BACKGROUNDConstruction of replication-deficient recombinant adenovirus expressing gag-pol and env genes of human immunodeficiency virus (HIV) in mice.

METHODSgag-polDelta and gp140TM genes were cloned into shuttle vector pAdTrack-CMV respectively, and then the plasmids containing gag-polDelta or gp140TM gene were cotransformed with the backbone of adenovirus into E.coli BJ5183. Transfections of the recombinants were performed to obtain recombinant adenoviruses. Its immunogenicity was evaluated by testing antibody levels of mice primed with DNA vaccines and boosted with recombinant adenoviruses.

RESULTSThe replication-deficient recombinant adenovirus could express Gp140TM, Gag P55 and P24 proteins correctly. The mice primed with DNA vaccines and boosted with recombinant adenoviruses elicited high titer of HIV-1-specific antibody compared with that inoculated with DNA vaccines only.

CONCLUSIONReplication-deficient recombinant adenovirus expressing gag-polDelta and gp140TM can elicit high titer HIV-1-specific antibodies.

AIDS Vaccines ; immunology ; Adenoviridae ; genetics ; Animals ; Female ; Fusion Proteins, gag-pol ; biosynthesis ; genetics ; Gene Products, env ; biosynthesis ; genetics ; HIV-1 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Transfection ; Vaccines, DNA ; immunology ; env Gene Products, Human Immunodeficiency Virus

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Bioluminescence Resonance Energy Transfer Techniques ; methods ; Fusion Proteins, gag-pol ; genetics ; metabolism ; HEK293 Cells ; HIV Protease ; metabolism ; physiology ; HIV-1 ; enzymology ; High-Throughput Screening Assays ; methods ; Humans ; Plasmids ; genetics ; Protein Precursors ; metabolism ; physiology ; Pyridazines ; pharmacology ; Transfection ; Virion ; growth & development ; Virus Assembly ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

OBJECTIVETo investigate the subtype distribution and the prevalence of sequence characteristics of HIV-1 strains in Beijing residents during 2006 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routines.

METHODSBlood samples from 32 new confirmed HIV-1 infected individuals from Beijing residents in 2006 and separated plasma specimens were collected. RNAs were extracted and the gag and env gene were amplified by RT-PCR and nest-PCR. PCR products were sequenced directly and phylogenetic analyses of gag and env gene were performed using the MEGA2 software.

RESULTSAmong 32 HIV-1 plasma samples, 22 gag and 4 env gene fragments were amplified and analyzed. Five HIV-1 subtypes or circulating recombinant forms(CRFs) of HIV-1 including Thai B (2 strains), B (9 strains), C (2 strains), CRF07_BC (5 strains), CRF01 AE (4 strains) were identified being circulated in Beijing. The gene divergences of gag gene inside the subtypes were 6.6%, 4.3%, 6.8%, 4.9% and 3.0% in subtype B, Thai B, C, CRF01_AE and CRF07_BC respectively. Subtypes B were predominant in Beijing, accounted for 40.9% among 22 samples.

CONCLUSIONFive HIV-1 subtypes were identified in Beijing and the surveillance of HIV-1 gene variation should be paid more attention to.

China ; HIV-1 ; classification ; genetics ; Humans ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; env Gene Products, Human Immunodeficiency Virus ; genetics ; gag Gene Products, Human Immunodeficiency Virus ; genetics

The 109 whole blood samples were collected from HIV-1 infected former blood donors in Henan and Shanxi. The RNA templates were extracted from plasma and used for the full gag gene amplification and sequencing. The sequences were divided into 3 groups according to sampling year. The Entropy software was used to identify the amino acids with composition difference among different groups of amino acid sequences. The results showed that there existed 8 and 13 amino acid sites with the statistical significance difference, respectively, in sequences in year 2004 and 2005, compared to those in 2002. Among them, there existed 5 amino acid sites in two groups. Of 16 amino acid sites, the increasing polymorphism and the decreasing polymorphism along the sampling year were observed in 10 and 6 amino acid sites respectively. Of 10 sites with increased polymorphism, 8 sites were located in the CTL epitopes recognized and presented by the main HLA alleles existed in Chinese population. The 6 sites with decreasing polymorphism all existed in main domains of Gag proteins.
Blood Donors ; China ; epidemiology ; Genetic Variation ; HIV-1 ; genetics ; Humans ; Polymorphism, Genetic ; gag Gene Products, Human Immunodeficiency Virus ; genetics

