豬生殖與呼吸道綜合症病毒疫苗之研發

The Development of Porcine Reproductive and Respiratory Syndrome Virus Vaccine

賈敏原(Min-Yuan Chia)

指導教授 ： 龐飛

共同指導教授 ： 鄭謙仁(Chian-Ren Jeng)

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00155

摘要

豬生殖與呼吸道綜合症病毒 (PRRSV) 自 90 年代末期至今對全世界養豬產業造成嚴重的經濟損失。目前只有少數商品化的死毒或減毒疫苗，此外亦有數種不同的實驗性 DNA 疫苗或是以 baculovirus、adenovirus、pseudorabies virus、vaccina virus 作為載體所發展的 PRRSV 次單位疫苗，但由於 PRRSV 本身在宿主所誘發的免疫反應極為複雜，加上其本身又具有規避宿主免疫系統捕捉的機制，導致發展有效對抗本病的疫苗仍具有相對的困難性。 PRRSV 的封套醣 (GP5) 和膜 (M) 蛋白被認為是誘發宿主免疫保護反應最主要的區段。研究顯示，DNA 疫苗單獨表現 GP5 或 M 蛋白時所誘發的中和抗體，比同時表現 GP5 和M 蛋白時為佳;再者以 vaccinia virus 作為載體系統的研究顯示，使用兩個啟動子同時表現 GP5 和 M所誘發的免疫原性，比只使用一個啟動子同時表現 GP5 和 M 蛋白作為融合蛋白 (fusion protein) 為佳，而 GP5/M 融合蛋白表現較差的原因，被推測是由於其自然結構於兩個蛋白融合時受到改變。在本研究中，我們嘗試在 GP5/M 融合蛋白間加入一段具有彎曲延展性的多肽連接子，期望能將 GP5 和 M 蛋白上所有自然結構的抗原決定區都能呈現給抗原呈現細胞。因此，本研究的第一個目的是評估以 glycine-proline-glycine-glycine (GPGP) 作為連接子時，是否能促進同時表現 GP5和M蛋白之 PRRSV DNA疫苗運用於小鼠和豬的免疫原性 (第二和第三章)。在本項研究中共構築了三組不同的 DNA，分別為 (1) 不具 GPGP 連接子的 pcDNA-56、(2) 具有 GPGP 連接子的 pcDNA-5L6、(3) 具有GPGP 連接子的 pcDNA-6L5。將上述三組構築載入真核表現系統之 pcDNA3.1/V5-His TOPO 載體內，作為 DNA 疫苗使用，將此三組疫苗以肌肉注射的方式免疫小鼠四次，每次間隔兩週 (第二章)，以及也將此三組疫苗以肌肉注射的方式免疫豬隻三次，每次間隔兩週，再於最後一次免疫的三週後，以 5 × 105 TCID50 PRRSV 進行攻毒 (第三章)，以評估該 DNA 疫苗於小鼠和豬隻所誘發之免疫及/或保護效果。結果顯示，小鼠和豬隻免疫 pcDNA-5L6 或 pcDNA-6L5 所誘發的 PRRSV 特異性血清 IgG 抗體、中和抗體及淋巴細胞增殖反應都比 pcDNA-56 組為佳，而攻毒後 pcDNA-5L6 或 pcDNA-6L5 組所出現的病毒血症和組織病毒分佈量均比 pcDNA-56 組低。此結果證實以 GPGP 連接子同時表現 GP5 和 M 蛋白時，可能因具有還原GP5/M 蛋白自然結構的功能，進而促進其在小鼠和豬隻的免疫原性。由於 PRRSV 感染豬隻時，主要是經由呼吸、生殖和消化道黏膜系統進入宿主體內，因此，若能藉由次單位口服疫苗的方式，進而活化黏膜免疫反應，則可於第一道免疫保護機制即阻斷 PRRSV 的感染。近年來分子生物學與植物生物科技日益發展，基因轉殖植物次單位口服疫苗，儼然成為發展新一代疫苗的趨勢，基因轉殖植物次單位口服疫苗具有價格便宜、容易保存、投予方便等優勢，且堅韌的植物細胞壁具有保護抗原蛋白免於被胃酸破壞的功用，使得抗原蛋白能夠到達腸道相關淋巴組織 (gut-associated lymphoid tissue; GALT)。另有研究發現，大腸桿菌熱溶解性次單位 B (Escherichia coli heat-labile enterotoxin B subunit; LTB) 是一種具有黏膜佐劑功能的細菌蛋白，故本研究第二個目的是評估以基因轉殖菸草同時表現 LTB 與 GP5 作為次單位疫苗的可行性及其在豬隻的免疫與保護效果 (第四章及第五章)。在此部分共設計了兩組基因轉殖菸草口服次單位疫苗，分別為 (1) 只表現 GP5 的基因轉殖菸草 (GP5-T) 及 (2) 同時表現 LTB 與GP5 的基因轉殖菸草 (LTB-GP5-T)。將此兩組基因轉殖菸草疫苗以口服的方式給予豬隻三次，每次間隔兩週，於最後一次免疫三週後，以 5 × 105 TCID50 PRRSV 進行攻毒，評估此疫苗於豬隻之免疫及/或保護效果。結果顯示，豬隻口服 LTB-GP5-T 或 GP5-T 所產生的 PRRSV 特異性抗體和細胞性免疫反應皆比口服一般菸草 (W-T) 的對照組顯著性高，同時攻毒後 LTB-GP5-T 或 GP5-T 組的病毒血症和組織病毒分佈量均比 W-T 組低，雖然 LTB-GP5-T 組所誘發的免疫反應比 GP5-T 組略高，但兩者之間並無統計上的顯著差異。總結來說，本研究中所建構的 DNA 疫苗和基因轉殖菸草口服疫苗，的確能誘發豬隻產生 PRRSV 特異性之體液性與細胞性免疫反應，然而，不論是 DNA 疫苗或基因轉殖菸草口服疫苗，在攻毒後皆不能完全避免 PRRSV 的持續性感染及呼吸道和淋巴組織的持續排毒。如何進一步針對 PRRS DNA 疫苗或基因轉殖植物口服疫苗在體液及細胞免疫反應的免疫效能上再做調理與強化為未來應努力的方向。

