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
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。
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 CRF_01AE 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.
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。
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 CRF_01AE 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.
引文
[1]UNAIDS, WHO. AIDS epidemic update:November 2009; 2009.
[2]卫生部新闻办公室.2009 卫生部介绍中国艾滋病疫情现状.2009 [cited; Available from: http://www.moh.gov.cn/publicfiles/business/htmlfiles/mohbgt/s3582/200911/44754.htm
[3]Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med,2009,60:471-484.
[4]Rambaut A, Posada D, Crandall KA, et al. The causes and consequences of HIV evolution. Nat Rev Genet,2004,5(1):52-61.
[5]HIV Sequence Database Web Site. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory,Los Alamos, NM:http://hiv-web.lanl.gov.
[6]Hemelaar J, Gouws E, Ghys PD, et al. Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004. Aids,2006,20(16):W13-23.
[7]Shao YM, Su L, XH S. Molecular epidemiology of HIV infection in China. XII International Conference on AIDS; 1998; Geneva, Switzerland.
[8]Ministry of Science and Technology C. Research progress:Nationwide HIV molecular epidemiology survey and its database setting. [cited; Available from:http://shgy.jhgl.org/shownews.asp?newsid=893.
[9]Rizzardini G, Trabattoni D, Saresella M, et al. Immune activation in HIV-infected African individuals. Italian-Ugandan AIDS cooperation program. Aids,1998,12(18):2387-2396.
[10]Murphy E, Korber B, Georges-Courbot MC, et al. Diversity of V3 region sequences of human immunodeficiency viruses type 1 from the central African Republic. AIDS Res Hum Retroviruses,1993, 9(10):997-1006.
[11]Carr JK, Salminen MO, Koch C, et al. Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand. J Virol,1996,70(9):5935-5943.
[12]Gao F, Robertson DL, Morrison SG, et al. The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin. J Virol,1996, 70(10):7013-7029.
[13]邢辉,梁浩,万卓越.中国CRF01_AE亚型人类免疫缺陷病毒毒株的分子流行病学研究.中华预防医学杂志,2004,38:300-304.
[14]尚红.中国艾滋病流行和检测及治疗现状与发展趋势.中华检验医学杂志,2008,31:1088-1090.
[15]Wang L, Wang N, Li D, et al. The 2007 Estimates for People at Risk for and Living With HIV in China:Progress and Challenges. J Acquir Immune Defic Syndr,2009,50(4):414-418.
[16]Lu L, Jia M, Ma Y, et al. The changing face of HIV in China. Nature,2008,455(7213):609-611.
[17]Wang W, Jiang S, Li S, et al. Identification of subtype B, multiple circulating recombinant forms and unique recombinants of HIV type 1 in an MSM cohort in China. AIDS Res Hum Retroviruses,2008, 24(10):1245-1254.
[18]任莉,仇超,黄相刚,等.HIV-1 B亚型gag-env融合基因的DNA疫苗构建和免疫原性分析.《中华微生物学和免疫学杂志》,2006,26(7):654-657.
[19]Wan Y, Wu L, Liu L, et al. Comparison of immunogenicity between codon optimized HIV-1 Thailand subtype B gp140 and gp145 vaccines. Vaccine,2007,25(26):4949-4959.
[20]孙健,李杰,吴南屏.HIV疫苗国内外研究进展.国际流行病学传染病学杂志,2009, 36(3)：183-187.
[21]程春林,冯毅,何翔.中国南方四省区流行的HIV-1 CRF01_AE病毒株基因特征研究.中华流行病学杂志,2009,30(7):720-725.
[22]Huang HL, Yan PP, Zheng J, et al. Genetic characterization of three CRF01_AE full-length HIV type 1 sequences from Fujian Province, China. Chin Med J (Engl),2006,119(19):1622-1628.
[23]邢辉,梁浩,洪坤学等.我国HIV-1主要流行株外膜蛋白(env)基因V3-V4区变异及其与生物学特性的关系.中华微生物学和免疫学杂志,2005,25(3):185-189.
[24]Letvin NL, Huang Y, Chakrabarti BK, et al. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. Journal of Virology,2004,78(14):7490-7497.
[25]Flynn NM, Forthal DN, Harro CD, et al. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis,2005,191(5):654-665.
[26]Pitisuttithum P, Gilbert P, Gurwith M, et al. Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis,2006,194(12):1661-1671.
[27]Russell ND, Graham BS, Keefer MC, et al. Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in combination with rgp120:negative results fail to trigger a phase 3 correlates trial. J Acquir Immune Defic Syndr,2007,44(2):203-212.
[28]Gnann JW, Jr., Nelson JA, Oldstone MB. Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J Virol,1987,61(8):2639-2641.
[29]Muster T, Guinea R, Trkola A, et al. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. J Virol,1994, 68(6):4031-4034.
[30]Chakrabarti BK, Kong WP, Wu BY, et al. Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. J Virol,2002, 76(11):5357-5368.
[31]Earl PL, Sugiura W, Montefiori DC, et al. Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140. J Virol,2001,75(2):645-653.
[32]Li Y, Cleveland B, Klots I, et al. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol,2008, 82(2):638-651.
[33]Yang ZY, Chakrabarti BK, Xu L, et al. Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope. J Virol,2004, 78(8):4029-4036.
[34]Luo M, Yuan F, Liu YX, et al. Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment. Vaccine,2006,24(4):435-442.
[35]Beddows S, Schulke N, Kirschner M, et al. Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. Journal of Virology,2005,79(14):8812-8827.
[36]Morner A, Douagi I, Forsell MNE, et al. Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein. Journal of Virology,2009,83(2):540-551.
[37]Fouts T, Godfrey K, Bobb K, et al. Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques. Proc Natl Acad Sci U S A,2002, 99(18):11842-11847.
[38]Yuste E, Bixby J, Lifson J, et al. Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies. J Virol,2008,82(24):12472-12486.
