豬生殖和呼吸道綜合症病毒GP5-M與大腸桿菌熱溶解性腸毒素次單位B(LTB)融合蛋白之基因轉殖菸草口服疫苗於豬隻之免疫效力評估

Evaluation of the immunogenicity and efficacy of a transgenic tobacco plant expressing the recombinant fusion protein of GP5-M of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pig

徐子婷(Tzu-Ting Hsu)

指導教授 ： 鄭謙仁

國立臺灣大學/獸醫專業學院/分子暨比較病理生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02842

摘要

豬生殖和呼吸綜合症病毒(porcine reproductive and respiratory syndrome Virus, PRRSV) 為一正向單股RNA病毒，屬於動脈病毒科（Arteriviridae）巢狀病毒目（Nidovirales）。自1980年代此病毒引起的疾病，便成為全世界養豬產業危害最嚴重的疾病之一，病毒感染造成母豬繁殖障礙及生長豬隻呼吸系統疾病。PRRSV疫苗早在1990年代中期即開始使用，但十多年來，無論死毒疫苗或馴化活毒疫苗，均無法有效預防或控制PRRS。由於PRRSV在宿主誘發之免疫反應相當複雜，且具迴避宿主免疫系統之機制，使得發展此病毒之疫苗具有相當困難性。之前的基因轉殖植物口服次單位疫苗GP5-LTB (Escherichia coli heat-labile enterotoxin B subunit; 大腸桿菌熱溶解性次單位B融合蛋白)已能激發有效的免疫反應且有一定的保護效力，唯其中和抗體力價仍有改善空間。本實驗設計共同表現GP5-M及LTB之基因轉殖菸草口服次單位疫苗(LTB-M-GP5-T)，並與商用MLV疫苗做比較，評估兩組疫苗於豬隻的安全性及免疫效力。將24頭5-6週齡PRRSV抗體陰性SPF豬隻分組，以兩週間隔進行三次GP5-M-LTB-T口服免疫，商用Ingelvac MLV疫苗組兩次肌肉注射免疫，於最後一次免疫後三週以5x105 TCID50 PRRSV MD001每頭動物給予2 ml攻毒進行動物實驗。本實驗檢測抗PRRSV抗體反應，以qrt-RT-PCR偵測病毒血症及唾液及組織病毒含量，並根據豬隻臨床生長表現(體重增長變化及平均日增重)評估疫苗安全性。本實驗結果顯示連續三次服用50公克劑量之菸草於豬隻生長表現並無影響。體液性免應反應結果顯示，GP5-M-LTB-T疫苗組在三次免疫中可偵測到血清PRRSV-specific IgG抗體反應並有逐漸上升趨勢，商用MLV疫苗組亦具有同樣的趨勢，兩組疫苗與控制組於統計上有顯著差異。GP5-M-LTB-T疫苗組於唾液有PRRSV-specific IgA抗體反應，但MLV商用疫苗組唾液無IgA抗體反應。GP5-M-LTB-T疫苗組組織病毒量普遍低於控制組且具有顯著差異，但是MLV商用疫苗組組織病毒含量具有相對更顯著的統計差異，然在病毒血症的部份，三組間並無統計顯著差異。總結本實驗結果，GP5-M-LTB-T疫苗可以啟動體液性及系統性免疫反應，基因轉殖菸草口服次單位疫苗及商用疫苗具有一定的保護效力但無法避免豬隻PRRSV感染。

關鍵字

豬隻生殖和呼吸道症候症病毒 ； 基因轉殖植物次單位疫苗 ； 黏膜免疫 ； 大腸桿菌熱溶解性次單位B融合蛋白 ； 基因轉殖菸草

並列摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive RNA virus, which belongs to the family Arteriviridae and is classified in the order of Nidovirales. PRRS has been one of the most economically significant diseases of swine industry since 1980 and is characterized by reproductive failure in sows and respiratory distress in growing pigs. Although PRRSV modified live-attenuated vaccines (MLVs) and inactivated vaccines have been available for more than a decade, the efficacy of both vaccines is limited. The complicate evading immune mechanism by PRRSV makes it difficult to develop vaccine, both effective and safe. The objective of the present study was to investigate the immunogenicity and efficacy of a transgenic tobacco PRRS subunit vaccine containing GP5-M protein and Escherichia coli heat-labile enterotoxin-B subunit (LTB) after oral administration. In addition, Ingelvac MLV vaccine was included in the study. Twenty-four 5-to 6-week old pigs, seronegative for PRRSV, were randomly divided into 4 groups. Pigs of group one were given orally three consecutive doses of equal concentration of recombinant GP5-M protein expressed in the leaves of tobacco (GP5-M-LTB-T) at a 2-week interval. Pigs from the group were inoculated intramuscularly with 2 ml of Ingelvac PRRS MLV vaccine two times. The pigs were challenged with 2 ml 5x105 TCID50 PRRSV MD001 at 8 weeks post-initial immunization (WPI). The efficacy of the two different types of vaccines was evaluated based on clinical parameters, immune responses as well as post-challenge virological profiles. Serologic and saliva responses were measured by ELISA; viremia, saliva and tissue viral loads upon necropsy were measured by quantitative real time PCR. The results of changes in the bodyweights and daily weight gain revealed no statistical significance among groups, and no clinical signs were observed throughout the study. The levels of serum PRRSV ORF5-specific IgG in GP5-M-LTB-T and MLV groups elevated gradually after three or two consecutive immunization, respectively, and were significantly higher than that of the control group. The level of saliva PRRSV ORF5-specific IgA elevated gradually in GP5-M-LTB-T, while the MLV group didn’t demonstrate IgA. The levels of tissue viral load in GP5-M-LTB-T did have statiscally significant differences from control group, while the MLV group generally showed lower mean values than those of control group and GP5-M-LTB-T group. On the contrary, the results of viremia showed no significant differences among the three groups. In summary, the present study has demonstrated that GP5-M-LTB-T transgenic tobacco leaves could induce mucosal and systemic immune responses in pigs, while neither GP5-M-LTB-T nor MLV group could prevent pigs from PRRSV infection. The preliminary results support the foreseeable potential of subunit vaccine development.

並列關鍵字

PRRSV ； subunit vaccine ； mucosal immunity ； LTB ； transgenic tobacco leaves

參考文獻

Albina, E., 1997. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet Microbiol 55, 309-316.

Ansari, I.H., Kwon, B., Osorio, F.A., Pattnaik, A.K., 2006. Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies. J Virol 80, 3994-4004.

Bautista, E.M., Faaberg, K.S., Mickelson, D., McGruder, E.D., 2002. Functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus. Virology 298, 258-270.

Bautista, E.M., Molitor, T.W., 1997. Cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine. Viral Immunol 10, 83-94.

Bautista, E.M., Suarez, P., Molitor, T.W., 1999. T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus. Arch Virol 144, 117-134.