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Important papers representing the advances in AIDS vaccine research were retrieved from Web of Sci-ence using HistCite.The citation chronological chart was plotted by analyzing the relation between their citation fre-quency and cited frequency , and analyzing their citation sequences , which shows the development rules and histori-cal development path in AIDS vaccine research , and provides a certain reference value for the researchers in related fields at home and abroad.
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Objective To establish the crystal violet plaque assay for detection of virus titer of recombined Tiantan vaccinia AIDS vaccine,and provide more stable method of virus titration for rTV AIDS Vaccine.Methods Optimized the concentration of Vero cells,the time and temperature of virus adsorption,and the time of determination for CPE,then established the crystal violet plaque assay for virus titer of rTV.Counting and analysis the plaques by BioSpot Reader,then analyzed the relativity of plaques counted with BioSpot Reader and manual; Several lots rTV AIDS Vaccine and Tiantan vaccinia were titrated by the method of plaque formation-hemadsorption assay,neutral red and crystal violet plaque assay,then analyzed the relativity of the results of three methods ; meanwhile,the virus titer of samples were determine repeatedly by the crystal violet plaque assay,then calculated the coefficient of variation( CV),and verified the precision of the method; SPSS17.0 was used in statistical analysis of the experimental results.Results When the concentration of Vero cells was 5.0×105-9.O×105 cells/ml,virus been adsorbesd 2 h at 37℃,then cultivated 72 h after adding the culture medium containing methyl cellulose.Plaques counted by BioSpot Reader was highly related with counted by manual (r =0.985),so BioSpot Reader counting can objectively reflect the virus plaques with various size,and reduce the error by manual counting; compared the virus titration for different lots of rTV AIDS vaccine and Tiantan vaccinia with three methods,the crystal violet plaque assay was highly related with plaque formation-hemadsorption assay (r =0.997,P＜0.01 ) and neutral red plaque assay(r=0.980,P＜0.01 ).Conclusion Crystal violet plaque assay was established for virus titration of rTV AIDS Vaccine.
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Objective To construct two DNA vaccines encoding Gag-Env fusion protein and Tat-Rev-Integrase(C-half)-Vif-Nef fusion protein derived from the first HIV-1 CRF01_AE isolate(AE2f) in Chi-na and to evaluate the immunogenicity in mice. Methods Two DNA vaccines were constructed by inserting the codon optimized and synthesized gag-env fusion gene and tat-rev-integrase(c-half)-vif-nef fusion gene de-rived from AE2f into mammalian expression vector pDRVISV1. 0, the generated DNA vaccines were desig-nated as pSVAE/GE and pSVAE/TRIVN, respectively, and their in vitro expression were determined by Western blot with transfected 293T cells. Mice were i. m. immunized with either pDRVI1.0 as mock control, pSVAE/GE or pSYAE/TRIVN for 4 times at two-week interval. Two weeks following the final im-munization, cellular responses to pool of HIV-1 Env, Gag, Tat, Rev, Intergrase, Vif and Nef peptides were evaluated by ELISPOT assay. Results The construction of DNA vaccine pSVAE/GE and pSVAE/TRIVN was validated by restriction enzyme digestion and bidirectional sequencing. Western blot showed a specific band at molecular mass 220×10~3 in lane of pSVAE/GE transfeeted 293T cell and a specific band at 95×10~3 in the lane of pSVAE/TRIVN. Both DNA vaccines mounted significant specific T cell responses with (3010 ± 566) SFC/10~6 splenocytes for DNA vaccine pSV AE/GE and (948 ± 737) SFC/10~6 spleno-cytes for DNA vaccine pSVAE/TRIVN, whereas the mock control of pDRVISV1.0 only raised marginal T cell responses. Conclusion Both pSVAE/GE and pSVAE/TRIVN were capable of expressing the inserted fusion immunogen genes and able to elicit vigorous cellular immune responses, therefore, these DNA vac-cines are highly immunogenic.
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A persistência de células T de memória funcionais é importante para garantir uma imunidade protetora na infecção pelo Vírus da Imunodeficiência Humana (HIV). As células T de memória têm sido subdivididas em memória central (TCM), memória efetora (TEM) e memória efetora altamente diferenciada (TEMRA) com base na expressão de moléculas de superfície como CCR7 e CD45RA, e na capacidade de produzir citocinas e proliferar. Recentemente, identificamos 18 peptídeos derivados de seqüências do consenso B do HIV-1, ligadores de múltiplas moléculas HLA-DR e amplamente reconhecidos por linfócitos T de sangue periférico de pacientes infectados pelo HIV. Diante disso e considerando a importância das células T de memória na manutenção da resposta imune específica, nosso objetivo foi caracterizar fenotípica e funcionalmente as subpopulações de células T de memória de indivíduos infectados pelo HIV envolvidas no reconhecimento in vitro desses epitopos. Foram incluídos 14 indivíduos controles sadios e 61 pacientes HIV+ com contagem de linfócitos T CD4+ maior que 250 células/mm3. Os pacientes HIV+ foram divididos em seis diferentes grupos clínicos de acordo com o estágio da infecção, carga viral (CV) plasmática e uso de terapia anti-retroviral (ART): não progressores por longo tempo (LTNP), avirêmicos em uso de ART (AV-ART), virêmicos em uso de ART (VI-ART), virêmicos sem uso de ART (VI sem ART), virêmicos recém infectados sem uso de ART (VI-RI) e controladores. Células mononucleares do sangue periférico dos indivíduos do estudo foram estimuladas com o conjunto de peptídeos do HIV-1 e com um conjunto de peptídeos do Citomegalovírus (CMV). A freqüência de células de memória produtoras de IFN- e IL-2 e a proliferação celular antígeno-específica foram detectadas por citometria de fluxo de multiparâmetros. Nossos resultados mostraram que o conjunto de peptídeos do HIV-1 foi capaz de ativar subpopulações funcionais de memória TCM, TEM e TEMRA secretoras de IFN- e IL-2 em 100%...
