【作者】 曾小玲；

【导师】 罗永文；

【作者基本信息】 华南农业大学 ， 兽医硕士（专业学位）， 2018， 硕士

【摘要】 狂犬病（rabies）是由狂犬病病毒（rabies virus,RABV）引起的具有高度神经嗜性的烈性人兽共患传染病,致死率达100%,已成为一个重大的公共卫生问题。该病目前无特效疗法,而通过暴露前预防（PrEP）或及时暴露后预防（PEP）可以完全预防感染。因此,探究狂犬病病毒的免疫防控机理,开发廉价高效的疫苗尤为重要。黏膜免疫是疫苗开发的一个重要方向,其中口服免疫可有效诱导机体产生免疫应答,操作简便快捷,是一种理想的免疫方式。单独口服抗原蛋白并不能产生良好的免疫反应,通常要与一定的佐剂或载体联合运用才能产生预期理想的免疫效果。大肠杆菌热敏肠毒素B亚基（heat-labile enterotoxin,LTB）已被证明是良好的黏膜免疫佐剂分子。本研究基于RABV弱毒株rHEP-Flury株的反向遗传操作平台上,成功构建了含狂犬病病毒糖蛋白胞外区（glycoprotein ectodomain,Get）和LTB基因的重组狂犬病病毒全长cDNA质粒pHEP-Get-LTB并成功拯救出重组病毒HEP-Get-LTB。对重组病毒HEP-Get-LTB的生物学特性进行研究,结果显示,重组病毒HEP-Get-LTB可以在NA细胞上稳定增殖,病毒滴度与亲本株rHEP-Flury无显著差异,均可达到较高水平。生长曲线表明,HEP-Get-LTB与rHEP-Flury有着相似的生长特性,当MOI=0.01和MOI=3时,两者均在72 h达到高峰。动物实验表明,重组病毒HEP-Get-LTB对乳鼠的LD₅₀较rHEP-Flury稍低,为10^-4.33/0.03 mL,颅内接种5⁶周龄成鼠,对其无致死性。通过口服或滴鼻方式接种小鼠,HEP-Get-LTB会造成成鼠体重在一定程度上降低,之后会回复正常。通过肌肉注射及颅内注射方式则无明显差异。在免疫效果评价上,口服或滴鼻免疫HEP-Get-LTB能够诱导小鼠产生中和抗体的水平显著高于rHEP-Flury,且其对小鼠免疫后的攻毒保护率更高;肌注免疫HEP-Get-LTB及rHEP-Flury均可诱导小鼠产生高于0.5 IU/mL的中和抗体,且免疫后对小鼠的保护率均达到80%以上。说明HEP-Get-LTB在注射免疫方式下与rHEP-Flury相似,均可诱导小鼠产生具有有效保护力水平的中和抗体,而在口服免疫方式下,其较亲本株rHEP-Flury株具有更好的免疫效力。在低剂量二次口服免疫小鼠后,HEP-Get-LTB比rHEP-Flury具有更好的免疫效果,但从产生的抗体水平和保护率上看免疫效果不如单次高剂量的免疫方式。因此可推测,口服免疫RABV活病毒要想获得较好的免疫效果对免疫的剂量有较大的依赖性。综上所述,本研究拯救得到的重组病毒HEP-Get-LTB复制能力良好,致病性弱,其口服免疫效果较rHEP-Flury有明显优势。下一步应在降低HEP-Get-LTB口服免疫对动物的影响以及优化其免疫剂量和免疫程序上进行研究。更多还原

【Abstract】 Rabies is caused by the rabies virus（RABV）which leads to fatal and highly neurotropic zoonotic infectious diseases.Its mortality is almost 100%and itIt has become a major public health issue.There is no effective therapy for this disease,however,complete prevention can be achieved through pre-or post-exposure prophylaxis.Therefore,it is critical to explore the replication and pathogenesis of the RABV,and develop the affordable and efficient vaccines.Mucosal immunity is an important goal of vaccine development because it can not only resist pathogens that invade through the mucosa,but also may be related to optimizing immunotherapy for tumors located on the mucosa.Oral vaccines are effective in inducing immune responses and immune tolerance in the body.It is an ideal way of immunity because Its operation is simple and quick.Oral antigenic protein alone does not produce good immunogenicity,and it is usually combined with a certain adjuvant or carrier to produce the desired,ideal immune effect.The Enterotoxin coli heat-labile enterotoxin B subunit（LTB）has proven to be a good mucosal immune adjuvant molecule.This study on the established reverse genetic operating platform in laboratory,the recombinant pfull-length cDNA lasmids pHEP-Get-LTB with Get gene and LTB gene have been constructed and successfully rescuing the recombined virus HEP-Get-LTB.The results of the biological characters study of the recombined virus HEP-Get-LTB indicated that the recombined virus HEP-Get-LTB could stably proliferate in NA cells.There was no significant difference of the virus titer with rHEP-Flury,both of which could reach higher levels.The growth curve showed that HEP-Get-LTB had similar growth characteristics with rHEP-Flury.At MOI of 0.01 and 3,the titers of three virus all peakde at72 h.Animal experiments showed that the LD₅₀ of recombinant virus HEP-Get-LTB on the neonatal 10^-4.33/0.03 mL,slightly lower than rHEP-Flury.Imtracranial vaccination 5⁶weeks of adult mice,shows no fatal.Through oral immunization and intranasal immunization,effect of HEP-Get-LTB on the weight of mice higher than the parent strain rHEP-Flury.However,There are no significant differences when through intramuscular injections and intracranial injections.The results of immunogenicity indicated that the recombined virus HEP-Get-LTB can induced the higher production of the rabies antibody in mice than the parent rHEP-Flury through oral immunization and intranasal immunization.Through Intramuscular injection,the recombined HEP-Get-LTB and the parent rHEP-Flury can Induce mice to produce neutralizing antibodies above 0.5 IU/mL and their protection rate is over 80%.It shows that HEP-Get-LTB is similar to rHEP-Flury when through conventional immunization that can induce mice to produce neutralizing antibodies with effective protective level.Moreover,It has better immunogenicity through oral immunization.In summary,the recombinant virus HEP-Get-LTB has good virus replication and Its pathogenicity,immunogenicity,and immune protection vary under different modes of immunization.There is a clear advantage over rHEP-Flury in oral immunization.It is necessary to further study its toxicity and adjust its immunization dose and immunization program to improve its applicability.更多还原