[39]Kwong PD, Doyle ML, Casper DJ, et al. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature,2002,420(6916):678-682.
[40]Mackett M, Smith GL, Moss B. Vaccinia virus:a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A,1982,79(23):7415-7419.
[41]Brochier B, Aubert MF, Pastoret PP, et al. Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America. Rev Sci Tech,1996,15(3):947-970.
[42]Kresge K. Leading AIDs vaccine researches gather to discuss balancing AIDS vaccine funding [2009-04-01].. http://wwwiavireportorg/arehives/2008/pages/IAVl-Report12(2)-balancingaspx.
[43]Letvin NL. Progress toward an HIV vaccine. Annu Rev Med,2005,56:213-223.
[44]Santra S, Sun Y, Parvani JG, et al. Heterologous prime/boost immunization of rhesus monkeys by using diverse poxvirus vectors. J Virol,2007,81(16):8563-8570.
[45]Liu L, Wan Y, Xu J, et al. Immunogenicity comparison between codon optimized HIV-1 CRF BC_07 gp140 and gp145 vaccines. AIDS Res Hum Retroviruses,2007,23(11):1396-1404.
[46]Gurunathan S, Klinman DM, Seder RA. DNA vaccines:immunology, application, and optimization*. Annu Rev Immunol,2000,18:927-974.
[47]Earl PL, Broder CC, Long D, et al. Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities. J Virol,1994,68(5):3015-3026.
[48]Richardson TM, Jr., Stryjewski BL, Broder CC, et al. Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein. J Virol,1996,70(2):753-762.
[49]Petry H, Dittmer U, Jones D, et al. Prechallenge high neutralizing antibodies and long-lasting immune reactivity to gp41 correlate with protection of rhesus monkeys against productive simian immunodeficiency virus infection or disease development. J Acquir Immune Defic Syndr Hum Retrovirol, 1998,19(5):441-450.
[50]Pantaleo G, Menzo S, Vaccarezza M, et al. Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N Engl J Med,1995,332(4):209-216.
[51]Ogg GS, Jin X, Bonhoeffer S, et al. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science,1998,279(5359):2103-2106.
[52]Planz O, Ehl S, Furrer E, et al. A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers. Proc Natl Acad Sci U S A,1997, 94(13):6874-6879.
[53]Jin X, Bauer DE, Tuttleton SE, et al. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med,1999,189(6):991-998.
[54]Bernstein HB, Tucker SP, Hunter E, et al. Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides. J Virol,1994,68(1):463-468.
[55]Haggerty S, Dempsey MP, Bukrinsky MI, et al. Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection. AIDS Res Hum Retroviruses,1991,7(6):501-510.
[56]Kolchinsky P, Kiprilov E, Bartley P, et al. Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops. J Virol,2001,75(7):3435-3443.
[57]Hansen JE, Clausen H, Nielsen C, et al. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies:peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization. J Virol,1990,64(6):2833-2840.
[58]Kang SM, Quan FS, Huang C, et al. Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies. Virology,2005,331(1):20-32.
[59]McCaffrey RA, Saunders C, Hensel M, et al. N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies. J Virol,2004,78(7):3279-3295.
[60]Haigwood NL, Nara PL, Brooks E, et al. Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons. J Virol, 1992,66(1):172-182.
[61]Bolmstedt A, Hinkula J, Rowcliffe E, et al. Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan. Vaccine,2001,20(3-4):397-405.
[62]Bolmstedt A, Sjolander S, Hansen JE, et al. Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response. J Acquir Immune Defic Syndr Hum Retrovirol,1996,12(3):213-220.
[63]Burke B, Derby NR, Kraft Z, et al. Viral evolution in macaques coinfected with CCR5-and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies. Virology,2006,355(2):138-151.
[64]Quinones-Kochs MI, Buonocore L, Rose JK. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein:effects on protein function and the neutralizing antibody response. J Virol, 2002,76(9):4199-4211.
[65]Mori K, Sugimoto C, Ohgimoto S, et al. Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model. J Virol,2005, 79(16):10386-10396.
[66]Wan Y, Liu L, Wu L, et al. Deglycosylation or partial removal of HIV-1 CN54 gp140 V1/V2 domain enhances env-specific T cells. AIDS Res Hum Retroviruses,2009,25(6):607-617.
[67]Hong PW, Nguyen S, Young S, et al. Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120. J Virol,2007,81(15):8325-8336.
[68]Lue J, Hsu M, Yang D, et al. Addition of a single gp120 glycan confers increased binding to dendritic cell-specific ICAM-3-grabbing nonintegrin and neutralization escape to human immunodeficiency virus type 1. J Virol,2002,76(20):10299-10306.
[69]Koch M, Pancera M, Kwong PD, et al. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition. Virology,2003,313(2):387-400.
[70]Amara RR, Smith JM, Staprans SI, et al. Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine. J Virol,2002,76(12):6138-6146.
[71]Kim YB, Han DP, Cao C, et al. Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies. Virology,2003, 305(1):124-137.
[72]Amara RR, Villinger F, Altman JD, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science,2001,292(5514):69-74.
[73]Robinson HL, Montefiori DC, Johnson RP, et al. Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations. Nat Med,1999,5(5):526-534.
[74]Hel Z, Tsai WP, Thornton A, et al. Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen. J Immunol, 2001,167(12):7180-7191.
[75]Kent SJ, Zhao A, Best SJ, et al. Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J Virol,1998,72(12):10180-10188.
[76]Franchini G, Gurunathan S, Baglyos L, et al. Poxvirus-based vaccine candidates for HIV:two decades of experience with special emphasis on canarypox vectors. Expert Rev Vaccines,2004,3(4 Suppl):S75-88.
[77]Dorrell L, Yang H, Ondondo B, et al. Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine. J Virol,2006,80(10):4705-4716.