To investigate the subtype distribution of human immunodeficiency virus-1(HIV-1) infection among men who have sex with men (MSM) in Zhengzhou, Henan Province, forty blood samples were collected from HIV-1 carriers, who acknowledged to have sex with men. The complete gag gene was amplified by RT-PCR and nested-PCR and sequenced. All sequences were edited by BioEdit and subtyped by genotyping software. Phylogenetic analysis of gag gene were then performed using the MEGA 3.1 software, the gene distances were calculated by Distance program. There were three different HIV-1 subtypes including B, CRF01-AE and CRF07-BC present among twenty four MSMs in Zhengzhou. Genotyping results showed that 33.33% (8/24) were B, 41.67% (10/24) were CRF01-AE and 25% (6/24) were CRF07-BC, and subtype CRF01-AE had become the most prevalent HIV-1 subtype in Zhengzhou, Henan province. In conclusion, recombinant HIV-1 strains are circulating in Henan province and the epidemiology is complicated.
Adult ; China ; HIV-1 ; classification ; genetics ; isolation & purification ; Homosexuality, Male ; Humans ; Male ; Middle Aged ; Phylogeny ; Sequence Analysis, DNA ; Young Adult ; gag Gene Products, Human Immunodeficiency Virus ; genetics

OBJECTIVETo study the characteristics of the variation of antigen epitopes and quasispecies group in the HIV-1 viruses in Henan province in China.

METHODSThe region of the p17-p24 of the HIV-1 gag gene was amplified by nested polymerse chain reaction (PCR), purified products were cloned into the vector, the obtained were analyzed by MEGA soft wares.

RESULTSB' subtype strains were predominant in Henan province, the mutations in antigen epitypes of the p17 region of the gag gene focus on E62g (55.8%), Y79f (48.9%), T84V (48.9), I44V (44.2%), the p24 region had not found the distinct mutation.

CONCLUSIONBoth the PCR sequences and clone strains sequences demonstrated that four antigen epitopes mutations in p17 region of the HIV B' subtype gag gene, and the region of p24 was more conservative, which was suitable for development of the epitope vaccien.

Antigenic Variation ; China ; Epitopes ; genetics ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Mutation ; Phylogeny ; gag Gene Products, Human Immunodeficiency Virus ; genetics

OBJECTIVETo estimate prevalence of HIV/AIDS among children and the transmission routes in a highly endemic villages of AIDS.

METHODSTotally 208 high-risk women of child bearing age and 159 of their children aged 0 - 14 years were investigated. Their medical histories of blood donation or transfusion were collected, blood samples were taken and sera were separated for HIV test. Enzyme-linked immunosorbent assay (ELISA) and Western blot assay were performed for HIV antibody. The Nested-polymerase chain reaction (PCR) assay amplifying gag gene p17 was performed on samples of children aged less than 18 months.

RESULTSThirty-seven HIV infected cases were found among 159 children aged 0 - 14 years of whom 33 were infected by mother-to-child transmission (89.2%, 33/37), 3 by blood transfusion (8.1%, 3/37) and one by iatrogenic route (2.7%, 1/37). Sixty seven mothers who were seropositive for HIV and their 86 children who were born after 1992 were investigated, 33 cases of them were infected with HIV. The rate of vertical transmission was 38.4% (33/86). The HIV vertical transmission rate among mothers with AIDS (68.8%, 22/32) was significantly greater than that among mothers with asymptomatic HIV infection (20.4%, 11/54, P < 0.05). The number of children infected with HIV through vertical transmission increased from 1993 to 2001. Among 37 children infected with HIV, 12 cases developed AIDS and 4 of them died, of whom 2 cases died from tuberculosis. The morbidity of AIDS was 27.3% (9/33). Ninety three point nine percent (31/33) of infected mothers didn't know their HIV seropositive status before pregnancy and delivery. Of 8 pregnant women infected with HIV, one had aggravation of AIDS, 2 miscarried, 2 terminated their pregnancy and 3 continued their pregnancy.