關鍵字

豬生殖與呼吸道綜合症 ； 疫苗

並列摘要

chapter I
Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry causing tremendous economic losses throughout the world since late 90’s. Currently, there are only a limited number of inactivated or modified-live PRRSV vaccines are available on the market. Several approaches have been used to develop subunit vaccines based on plasmid DNA, baculovirus, adenovirus, pseudorabies virus, and vaccinia virus systems. However, the complexity of the host immune response to PRRSV and the ability of the virus to escape or modulate the host’s immune system make it difficult to develop a vaccine that could eradicate this disease. The glycoprotein 5 (GP5) and membrane protein (M) of PRRSV are essential in inducing host protective immunity. It has been demonstrated that DNA vaccines expressing GP5 or M alone induced relatively weak NA responses when compared to that co-expressing GP5 and M as a fusion protein. Moreover, recombinant vectored vaccinia virus vaccine co-expressing GP5 and M proteins of PRRSV with two different promoters displayed more immunogenicity than that co-expressing GP5 and M as a fusion protein; and it was speculated due to altered natural conformation of the fusion protein. A flexible linker such as the twelve-base-pair oligonucleotide sequence-encoded flexible glycine-proline-glycine-proline (GPGP) linker may preserve the intact neutralizing epitopes of these two proteins and present them to antigen-presenting cells. Thus, the first objective of the present study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in mice and pigs (Chapters II and III). Three different DNA constructs, one expressing GP5/M without GPGP linker (pcDNA-56) and two expressing GP5/M conjugated by GPGP linker (pcDNA-5L6 and pcDNA-6L5) but with different tandem orientation, were established. These constructs were then inserted into an eukaryotic expression vector, pcDNA3.1/V5-His TOPO®, as DNA vaccines to immunize mice intramuscularly for four times at a 2-week interval (Chapter II) and to immunize pigs intramuscularly for three times at a 2-week interval followed by challenge with 5 × 105 TCID50 PRRSV at three weeks after final immunization (Chapter III). The results showed that pcDNA-5L6 and pcDNA-6L5 induced higher PRRSV-specific IgG and neutralizing antibody (NA) responses, greater PRRSV-specific lymphocyte proliferation responses, and lower viremia and tissue viral load than did pcDNA-56. The results suggest that the GPGP linker may indeed preserve the natural conformation and immunogenicity of both GP5 and M proteins. It is known that PRRSV enters the host via the mucosa of gastrointestinal, respiratory, and reproductive tracts. Thus, it may be possible to activate the common mucosal immunity by using subunit oral vaccine to prevent PRRSV infection at the first line of defense. In recent years, with the development of genetic molecular biology and plant biotechnology, the genetic engineering subunit vaccine is taking on a prosperous evolvement. Expressing subunit vaccine candidates in plants opens a new avenue for producing oral/edible vaccines. Transgenic plant vaccines have advantages such as low cost, easiness in storage, and convenience in inoculation. The rigid plant cell wall can also protect the antigenic proteins from gastric acidic environment, allowing the antigens to reach gut-associated lymphoid tissue intactly. Escherichia coli heat-labile enterotoxin B subunit (LTB) is a well-characterized bacterial protein having a strong potential as a mucosal adjuvant. The antigen can be delivered across the mucosa epithelium to the underlying mucosa-associated lymphoid tissue if the antigen is genetically fused with LTB to form a pentamer. Thus, the second objective of this study was to evaluate the feasibility of co-expressing of LTB and PRRSV GP5 in transgenic tobacco plant and its immunogenicity and protective efficacy in pigs (Chapters IV and V). Two different transgenic tobacco plants, one expressing PRRSV GP5 (GP5-T) and the other co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T), were constructed. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at three weeks after final oral feeding. Pigs receiving LTB-GP5-T or GP5-T developed significantly higher PRRSV-specific antibody- (AMI) and cell- (CMI) mediated immunity and showed significantly lower viremia, tissue viral load, and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant. In summary, the DNA constructs and transgenic tobacco plants indeed developed specific AMI and CMI responses in pigs against PRRSV infection. However, neither the DNA constructs nor the transgenic tobacco plants could completely eliminate the persistent infection and shedding of PRRSV in the respiratory tract and lymphoid tissue after challenge. Effects are required to further effectively improve the immunogenicity in both AMI and CMI responses of either DNA constructs or transgenic plants.

並列關鍵字

PRRSV ； vaccine

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