Background To effectively block the invasion of human immunodeficiency virus （HIV）-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because...Background To effectively block the invasion of human immunodeficiency virus （HIV）-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction.Methods A murine model was employed with nonoxynol-9 （N-9） as the topical stimulant within the vagina. Different concentrations of N-9 （1%, 3%, and 4%） were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 （IL-2）, IL-4, IL-6, IL-17A, interferon-Y （IFN-Y）, tumor necrosis factor-α （TNF-α）, and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array （CBA） and reverse transcript （RT） real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis.Results All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice （sores： 10-13） and moderate damage in one mouse （score： 8）, which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α （r=0.739; P ＜0.05）, anti-inflammatory cytokines IL-10 （r=0.804; P ＜0.01） and IL-4 （r=0.668; P ＜0.05）.Conclusions Our data demonstrate that a panel of cytokines （IL-17A, IL-6, IL-4 and IL-10） could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.展开更多
The need for AIDS vaccine has been emphasized by the increase of HIV-1 prevalence in sexual transmission which bridges the spreading of HIV-1 from high-risk population to other populations. After more than two-decade ...The need for AIDS vaccine has been emphasized by the increase of HIV-1 prevalence in sexual transmission which bridges the spreading of HIV-1 from high-risk population to other populations. After more than two-decade intensive effort on the AIDS vaccine development, it remains elusive whether and how an effective vaccine will be achieved. Recent data released from a phase Ⅲ trial in Thailand showed a partial protection might be accomplishable by（R）the ＂prime-boost＂combination of two vaccines： ALVAC（R） HIV vaccine （the prime）, and AIDSVAX（R） B/E vaccine （the boost）.1 This unprecedented large clinical trial observed that the prime-boost combination lowered the rate of HIV infection by 31.2% compared to placebo based on the modified intent-to-treat population （n=51 vs. n=74,respectively; P=0.04）. However, debating on the efficacy interpretation of this trial arose among field scientists.Furthermore, how to improve the efficacy will become the most important question to be tackled. Here we reviewed the recent publications and summarized the major progress achieved.展开更多
Background Cholera toxin B subunit （CTB） was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter...Background Cholera toxin B subunit （CTB） was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine. Methods Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ （IFN-γ） enzyme-linked immunospot （Elispot） assay was used to read out the specific T-cell immunity. Results Chloramphenicol was essential for the efficient expression of recombinant CTB （rCTB） in pET-30a/BL21 （DE3） system and could be optimized at the concentration of 0.625 μg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group （（734±240） spot forming cells/106 splenocytes） was higher than that induced by non-adjuvanted （（520±150） spot forming cells/10e splenocytes）, all responses against different antigens were enhanced in parallel. Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancincl the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.展开更多
文摘Background To effectively block the invasion of human immunodeficiency virus （HIV）-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction.Methods A murine model was employed with nonoxynol-9 （N-9） as the topical stimulant within the vagina. Different concentrations of N-9 （1%, 3%, and 4%） were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 （IL-2）, IL-4, IL-6, IL-17A, interferon-Y （IFN-Y）, tumor necrosis factor-α （TNF-α）, and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array （CBA） and reverse transcript （RT） real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis.Results All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice （sores： 10-13） and moderate damage in one mouse （score： 8）, which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α （r=0.739; P ＜0.05）, anti-inflammatory cytokines IL-10 （r=0.804; P ＜0.01） and IL-4 （r=0.668; P ＜0.05）.Conclusions Our data demonstrate that a panel of cytokines （IL-17A, IL-6, IL-4 and IL-10） could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.
文摘The need for AIDS vaccine has been emphasized by the increase of HIV-1 prevalence in sexual transmission which bridges the spreading of HIV-1 from high-risk population to other populations. After more than two-decade intensive effort on the AIDS vaccine development, it remains elusive whether and how an effective vaccine will be achieved. Recent data released from a phase Ⅲ trial in Thailand showed a partial protection might be accomplishable by（R）the ＂prime-boost＂combination of two vaccines： ALVAC（R） HIV vaccine （the prime）, and AIDSVAX（R） B/E vaccine （the boost）.1 This unprecedented large clinical trial observed that the prime-boost combination lowered the rate of HIV infection by 31.2% compared to placebo based on the modified intent-to-treat population （n=51 vs. n=74,respectively; P=0.04）. However, debating on the efficacy interpretation of this trial arose among field scientists.Furthermore, how to improve the efficacy will become the most important question to be tackled. Here we reviewed the recent publications and summarized the major progress achieved.
基金This study was supported by the grants from the Chinese National Grand Program on Key Infectious Disease Control （No. 2008ZX10001-012 and -002）, the National Natural Science Foundation of China （No. 81072496H1014） and the Fund for Young Scientists from Fudan University （No. 07L03）.
文摘Background Cholera toxin B subunit （CTB） was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine. Methods Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ （IFN-γ） enzyme-linked immunospot （Elispot） assay was used to read out the specific T-cell immunity. Results Chloramphenicol was essential for the efficient expression of recombinant CTB （rCTB） in pET-30a/BL21 （DE3） system and could be optimized at the concentration of 0.625 μg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group （（734±240） spot forming cells/106 splenocytes） was higher than that induced by non-adjuvanted （（520±150） spot forming cells/10e splenocytes）, all responses against different antigens were enhanced in parallel. Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancincl the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.
