HIV-1 AE重组型包膜蛋白疫苗构建及免疫原改造和免疫策略研究

【作者】 张聪优；

【导师】 许雪梅； 徐建青；

【作者基本信息】 中国协和医科大学 ， 病原生物学， 2010， 硕士

【摘要】 人免疫缺陷病毒I型(Human Immunodeficiency Virus TypeⅠ, HIV-1)感染可以导致获得性免疫缺陷综合症(acquired immunodeficiency syndrome, AIDS),即艾滋病。艾滋病已成为有史以来最具破坏性的传染病之一：目前全球共存活艾滋病病毒感染者和病人(HIV/AIDS)约3340万人(约占总人口的0.6%),在中国这个数字也有是74万,且2009年当年中国新发艾滋病病毒感染者就有4.8万人。HIV-1一旦感染,便很难被机体清除；结合历史上多次传染病控制经验,研发有效安全的疫苗将是控制HIV-1大规模流行的最佳途径。流行病学调查显示我国HIV-1最主要的流行亚型或流行重组型是CRF07-BC,CRF08-BC,B’(泰国B)和CRF01_AE。其中AE重组型病毒主要通过性接触途径传播,而性接触是已成为我国新发感染的主要途径,伴随这一情况,AE重组型在新发感染中的比例也呈逐年上升的态势。因此研发针对HIV-1 AE重组型的预防性疫苗,对于控制我国HIV-1的传播有重要意义。包膜蛋白是设计HIV-1抗体疫苗时选择的主要免疫原。但是,天然的HIV-1包膜蛋白由于高度糖基化导致一些保守抗原表位遮蔽,因而天然HIV-1膜蛋白难于有效地活化广谱中和抗体。有研究显示,通过对包膜蛋白糖基化位点的修饰改造可以消除这些不利因素,提高HIV-1膜蛋白活化中和抗体的能力。所以在本研究的第-部分,我们构建了表达HIV-1 AE重组型膜蛋白gp140的DNA疫苗,并在此基础上通过定点突变方法分别突变了gp140 V1/V3的157/161和V4区的382/388糖基化位点,并构建成相应DNA疫苗m157/161和m157/161。Western Blot结果显示,改造前后的质粒均可以高效表达目的蛋白,其中包括大量以分泌形式存在的蛋白;改造对蛋白的表达量无明显影响。为了评价上述几种DNA疫苗活化的免疫反应特征,我们用所构建的DNA疫苗肌肉注射免疫小鼠4次,最后一次免疫两周后处死小鼠进行检测。基于IFN-γ检测的ELISPOT实验显示,gp140免疫组小鼠的特异性T细胞反应为(2432±586)SFCs/106脾细胞,m157/161组为(2682±893)SFCs/106脾细胞,m382/388组为(2360±560)SFCs/106脾细胞,各实验组间无统计学差异。说明疫苗可以活化高水平T细胞反应,且糖基化改造对疫苗活化的特异性T细胞反应强度没有显著影响。ELISA实验检测小鼠血清中的结合抗体,结果显示,gp140组平均抗体滴度是4800,m157/161组是1600,m382/388组是1200。两个糖基化改造组显著低于未改造组,p<0.02。说明糖基化位点突变后,膜蛋白疫苗活化结合抗体反应的能力明显下降。最后,用体外中和实验评价小鼠血清对3株B’/C重组型HIV-1临床分离株(XBC6371,XBC0793和XBC6431)的中和能力。数据显示：对XBC6371和XBC6431病毒株,gp140免疫组的血清几乎没有中和作用,m157/161和m382/388组有个别小鼠出现6%-16%的中和活性；中和XBC0793病毒时,m157/161组平均病毒抑制率为47%,显著高于gp140免疫组(17.7%),p=0.023：m382/388平均病毒抑制率为49%,也显著高于gp140免疫组,p=0.013。说明157/161和382/388位点的糖基化改造可以提高HIV-1AE亚型gp140疫苗中和异源型别病毒能力,并且中和活性的提高并不是因为结合抗体的活化导致的。这部分实验结果说明,本实验构建了可以有效活化结合抗体和T细胞反应的AE亚型膜蛋白疫苗；本实验开发的糖基化位点改造方案为增强HIV膜蛋白疫苗活化中和抗体能力提供了一定思路。后续应选择更多不同来源病毒株对其中和能力进行进一步验证,其中和能力提高的原因也需要进行进一步研究。另一方面,合适的载体是提高疫苗效果的有效途径之一。经过多年临床使用的、安全性好的复制型痘苗病毒载体疫苗将是艾滋病载体疫苗未来发展的主流方向之一。相对于DNA疫苗或痘病毒疫苗单独免疫,DNA初免-痘病毒加强策略可以提高免疫原的免疫原性,并且避免了加强针对载体的免疫反应。因而本研究第二部分中,我们构建了表达HIV-1 AE亚型包膜蛋白的复制型天坛株痘苗病毒载体疫苗,并使用DNA疫苗初免/痘苗病毒加强策略,在小鼠模型评价疫苗的免疫效果。以往研究发现,包膜蛋白去除胞内区和跨膜区的截短型gp140作为可分泌抗原,可以活化高水平的抗体反应；而保留跨膜区,仅去除胞内区的另一种截短型包膜蛋白gp145被证明相对gp140可以更好的活化T细胞反应。我们设想,在DNA初免/痘苗病毒加强策略下,用gp140和gp145先后免疫同一个体是否可以同时活化高水平的T细胞反应和抗体反应?于是在第二部分研究中,我们设计了gp140和gp145疫苗异源交叉初免/加强的实验方案,即用一种截短型膜蛋白DNA免疫3次后,用另一种截短型膜蛋白重组痘苗病毒加强免疫1次,以探讨不同免疫方案活化的免疫效果,并选择最优方案。我们首先用ELISA实验方法检测了疫苗活化的结合抗体,结果显示gp140DNA-gp140痘苗免疫组和gp140DNA-gp145痘苗免疫组活化了很高的抗体反应,滴度的几何平均值分别为12800和19401；而gp145 DNA-gp145痘苗免疫组和gp145DNA-gp140痘苗免疫组活化的结合抗体滴度的几何平均值分别只有566和606。本结果证实了gp140初免活化抗体的能力高于gp145初免；同时发现在本免疫策略下,初免选择的DNA免疫原类型主要决定了小鼠最终的抗体滴度。接着我们用基于IFN-γ检测的ELISPOT实验方法评价了各实验组活化的特异性T细胞反应水平。结果显示,gp145疫苗初免加强组活化的T细胞反应((3424±650)SFCs/106脾细胞),高于gp140疫苗初免/加强组((1918±442)SFCs/106脾细胞)。并且,gp140 DNA初免的小鼠,用gp145痘苗异源加强可以活化比用gp140加强更高一些的T细胞反应;同样,用gp145 DNA初免的小鼠,用gp145加强也比用gp140加强活化的T细胞反应略高。这部分数据说明,相对于gp140,gp145无论用做初免还是加强,都可以更好的活化T细胞反应。结合以上两部分数据可以看出,gp140 DNA初免/gp145痘苗病毒加强是可以同时活化高水平的T细胞反应和结合抗体反应的免疫方案。