[78]Sadagopal S, Amara RR, Montefiori DC, et al. Signature for long-term vaccine-mediated control of a Simian and human immunodeficiency virus 89.6P challenge:stable low-breadth and low-frequency T-cell response capable of coproducing gamma interferon and interleukin-2. J Virol,2005,79(6):3243-3253.
[79]Santra S, Barouch DH, Korioth-Schmitz B, et al. Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses. Proc Natl Acad Sci U S A,2004, 101(30):11088-11093.
[80]Cole KS, Paliotti MJ, Murphey-Corb M, et al. Maturation of envelope-specific antibody responses to linear determinants in monkeys inoculated with attenuated SIV. J Med Primatol,2000,29(3-4):220-230.
[81]Cole KS, Rowles JL, Jagerski BA, et al. Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency virus and its association with the development of protective immunity. J Virol,1997,71(7):5069-5079.
[82]Nguyen M, Pean P, Lopalco L, et al. HIV-specific antibodies but not t-cell responses are associated with protection in seronegative partners of HIV-1-infected individuals in Cambodia. J Acquir Immune Defic Syndr,2006,42(4):412-419.
[83]Rao SS, Gomez P, Mascola JR, et al. Comparative evaluation of three different intramuscular delivery methods for DNA immunization in a nonhuman primate animal model. Vaccine,2006,24(3):367-373.
[84]Guerra S, Lopez-Fernandez LA, Pascual-Montano A, et al. Host response to the attenuated poxvirus vector NYVAC:upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells. J Virol,2006,80(2):985-998.
[85]Najera JL, Gomez CE, Domingo-Gil E, et al. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene. J Virol,2006, 80(12):6033-6047.
[86]Inaba K, Turley S, Yamaide F, et al. Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med,1998, 188(11):2163-2173.
[87]Albert ML. Death-defying immunity:do apoptotic cells influence antigen processing and presentation? Nat Rev Immunol,2004,4(3):223-231.
[88]Palmowski MJ, Choi EM, Hermans IF, et al. Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol,2002,168(9):4391-4398.
[89]Gomez CE, Najera JL, Jimenez EP, et al. Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1B X08 gp120 and HIV-1(ⅢB) Gag-Pol-Nef proteins of clade B. Vaccine,2007, 25(15):2863-2885.
[90]Rodriguez F, Harkins S, Slifka MK, et al. Immunodominance in virus-induced CD8(+) T-cell responses is dramatically modified by DNA immunization and is regulated by gamma interferon. J Virol, 2002,76(9):4251-4259.
[91]Barouch DH, Kunstman J, Kuroda MJ, et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature,2002,415(6869):335-339.
[92]Xu J, Ren L, Huang X, et al. Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes. AIDS,2006,20(18):2293-2303.
[1]万延民,吴岚,徐建青,et al.HIV-1 膜蛋白抗原优化改造研究进展.国际病毒学杂志,2007, 14(2):49-53.
[2]Kwong PD,Wyatt R,Robinson J,et al.Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.Nature,1998,393(6686):648-659.
[3]Reitter JN,Means RE,Desrosiers RC.A role for carbohydrates in immune evasion in AIDS.Nat Med, 1998,4(6):679-684.
[4]Burton DR,Desrosiers RC,Doms RW,et al.HIV vaccine design and the neutralizing antibody problem.Nat Immunol,2004,5(3):233-236.
[5]Flynn NM,Forthal DN,Harro CD,et a1.Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection.J Infect Dis,2005,191(5):654-665.
[6]Pitisuttithum P,Gilbert P,Gurwith M,et al.Randomized,double-blind,placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand.J Infect Dis,2006,194(12):1661-1671.
[7]Russell ND,Graham BS,Keefer MC,et al.Phase 2 study of an HIV-1 canarypox vaccine(vCP1452) alone and in combination with rgp120:negative results fail to trigger a phase 3 correlates trial.J Acquir Immune Defic Syndr,2007,44(2):203-212.
[8]Quinnan GV, Jr., Zhang PF, Fu DW, et al. Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response. AIDS Res Hum Retroviruses, 1999,15(6):561-570.
[9]Zhang PF, Cham F, Dong M, et al. Extensively cross-reactive anti-HIV-1 neutralizing antibodies induced by gp 140 immunization. Proc Natl Acad Sci U S A,2007,104(24):10193-10198.
[10]Zhang PF, Bouma P, Park EJ, et al. A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response. J Virol, 2002,76(2):644-655.
[11]Choudhry V, Zhang MY, Sidorov IA, et al. Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies. Virology,2007,363(1):79-90.
[12]McBurney SP, Ross TM. Viral sequence diversity:challenges for AIDS vaccine designs. Expert Rev Vaccines,2008,7(9):1405-1417.
[13]Liao HX, Sutherland LL, Xia SM, et al. A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and CHIV-1 primary viruses. Virology,2006, 353(2):268-282.
[14]Doria-Rose NA, Learn GH, Rodrigo AG, et al. Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope. J Virol,2005,79(17):11214-11224.
[15]Kothe DL, Li Y, Decker JM, et al. Ancestral and consensus envelope immunogens for HIV-1 subtype C. Virology,2006,352(2):438-449.
[16]Rolland M, Jensen MA, Nickle DC, et al. Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol,2007,81(16):8507-8514.
[17]Kong WP, Wu L, Wallstrom TC, et al. Expanded breadth of the T-cell response to mosaic human immunodeficiency virus type 1 envelope DNA vaccination. J Virol,2009,83(5):2201-2215.
[18]Xu J, Ren L, Huang X, et al. Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes. Aids,2006,20(18):2293-2303.
[19]Chohan B, Lang D, Sagar M, et al. Selection for human immunodeficiency virus type 1 envelope glycosylation variants with shorter VI-V2 loop sequences occurs during transmission of certain genetic subtypes and may impact viral RNA levels. J Virol,2005,79(10):6528-6531.