The persistence of functional memory T cell is important to ensure a protective immunity to Human Immunodeficiency Virus (HIV) infection. Memory T cells have been subdivided into central memory (TCM), effector memory (TEM) and highly differentiated effector memory (TEMRA) based on the expression of surface molecules such as CCR7 and CD45RA, and the ability to produce cytokines and proliferate. Recently, we identified 18 peptides derived from B consensus sequences of HIV-1 that bind to multiple HLA-DR molecules and are widely recognized by peripheral blood T lymphocytes from HIV-infected patients. Given this and considering the importance of memory T cells in the maintenance of specific immune response, our objective was to characterize phenotypic and functionally memory T cell subsets from HIV-infected individuals involved in the recognition of these epitopes in vitro. The study included 14 healthy control subjects and 61 HIV+ patients with CD4+ lymphocytes counts higher than 250 cells/mm³. The HIV+ patients were divided into six different clinical groups according to the stage of infection, plasma viral load (VL) and antiretroviral therapy use (ART): long-term non-progressors (LTNP), aviremic under ART (AV-ART), viremic under ART (VI-ART), viremic without using ART (VI without ART), recently infected viremic without using ART (VI-RI) and controllers. Peripheral blood mononuclear cells from study subjects were stimulated with HIV-1 peptide pool and with a cytomegalovirus (CMV) peptide pool. The frequencies of IFN- and IL-2 producing memory cells and antigenspecific cell proliferation were detected by multiparametric flow cytometry. Our results showed that the HIV-1 set of peptides was able to activate TCM, TEM and TEMRA functional memory subsets that secrete IFN- and IL-2 in 100% of the HIV patients from the different clinical groups. The HIV-1 set of peptides also induced memory T lymphocyte subsets proliferation...
Subject(s)
Humans , Male , Female , Adult , Middle Aged , AIDS Vaccines , Epitopes, T-Lymphocyte , HIV , HIV-1 , HIV Infections/immunology , T-Lymphocytes/immunology , Acquired Immunodeficiency Syndrome/prevention & control , T-LymphocytesABSTRACT
It has been estimated that by 2010, there might be more people living with HIV in Asia than in Africa. There is an urgent need for a safe, effective, accessible and affordable AIDS vaccine suitable for use in Asia. Factors that may hinder the development of AIDS vaccines in Asia include: 1) difficulty in recruiting adequate number of trial participants due to the low incidence of HIV infection in the general population and in defined population groups at high risk for HIV; 2) circulation of multiple HIV genetic subtypes and recombinant forms, and 3) unique geographical diversity of populations, cultures, social and political backgrounds. A proposed strategy to accelerate the development of an effective AIDS vaccine for Asia could be the constitution of a collaborative regional network in support of AIDS vaccine research and development. Collaborations would include 1) promoting the conduct of additional epidemiological studies and establishment of regional vaccine trial cohorts to reach adequate sample size for efficacy trials, 2) developinga regional platform for the conduct of clinical trials at multiple sites and harmonization of legal, regulatory and ethical frameworks to facilitate the review and approval processes, 3) strengthening the regional clinical research capacities and human resources for efficient development and testing of various vaccine candidates; and 4) promoting the development of regional capacities and infrastructures for vaccine production for the conduct of all phases of clinical trials, licensing and future public health use. A collaborative regional network in support of AIDS vaccine research and development will also require strong political commitment and leadership by all regional and international partners playing a significant role in the region.
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Estimated number of adults and children newly infected with HIV-1 during 2001 alone is 5 million in total. An effective vaccine, in addition to education & public health approaches, has been believed to be the best option to stop the HIV-1 transmission, especially for developing countries. Among AIDS vaccine candidates, DNA vaccine is relatively safe and, in a certain extent, mimics some attributes of live attenuated vaccine, with regard to in vivo gene expression & the type of immunity induced. We recently demonstrated that DNA vaccines expressing SIVmac239 structural and regulatory genes, augmented with coadministration of IL-12 mutant induced the strongest T cell responses, resulting in low to undetectable setpoint viral loads, stable CD4+ T cell counts, and no evidence of clinical diseases or mortality by day 420 after challenge. This finding is the second demonstration, following the protective result of live attenuated SIV vaccine in SIVmac-rhesus monkey model, which was known to have safety problem. So, our DNA vaccines could give a significant impact on HIV-1 epidemic by slowing or stopping the spread of HIV-1, leading to eventual eradication of HIV-1 and AIDS in the population.