为了深入了解该疫苗活化的T细胞反应特征,本部分进一步研究了AE亚型膜蛋白疫苗在BALB/c (H-2d)小鼠模型上的T细胞表位分布,用肽库作图筛选法,我们定位到几个可以活化相对较强T细胞反应的肽表位：35号肽(NSNNTTNGPNKIGNI)、16号肽(ETEVHNVWATHACVP)、136号肽(QQQSNLLRAIEAQQH)和106号肽(GQAMYAPPISGRINC).其中,第35号肽是一个很强的T细胞免疫优势表位,针对这个表位的T细胞反应强度占到总T细胞反应的61.3%。针对以上几个肽表位的T细胞反应占总T细胞反应的81.7%,说明本疫苗在BALB/c小鼠体内活化的T细胞免疫反应主要集中于几个优势表位上。经过与Los Alamos HIV数据库和其他研究文章的比对,我们发现本实验定位到的35号肽表位为本实验首次发现到的H-2d限制性T细胞表位。该研究将为评价HIV疫苗在H-2d型遗传背景下的免疫效果提供数据支持。本研究也存在一定的局限之处,期待对本研究的疫苗和免疫策略在非人灵长类大动物模型的进一步评价及对其活化中和抗体能力的进一步评价；也提示了开发可以活化针对更多表位,尤其是保守表位的T细胞反应免疫策略的重要性。更多还原

【Abstract】 Human Immunodeficiency Virus Type I （HIV-1） is the etiologic agent for acquired immunodeficiency syndrome （AIDS） and there are 33.4 million people reported living with HIV/AIDS world widely （the prevalence rate is 6%o approximately）, including around 700,000 cases from China. Once HIV-1 infection occurs, it is hardly to be eradicated by host immunity. The history of containing infectious diseases surmises that a safe and effective vaccine for HIV is the cost-effective way to control its pandemic. Epidemiological data showed that several subtypes and CRFs of HIV-1 circulate in China, CRF07_BC, CRF08_BC, B’（Thailand B） and CRF01_AE are the major forms. Currently, sexual contact has become the dominant risk factor of HIV infection in China. CRF01_AE usually spread with sexual transmitted population is on the rise consequently. Therefore, to develop a prophylactic vaccine against CRF_AE is desirable for controlling HIV-1 pandemic in China.Envelope protein of HIV-1 （Env） is a major immunogen for antibody based vaccine and has been tested in several vaccines in clinical trials. However, the natural form of Env is incapable of inducing broadly neutralizing antibodies because of its highly glycosylation. Several studies have shown that introducing particular modifications at the glycosylation sites of Env can improve its ability to elicit neutralizing antibodies. In the first section of this thesis, we constructed a DNA vaccine expressing human codon-optimized gp140 of CRF01_AE, a truncated Env without transmembrane and intracellular regions, and then further introduced N to Q mutations at the sites of N 157 and N 161 in the V1/V2 （m157/161） or of N 382 and N 388 in the V4 （m382/388） loops. Western Blot showed that all DNA vaccines can express gp140 effectively in a secreting form at a similar level and is detectable in both cell lysate and culture supernatant.To determine the immune response elicited by these DNA vaccines, we immunized mice four times at 2-week interval, and immune responses were examined in 2 weeks after the final injection. ELISPOT based on IFN-y secreting showed that the specific T cell responses elicited by gp140 （2432±586 SFCs/106 splenocytes）, m157/161 （2682±893 SFCs/106 splenocytes）, and m382/388 （2360±560 SFCs/106 