[20]Derdeyn CA, Decker JM, Bibollet-Ruche F, et al. Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science,2004,303(5666):2019-2022.
[21]Blish CA, Nedellec R, Mandaliya K, et al. HIV-1 subtype A envelope variants from early in infection have variable sensitivity to neutralization and to inhibitors of viral entry. Aids,2007,21(6):693-702.
[22]Blish CA, Nguyen MA, Overbaugh J. Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41. PLoS Med,2008,5(1):e9.
[23]Letvin NL, Huang Y, Chakrabarti BK, et al. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. Journal of Virology,2004,78(14):7490-7497.
[24]Laird ME, Igarashi T, Martin MA, et al. Importance of the V1/V2 loop region of simian-human immunodeficiency virus envelope glycoprotein gp120 in determining the strain specificity of the neutralizing antibody response. J Virol,2008,82(22):11054-11065.
[25]Yang ZY, Chakrabarti BK, Xu L, et al. Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope. J Virol,2004, 78(8):4029-4036.
[26]Cherpelis S, Jin X, Gettie A, et al. DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques. Immunol Lett,2001,79(1-2):47-55.
[27]Wan Y, Liu L, Wu L, et al. Deglycosylation or partial removal of HIV-1 CN54 gp140 V1/V2 domain enhances env-specific T cells. AIDS Res Hum Retroviruses,2009,25(6):607-617.
[28]Chen Z, Huang Y, Zhao X, et al. Design, construction, and characterization of a multigenic modified vaccinia Ankara candidate vaccine against human immunodeficiency virus type 1 subtype C/B' . J Acquir Immune Defic Syndr,2008,47(4):412-421.
[29]Mori K, Sugimoto C, Ohgimoto S, et al. Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model. J Virol,2005, 79(16):10386-10396.
[30]Li Y, Cleveland B, Klots I, et al. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol,2008, 82(2):638-651.
[31]Yuste E, Bixby J, Lifson J, et al. Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies. J Virol,2008,82(24):12472-12486.
[32]Quinones-Kochs MI, Buonocore L, Rose JK. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein:effects on protein function and the neutralizing antibody response. J Virol, 2002,76(9):4199-4211.
[33]Garrity RR, Rimmelzwaan G, Minassian A, et al. Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope. J Immunol,1997,159(l):279-289.
[34]Trujillo JD, Kumpula-McWhirter NM, Hotzel KJ, et al. Glycosylation of immunodominant linear epitopes in the carboxy-terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses. J Virol,2004, 78(17):9190-9202.
[35]Braibant M, Brunet S, Costagliola D, et al. Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors:prevalence and association with neutralizing activity. Aids,2006, 20(15):1923-1930.
[36]Suarez T, Nir S, Goni FM, et al. The pre-transmembrane region of the human immunodeficiency virus type-1 glycoprotein:a novel fusogenic sequence. Febs Letters,2000,477(1-2):145-149.
[37]Coeffier E, Clement JM, Cussac V, et al. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine,2000,19(7-8):684-693.
[38]Liang XP, Munshi S, Shendure J, et al. Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein. Vaccine,1999,17(22):2862-2872.
[39]Eckhart L, Raffelsberger W, Ferko B, et al. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus. Journal of General Virology,1996,77:2001-2008.
[40]Ho J, MacDonald KS, Barber BH. Construction of recombinant targeting immunogens incorporating an HIV-1 neutralizing epitope into sites of differing conformational constraint. Vaccine,2002, 20(7-8):1169-1180.
[41]Luo M, Yuan F, Liu YX, et al. Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment. Vaccine,2006,24(4):435-442.
[42]Srivastava IK, Stamatatos L, Kan E, et al. Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate. Journal of Virology,2003,77(20):11244-11259.
[43]Dey AK, David KB, Lu M, et al. Biochemical and biophysical comparison of cleaved and uncleaved soluble, trimeric HIV-1 envelope glycoproteins. Virology,2009,385(1):275-281.
[44]Binley JM, Sanders RW, Clas B, et al. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gpl20 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. Journal of Virology,2000,74(2):627-643.
[45]Edinger AL, Blanpain C, Kunstman KJ, et al. Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains. Journal of Virology,1999, 73(5):4062-4073.
[46]Beddows S, Schulke N, Kirschner M, et al. Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. Journal of Virology,2005,79(14):8812-8827.
[47]Morner A, Douagi I, Forsell MNE, et al. Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein. Journal of Virology,2009,83(2):540-551.
[48]Fouts T, Godfrey K, Bobb K, et al. Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques. Proc Natl Acad Sci U S A,2002, 99(18):11842-11847.
[49]LaBranche CC, Hoffman TL, Romano J, et al. Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity. J Virol, 1999,73(12):10310-10319.
[50]Kolchinsky P, Mirzabekov T, Farzan M, et al. Adaptation of a CCR5-using, primary human immunodeficiency virus type 1 isolate for CD4-independent replication. J Virol,1999,73(10):8120-8126.
[51]He Y, D'Agostino P, Pinter A. Analysis of the immunogenic properties of a single-chain polypeptide analogue of the HIV-1 gp120-CD4 complex in transgenic mice that produce human immunoglobulins. Vaccine,2003,21 (27-30):4421-4429.
[52]Yang X, Tomov V, Kurteva S, et al. Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1. J Virol,2004,78(23):12975-12986.
[53]Zhou T, Xu L, Dey B, et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature,2007,445(7129):732-737.
[54]Wu L, Zhou T, Yang ZY, et al. Enhanced exposure of the CD4-binding site to neutralizing antibodies by structural design of a membrane-anchored human immunodeficiency virus type 1 gp120 domain. J Virol, 2009,83(10):5077-5086.
[55]Hessell AJ, Poignard P, Hunter M, et al. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med,2009,15(8):951-954.
[56]Hessell AJ, Rakasz EG, Poignard P, et al. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog, 2009,5(5):e1000433.
