过传统FMD疫苗所提供的那些，应该对持久性和携带者状态的抑制具有直接的有益属性。这在地方病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。编码“空衣壳”沿着非结构蛋白2A、3C和3D的FMD DNA疫苗已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，其诱导极强的体液和细胞应答并在猪和羊中赋予保护作用。进一步优化进一步扩增了特异性和重要的中和抗体应答，并导致细胞免疫应答，检测为T细胞增殖。当FMD灭活的同源病毒抗原和3D重组蛋白用作最终加强和IFN-γ的产生时，IFN-a、IL-2、4、6、8和10证实先天性和特异性免疫应答都被激活。事实上，DNA疫苗现在被认为是作为初免加强策略的一部分的用于引发广泛免疫库的极好工具，并且存在许多这样的实例，其中这在宿主如牛、山羊和马中是成功的。使用这种策略对牛疱疹病毒-1导致增强和保护细胞和体液免疫和病毒脱落显着减少。最近，间隔14天的DNA初免-蛋白质加强或反之亦然的两剂量方案足以刺激牛中针对FMDV的增强的免疫力。因此，有可能缩短初免和加强之间的间隔，并在紧急情况下检查DNA与蛋白抗原的同时接种。DNA疫苗接种制度，以增强免疫反应的能力将被利用，以提供一个更全面的了解那些重要的保护，可以利用或更好地促进新的疫苗，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.