splenocytes） did not reach any significant differences. This implies that each of the three DNA vaccines can elicit a robust specific T cell response, and the modification of selected glycans does not significantly affect specific T cell response. Then ELISA assay was performed to test the titer of binding antibody. The average antibody titer of gp140 group, m157/16, m382/388 is 4800,1600 and 1200, respectively. It’s significantly decreased in the glycans deleted vaccine immunized groups （ml57/16 VS gp140 p<0.02, m382/388 VS gp140）. This result implies that deglycosylation of the VI/V2 or V4 loops failed to enhance the levels of binding antibodies to its wild-type counterpart.We also tested the neutralizing activity of mice sera against three primary isolates of CRF07_BC （XBC6371, XBC0793, and XBC6431）. Our data showed that sera of mice inoculated with gp140 did not have any neutralizing activities against XBC6371 and XBC6431, whereas several mice from glycosylation deleted groups showed a virus inhibition activity of 6%-16%. In contrast, for XBC0793, the mean inhibition of m157/161 group was 47% and m382/388 group was 49%, both are significantly higher than gp140 group （17.7%,p<0.05）. It indicated m157/161 and m382/388 could enhance capacity of CRF₀1AE gp140 to elicit neutralizing antibodies against heterologous viruses, and the increased neutralizing activity was not associated with binding antibody activity. In conclusion, we successfully constructed a CRF01_AE Env vaccine that can effectively induce binding antibodies and T cell responses; the deglycosylation forms we tested here also provide prospects for designing vaccine aiming broad neutralizing antibody activities. Moreover, different subtypes of viruses should be further tested to confirm the spectrum of neutralization and the mechanism underlying the promotion of neutralizing activity should be further explored.As known, a proper vector can significantly improve a vaccine’s efficiency. The clinically tested, safety proved replication-competent vaccinia virus will be one of the candidates for HIV-1 vaccine. Comparing with the strategy to use DNA or poxvirus alone, the DNA prime-poxvirus boost strategy is more widely used for its capability of inducing higher antigen specific immune responses without increasing of immunity against vector. In the second section of this thesis, we constructed recombinant replication-competent vaccinia vaccines expressing HIV-1 CRF01_AE Env, and the DNA prime/recombinant vaccinia boost strategy was used in a Balb/c mice model to test immunogenicity of this immunization strategy. Consistent with previous studies, g high level of B cell responses was elicited by secreting formed gp140, in contrast the membrane anchored gp145 with transmembrane region was capable of inducing high level T cell