[2]卫生部新闻办公室.2009 卫生部介绍中国艾滋病疫情现状.2009 [cited; Available from: http://www.moh.gov.cn/publicfiles/business/htmlfiles/mohbgt/s3582/200911/44754.htm
[3]Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med,2009,60:471-484.
[4]Rambaut A, Posada D, Crandall KA, et al. The causes and consequences of HIV evolution. Nat Rev Genet,2004,5(1):52-61.
[5]HIV Sequence Database Web Site. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory,Los Alamos, NM:http://hiv-web.lanl.gov.
[6]Hemelaar J, Gouws E, Ghys PD, et al. Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004. Aids,2006,20(16):W13-23.
[7]Shao YM, Su L, XH S. Molecular epidemiology of HIV infection in China. XII International Conference on AIDS; 1998; Geneva, Switzerland.
[8]Ministry of Science and Technology C. Research progress:Nationwide HIV molecular epidemiology survey and its database setting. [cited; Available from:http://shgy.jhgl.org/shownews.asp?newsid=893.
[9]Rizzardini G, Trabattoni D, Saresella M, et al. Immune activation in HIV-infected African individuals. Italian-Ugandan AIDS cooperation program. Aids,1998,12(18):2387-2396.
[10]Murphy E, Korber B, Georges-Courbot MC, et al. Diversity of V3 region sequences of human immunodeficiency viruses type 1 from the central African Republic. AIDS Res Hum Retroviruses,1993, 9(10):997-1006.
[11]Carr JK, Salminen MO, Koch C, et al. Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand. J Virol,1996,70(9):5935-5943.
[12]Gao F, Robertson DL, Morrison SG, et al. The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin. J Virol,1996, 70(10):7013-7029.
[13]邢辉,梁浩,万卓越.中国CRF01_AE亚型人类免疫缺陷病毒毒株的分子流行病学研究.中华预防医学杂志,2004,38:300-304.
[14]尚红.中国艾滋病流行和检测及治疗现状与发展趋势.中华检验医学杂志,2008,31:1088-1090.
[15]Wang L, Wang N, Li D, et al. The 2007 Estimates for People at Risk for and Living With HIV in China:Progress and Challenges. J Acquir Immune Defic Syndr,2009,50(4):414-418.
[16]Lu L, Jia M, Ma Y, et al. The changing face of HIV in China. Nature,2008,455(7213):609-611.
[17]Wang W, Jiang S, Li S, et al. Identification of subtype B, multiple circulating recombinant forms and unique recombinants of HIV type 1 in an MSM cohort in China. AIDS Res Hum Retroviruses,2008, 24(10):1245-1254.
[18]任莉,仇超,黄相刚,等.HIV-1 B亚型gag-env融合基因的DNA疫苗构建和免疫原性分析.《中华微生物学和免疫学杂志》,2006,26(7):654-657.
[19]Wan Y, Wu L, Liu L, et al. Comparison of immunogenicity between codon optimized HIV-1 Thailand subtype B gp140 and gp145 vaccines. Vaccine,2007,25(26):4949-4959.
[20]孙健,李杰,吴南屏.HIV疫苗国内外研究进展.国际流行病学传染病学杂志,2009, 36(3)：183-187.
[21]程春林,冯毅,何翔.中国南方四省区流行的HIV-1 CRF01_AE病毒株基因特征研究.中华流行病学杂志,2009,30(7):720-725.
[22]Huang HL, Yan PP, Zheng J, et al. Genetic characterization of three CRF01_AE full-length HIV type 1 sequences from Fujian Province, China. Chin Med J (Engl),2006,119(19):1622-1628.
[23]邢辉,梁浩,洪坤学等.我国HIV-1主要流行株外膜蛋白(env)基因V3-V4区变异及其与生物学特性的关系.中华微生物学和免疫学杂志,2005,25(3):185-189.
[24]Letvin NL, Huang Y, Chakrabarti BK, et al. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. Journal of Virology,2004,78(14):7490-7497.
[25]Flynn NM, Forthal DN, Harro CD, et al. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis,2005,191(5):654-665.
[26]Pitisuttithum P, Gilbert P, Gurwith M, et al. Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis,2006,194(12):1661-1671.
[27]Russell ND, Graham BS, Keefer MC, et al. Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in combination with rgp120:negative results fail to trigger a phase 3 correlates trial. J Acquir Immune Defic Syndr,2007,44(2):203-212.
[28]Gnann JW, Jr., Nelson JA, Oldstone MB. Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J Virol,1987,61(8):2639-2641.
[29]Muster T, Guinea R, Trkola A, et al. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. J Virol,1994, 68(6):4031-4034.
[30]Chakrabarti BK, Kong WP, Wu BY, et al. Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. J Virol,2002, 76(11):5357-5368.
[31]Earl PL, Sugiura W, Montefiori DC, et al. Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140. J Virol,2001,75(2):645-653.
[32]Li Y, Cleveland B, Klots I, et al. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol,2008, 82(2):638-651.
[33]Yang ZY, Chakrabarti BK, Xu L, et al. Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope. J Virol,2004, 78(8):4029-4036.
[34]Luo M, Yuan F, Liu YX, et al. Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment. Vaccine,2006,24(4):435-442.
[35]Beddows S, Schulke N, Kirschner M, et al. Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. Journal of Virology,2005,79(14):8812-8827.
[36]Morner A, Douagi I, Forsell MNE, et al. Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein. Journal of Virology,2009,83(2):540-551.
[37]Fouts T, Godfrey K, Bobb K, et al. Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques. Proc Natl Acad Sci U S A,2002, 99(18):11842-11847.
[38]Yuste E, Bixby J, Lifson J, et al. Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies. J Virol,2008,82(24):12472-12486.
[39]Kwong PD, Doyle ML, Casper DJ, et al. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature,2002,420(6916):678-682.
[40]Mackett M, Smith GL, Moss B. Vaccinia virus:a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A,1982,79(23):7415-7419.
[41]Brochier B, Aubert MF, Pastoret PP, et al. Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America. Rev Sci Tech,1996,15(3):947-970.
[42]Kresge K. Leading AIDs vaccine researches gather to discuss balancing AIDS vaccine funding [2009-04-01].. http://wwwiavireportorg/arehives/2008/pages/IAVl-Report12(2)-balancingaspx.
[43]Letvin NL. Progress toward an HIV vaccine. Annu Rev Med,2005,56:213-223.
[44]Santra S, Sun Y, Parvani JG, et al. Heterologous prime/boost immunization of rhesus monkeys by using diverse poxvirus vectors. J Virol,2007,81(16):8563-8570.
[45]Liu L, Wan Y, Xu J, et al. Immunogenicity comparison between codon optimized HIV-1 CRF BC_07 gp140 and gp145 vaccines. AIDS Res Hum Retroviruses,2007,23(11):1396-1404.
[46]Gurunathan S, Klinman DM, Seder RA. DNA vaccines:immunology, application, and optimization*. Annu Rev Immunol,2000,18:927-974.
[47]Earl PL, Broder CC, Long D, et al. Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities. J Virol,1994,68(5):3015-3026.
[48]Richardson TM, Jr., Stryjewski BL, Broder CC, et al. Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein. J Virol,1996,70(2):753-762.
[49]Petry H, Dittmer U, Jones D, et al. Prechallenge high neutralizing antibodies and long-lasting immune reactivity to gp41 correlate with protection of rhesus monkeys against productive simian immunodeficiency virus infection or disease development. J Acquir Immune Defic Syndr Hum Retrovirol, 1998,19(5):441-450.
[50]Pantaleo G, Menzo S, Vaccarezza M, et al. Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N Engl J Med,1995,332(4):209-216.
[51]Ogg GS, Jin X, Bonhoeffer S, et al. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science,1998,279(5359):2103-2106.
[52]Planz O, Ehl S, Furrer E, et al. A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers. Proc Natl Acad Sci U S A,1997, 94(13):6874-6879.
[53]Jin X, Bauer DE, Tuttleton SE, et al. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med,1999,189(6):991-998.
[54]Bernstein HB, Tucker SP, Hunter E, et al. Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides. J Virol,1994,68(1):463-468.
[55]Haggerty S, Dempsey MP, Bukrinsky MI, et al. Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection. AIDS Res Hum Retroviruses,1991,7(6):501-510.
[56]Kolchinsky P, Kiprilov E, Bartley P, et al. Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops. J Virol,2001,75(7):3435-3443.
[57]Hansen JE, Clausen H, Nielsen C, et al. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies:peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization. J Virol,1990,64(6):2833-2840.
[58]Kang SM, Quan FS, Huang C, et al. Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies. Virology,2005,331(1):20-32.
[59]McCaffrey RA, Saunders C, Hensel M, et al. N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies. J Virol,2004,78(7):3279-3295.
[60]Haigwood NL, Nara PL, Brooks E, et al. Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons. J Virol, 1992,66(1):172-182.
[61]Bolmstedt A, Hinkula J, Rowcliffe E, et al. Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan. Vaccine,2001,20(3-4):397-405.
[62]Bolmstedt A, Sjolander S, Hansen JE, et al. Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response. J Acquir Immune Defic Syndr Hum Retrovirol,1996,12(3):213-220.
[63]Burke B, Derby NR, Kraft Z, et al. Viral evolution in macaques coinfected with CCR5-and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies. Virology,2006,355(2):138-151.
[64]Quinones-Kochs MI, Buonocore L, Rose JK. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein:effects on protein function and the neutralizing antibody response. J Virol, 2002,76(9):4199-4211.
[65]Mori K, Sugimoto C, Ohgimoto S, et al. Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model. J Virol,2005, 79(16):10386-10396.
[66]Wan Y, Liu L, Wu L, et al. Deglycosylation or partial removal of HIV-1 CN54 gp140 V1/V2 domain enhances env-specific T cells. AIDS Res Hum Retroviruses,2009,25(6):607-617.
[67]Hong PW, Nguyen S, Young S, et al. Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120. J Virol,2007,81(15):8325-8336.
[68]Lue J, Hsu M, Yang D, et al. Addition of a single gp120 glycan confers increased binding to dendritic cell-specific ICAM-3-grabbing nonintegrin and neutralization escape to human immunodeficiency virus type 1. J Virol,2002,76(20):10299-10306.
[69]Koch M, Pancera M, Kwong PD, et al. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition. Virology,2003,313(2):387-400.
[70]Amara RR, Smith JM, Staprans SI, et al. Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine. J Virol,2002,76(12):6138-6146.
[71]Kim YB, Han DP, Cao C, et al. Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies. Virology,2003, 305(1):124-137.
[72]Amara RR, Villinger F, Altman JD, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science,2001,292(5514):69-74.
[73]Robinson HL, Montefiori DC, Johnson RP, et al. Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations. Nat Med,1999,5(5):526-534.
[74]Hel Z, Tsai WP, Thornton A, et al. Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen. J Immunol, 2001,167(12):7180-7191.
[75]Kent SJ, Zhao A, Best SJ, et al. Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J Virol,1998,72(12):10180-10188.
[76]Franchini G, Gurunathan S, Baglyos L, et al. Poxvirus-based vaccine candidates for HIV:two decades of experience with special emphasis on canarypox vectors. Expert Rev Vaccines,2004,3(4 Suppl):S75-88.
[77]Dorrell L, Yang H, Ondondo B, et al. Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine. J Virol,2006,80(10):4705-4716.
[78]Sadagopal S, Amara RR, Montefiori DC, et al. Signature for long-term vaccine-mediated control of a Simian and human immunodeficiency virus 89.6P challenge:stable low-breadth and low-frequency T-cell response capable of coproducing gamma interferon and interleukin-2. J Virol,2005,79(6):3243-3253.