responses. As a result, we presumed that inoculated one animal with the two differently truncated Env forms as prime/boost immunogens may efficiently elicit both arms of the adaptive immunity. Therefore, in the second section of this thesis, we tested our hypothesis to select out the best combination. Firstly, we tested binding antibody titers elicited by vaccines using ELISA assay. Data showed that both groups priming with gp140 raised high-level of binding antibody titers with a geometric-mean of 128 00 in gp140 prime/gp140 boost group and 19 401 in gp140 prime/gp145 boost group respectively. On the contrary, in gp145 primed groups, only low antibody titers were elicited with a geometric-mean of 566 in gp145 prime/ gp145 boost group and 606 in gp145 prime/gp140 boost group respectively. Our data confirmed the fact that gp140 elicits better antibody responses than gp145, and we observed that the priming immunogen determines the final antibody level. We quantified vaccine activated specific T cell responses using IFN-γbased ELISPOT assay. Our data showed, T cell response elicited by gp145 prime/gp145 boost （（3424±650） SFCs/106 splenocytes） is higher than that elicited by gp140 prime/gp140 boost （（1918±442） SFCs/106 splenocytes）. Furthermore, in heterologous prime/boost groups, gp145 also primed or boosted slightly higher T cell response than gp140. These data indicated that gp145 induced higher T cell response either as priming immunogen or as boosting immunogen than gp140 in DNA prime/recombinant vaccinia virus boost regimen. Taken together, we draw a conclusion that gp140 DNA prime/gp145 vaccinia virus boost strategy will be the best one for inducing both high-level of binding antibody and T cell responses among all regimens we tested.For further defining of the specific T cell responses, we mapped T cell epitopes by using HIV-1 Env derived peptides in Balb/c （H-2d） mice. Several epitopes were defined in peptides as following:No.35 （NSNNTTNGPNKIGNI）, No.16 （ETEVHNVWATHACVP）, No.136 （QQQSNLLRAIEAQQH） and No.106 （GQAMYAPPISGRINC）. The No.35 peptide is a dominant T cell epitope, T cell responses induced by this epitope accounts for 61.3% of total T cell responses, and by the five epitopes accounts totally for 81.7% of the total T cell response. After blasting in Los Alamos database and comparing with other studies, we identified that the No.35 peptideis a newly discovered H-2d restricted T cell epitope. These results would be reference for analyzing immune responses elicited in Balb/c mice and other with H-2d genetic backgrounds. The limitation of our immunization strategy is that it failed to elicit broad T cell responses against its counterparts derived from different clades. The vaccines and immunization strategy should also be further explored in non-human private models.更多还原