[79]Santra S, Barouch DH, Korioth-Schmitz B, et al. Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses. Proc Natl Acad Sci U S A,2004, 101(30):11088-11093.
[80]Cole KS, Paliotti MJ, Murphey-Corb M, et al. Maturation of envelope-specific antibody responses to linear determinants in monkeys inoculated with attenuated SIV. J Med Primatol,2000,29(3-4):220-230.
[81]Cole KS, Rowles JL, Jagerski BA, et al. Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency virus and its association with the development of protective immunity. J Virol,1997,71(7):5069-5079.
[82]Nguyen M, Pean P, Lopalco L, et al. HIV-specific antibodies but not t-cell responses are associated with protection in seronegative partners of HIV-1-infected individuals in Cambodia. J Acquir Immune Defic Syndr,2006,42(4):412-419.
[83]Rao SS, Gomez P, Mascola JR, et al. Comparative evaluation of three different intramuscular delivery methods for DNA immunization in a nonhuman primate animal model. Vaccine,2006,24(3):367-373.
[84]Guerra S, Lopez-Fernandez LA, Pascual-Montano A, et al. Host response to the attenuated poxvirus vector NYVAC:upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells. J Virol,2006,80(2):985-998.
[85]Najera JL, Gomez CE, Domingo-Gil E, et al. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene. J Virol,2006, 80(12):6033-6047.
[86]Inaba K, Turley S, Yamaide F, et al. Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med,1998, 188(11):2163-2173.
[87]Albert ML. Death-defying immunity:do apoptotic cells influence antigen processing and presentation? Nat Rev Immunol,2004,4(3):223-231.
[88]Palmowski MJ, Choi EM, Hermans IF, et al. Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol,2002,168(9):4391-4398.
[89]Gomez CE, Najera JL, Jimenez EP, et al. Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1B X08 gp120 and HIV-1(ⅢB) Gag-Pol-Nef proteins of clade B. Vaccine,2007, 25(15):2863-2885.
[90]Rodriguez F, Harkins S, Slifka MK, et al. Immunodominance in virus-induced CD8(+) T-cell responses is dramatically modified by DNA immunization and is regulated by gamma interferon. J Virol, 2002,76(9):4251-4259.
[91]Barouch DH, Kunstman J, Kuroda MJ, et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature,2002,415(6869):335-339.
[92]Xu J, Ren L, Huang X, et al. Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes. AIDS,2006,20(18):2293-2303.
[1]万延民,吴岚,徐建青,et al.HIV-1 膜蛋白抗原优化改造研究进展.国际病毒学杂志,2007, 14(2):49-53.
[2]Kwong PD,Wyatt R,Robinson J,et al.Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.Nature,1998,393(6686):648-659.
[3]Reitter JN,Means RE,Desrosiers RC.A role for carbohydrates in immune evasion in AIDS.Nat Med, 1998,4(6):679-684.
[4]Burton DR,Desrosiers RC,Doms RW,et al.HIV vaccine design and the neutralizing antibody problem.Nat Immunol,2004,5(3):233-236.
[5]Flynn NM,Forthal DN,Harro CD,et a1.Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection.J Infect Dis,2005,191(5):654-665.
[6]Pitisuttithum P,Gilbert P,Gurwith M,et al.Randomized,double-blind,placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand.J Infect Dis,2006,194(12):1661-1671.
[7]Russell ND,Graham BS,Keefer MC,et al.Phase 2 study of an HIV-1 canarypox vaccine(vCP1452) alone and in combination with rgp120:negative results fail to trigger a phase 3 correlates trial.J Acquir Immune Defic Syndr,2007,44(2):203-212.
[8]Quinnan GV, Jr., Zhang PF, Fu DW, et al. Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response. AIDS Res Hum Retroviruses, 1999,15(6):561-570.
[9]Zhang PF, Cham F, Dong M, et al. Extensively cross-reactive anti-HIV-1 neutralizing antibodies induced by gp 140 immunization. Proc Natl Acad Sci U S A,2007,104(24):10193-10198.
[10]Zhang PF, Bouma P, Park EJ, et al. A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response. J Virol, 2002,76(2):644-655.
[11]Choudhry V, Zhang MY, Sidorov IA, et al. Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies. Virology,2007,363(1):79-90.
[12]McBurney SP, Ross TM. Viral sequence diversity:challenges for AIDS vaccine designs. Expert Rev Vaccines,2008,7(9):1405-1417.
[13]Liao HX, Sutherland LL, Xia SM, et al. A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and CHIV-1 primary viruses. Virology,2006, 353(2):268-282.
[14]Doria-Rose NA, Learn GH, Rodrigo AG, et al. Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope. J Virol,2005,79(17):11214-11224.
[15]Kothe DL, Li Y, Decker JM, et al. Ancestral and consensus envelope immunogens for HIV-1 subtype C. Virology,2006,352(2):438-449.
[16]Rolland M, Jensen MA, Nickle DC, et al. Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol,2007,81(16):8507-8514.
[17]Kong WP, Wu L, Wallstrom TC, et al. Expanded breadth of the T-cell response to mosaic human immunodeficiency virus type 1 envelope DNA vaccination. J Virol,2009,83(5):2201-2215.
[18]Xu J, Ren L, Huang X, et al. Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes. Aids,2006,20(18):2293-2303.
[19]Chohan B, Lang D, Sagar M, et al. Selection for human immunodeficiency virus type 1 envelope glycosylation variants with shorter VI-V2 loop sequences occurs during transmission of certain genetic subtypes and may impact viral RNA levels. J Virol,2005,79(10):6528-6531.
[20]Derdeyn CA, Decker JM, Bibollet-Ruche F, et al. Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science,2004,303(5666):2019-2022.
[21]Blish CA, Nedellec R, Mandaliya K, et al. HIV-1 subtype A envelope variants from early in infection have variable sensitivity to neutralization and to inhibitors of viral entry. Aids,2007,21(6):693-702.
[22]Blish CA, Nguyen MA, Overbaugh J. Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41. PLoS Med,2008,5(1):e9.
[23]Letvin NL, Huang Y, Chakrabarti BK, et al. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. Journal of Virology,2004,78(14):7490-7497.
[24]Laird ME, Igarashi T, Martin MA, et al. Importance of the V1/V2 loop region of simian-human immunodeficiency virus envelope glycoprotein gp120 in determining the strain specificity of the neutralizing antibody response. J Virol,2008,82(22):11054-11065.
[25]Yang ZY, Chakrabarti BK, Xu L, et al. Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope. J Virol,2004, 78(8):4029-4036.
[26]Cherpelis S, Jin X, Gettie A, et al. DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques. Immunol Lett,2001,79(1-2):47-55.
[27]Wan Y, Liu L, Wu L, et al. Deglycosylation or partial removal of HIV-1 CN54 gp140 V1/V2 domain enhances env-specific T cells. AIDS Res Hum Retroviruses,2009,25(6):607-617.
[28]Chen Z, Huang Y, Zhao X, et al. Design, construction, and characterization of a multigenic modified vaccinia Ankara candidate vaccine against human immunodeficiency virus type 1 subtype C/B' . J Acquir Immune Defic Syndr,2008,47(4):412-421.
[29]Mori K, Sugimoto C, Ohgimoto S, et al. Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model. J Virol,2005, 79(16):10386-10396.
[30]Li Y, Cleveland B, Klots I, et al. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol,2008, 82(2):638-651.
[31]Yuste E, Bixby J, Lifson J, et al. Glycosylation of gp41 of simian immunodeficiency virus shields epitopes that can be targets for neutralizing antibodies. J Virol,2008,82(24):12472-12486.
[32]Quinones-Kochs MI, Buonocore L, Rose JK. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein:effects on protein function and the neutralizing antibody response. J Virol, 2002,76(9):4199-4211.
[33]Garrity RR, Rimmelzwaan G, Minassian A, et al. Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope. J Immunol,1997,159(l):279-289.
[34]Trujillo JD, Kumpula-McWhirter NM, Hotzel KJ, et al. Glycosylation of immunodominant linear epitopes in the carboxy-terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses. J Virol,2004, 78(17):9190-9202.
[35]Braibant M, Brunet S, Costagliola D, et al. Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors:prevalence and association with neutralizing activity. Aids,2006, 20(15):1923-1930.
[36]Suarez T, Nir S, Goni FM, et al. The pre-transmembrane region of the human immunodeficiency virus type-1 glycoprotein:a novel fusogenic sequence. Febs Letters,2000,477(1-2):145-149.
[37]Coeffier E, Clement JM, Cussac V, et al. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine,2000,19(7-8):684-693.
[38]Liang XP, Munshi S, Shendure J, et al. Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein. Vaccine,1999,17(22):2862-2872.
[39]Eckhart L, Raffelsberger W, Ferko B, et al. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus. Journal of General Virology,1996,77:2001-2008.
[40]Ho J, MacDonald KS, Barber BH. Construction of recombinant targeting immunogens incorporating an HIV-1 neutralizing epitope into sites of differing conformational constraint. Vaccine,2002, 20(7-8):1169-1180.
[41]Luo M, Yuan F, Liu YX, et al. Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment. Vaccine,2006,24(4):435-442.
[42]Srivastava IK, Stamatatos L, Kan E, et al. Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate. Journal of Virology,2003,77(20):11244-11259.
[43]Dey AK, David KB, Lu M, et al. Biochemical and biophysical comparison of cleaved and uncleaved soluble, trimeric HIV-1 envelope glycoproteins. Virology,2009,385(1):275-281.
[44]Binley JM, Sanders RW, Clas B, et al. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gpl20 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. Journal of Virology,2000,74(2):627-643.
[45]Edinger AL, Blanpain C, Kunstman KJ, et al. Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains. Journal of Virology,1999, 73(5):4062-4073.
[46]Beddows S, Schulke N, Kirschner M, et al. Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. Journal of Virology,2005,79(14):8812-8827.
[47]Morner A, Douagi I, Forsell MNE, et al. Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein. Journal of Virology,2009,83(2):540-551.
[48]Fouts T, Godfrey K, Bobb K, et al. Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques. Proc Natl Acad Sci U S A,2002, 99(18):11842-11847.
[49]LaBranche CC, Hoffman TL, Romano J, et al. Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity. J Virol, 1999,73(12):10310-10319.
[50]Kolchinsky P, Mirzabekov T, Farzan M, et al. Adaptation of a CCR5-using, primary human immunodeficiency virus type 1 isolate for CD4-independent replication. J Virol,1999,73(10):8120-8126.
[51]He Y, D'Agostino P, Pinter A. Analysis of the immunogenic properties of a single-chain polypeptide analogue of the HIV-1 gp120-CD4 complex in transgenic mice that produce human immunoglobulins. Vaccine,2003,21 (27-30):4421-4429.
[52]Yang X, Tomov V, Kurteva S, et al. Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1. J Virol,2004,78(23):12975-12986.
[53]Zhou T, Xu L, Dey B, et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature,2007,445(7129):732-737.
[54]Wu L, Zhou T, Yang ZY, et al. Enhanced exposure of the CD4-binding site to neutralizing antibodies by structural design of a membrane-anchored human immunodeficiency virus type 1 gp120 domain. J Virol, 2009,83(10):5077-5086.
[55]Hessell AJ, Poignard P, Hunter M, et al. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med,2009,15(8):951-954.
[56]Hessell AJ, Rakasz EG, Poignard P, et al. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog, 2009,5(5):e1000433.