CONCLUSIONMother-to-child transmission of HIV was the major route of HIV/AIDS transmission to the children. The main reason leading to HIV infection in children was the lack of prenatal HIV counseling and testing for the high-risk women of childbearing age and lake of interventions. The countermeasures must be taken to control the further transmission of AIDS in order to protect the health of women and children in the highly endemic areas of AIDS.

Acquired Immunodeficiency Syndrome ; diagnosis ; epidemiology ; transmission ; Adolescent ; Adult ; Antibodies, Viral ; blood ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Gene Products, gag ; genetics ; HIV Antigens ; genetics ; HIV Infections ; diagnosis ; epidemiology ; transmission ; HIV-1 ; genetics ; immunology ; Humans ; Infant ; Infectious Disease Transmission, Vertical ; Male ; Polymerase Chain Reaction ; Prevalence ; Viral Proteins ; gag Gene Products, Human Immunodeficiency Virus

BACKGROUNDDeveloping an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy.

METHODSWe constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy.

RESULTSOur data demonstrated that the rTTV-lucgag was able to express high level of luciferase (< or = 10(6) relative luciferase units (RLU)/mg protein) and HIV-1 Gag (> 3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r(2) = 0.71, P < 0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006 +/- 3141) RLU/mg protein in control group to (1538 +/- 463) RLU/mg protein in vaccine group (P = 0.1969).

CONCLUSIONSThe luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

AIDS Vaccines ; genetics ; Animals ; Female ; HIV Infections ; immunology ; prevention & control ; HIV-1 ; genetics ; Humans ; Kinetics ; Luciferases ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Poxviridae ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Virus Replication ; genetics ; gag Gene Products, Human Immunodeficiency Virus ; genetics

OBJECTIVETo observe the level of anti-adenovirus neutralizing antibodies and Gag specific cellular immune responses in Macaca fascicularis immunized with different dosage of recombinant adenovirus vaccine Ad5-HIVgag by repeated intramuscular injection.

METHODSThe Macaca fascicularis were randomly divided into four groups of 6. Different amount of the purified Ad5-HIVgag (0.99 x 10(11) VP, 4.94 x 10(11) VP, 24.68 x 10(11) VP) or PBS were administered in 3 weeks interval and five times. The level of anti-adenovirus neutralizing antibodies and Gag-specific cellular immune responses at different time points were detected by neutralization assay and Elispot assay respectively.

RESULTSHigh level of anti-adenovirus neutralizing antibodies could be detected in three groups immunized with Ad5-HIVgag at 3 weeks after first immunization. The neutralizing antibodies reached peak at 8 weeks after primary immunization, and declined slightly at late time. Significant HIV-1 Gag-specific cellular immune responses were detected in all Ad5-HIVgag immunized groups at 5 weeks post first immunization. The Gag-specific cellular immune responses declined at 12 weeks and then increased with time.

CONCLUSIONAnti-adenovirus neutralizing antibodies could be induced in Macaca fascicularis immunized with Ad5-HIVgag by repeated intramuscular injection. And the Gag-specific cellular immune responses tended to increase with the injection times. The presence of anti-adenovirus neutralizing antibodies induced by vaccination with adenovirus vectors in Ad5-naive animals did not further reduce Gag-specific cellular immune responses.

AIDS Vaccines ; immunology ; Adenoviridae ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Female ; Immunity, Cellular ; Immunization ; Macaca fascicularis ; Male ; Vaccines, Synthetic ; immunology ; gag Gene Products, Human Immunodeficiency Virus ; immunology

OBJECTIVETo investigate the characteristic of subtypes and genetic diversity of HIV-1 circulating in Hubei province and its molecular epidemiological linkages with regard to risk factors of viral transmission.

METHODSplasma samples of 80 diagnosed individuals was characterized. The gene fragments of gag were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and HIV-1 genotypes were determined based on the nucleotide sequences of gag region.

RESULTSSeven HIV-1 group M subtypes or CRF including B, B', G, CRF01-AE, CRF07-BC, CRF08-BC and CRF15-01B were identified. CRF01-AE was found to be the most dominant subtype (48.4%) followed by CRF7-BC (22.6%) and B' (12.9%).

CONCLUSIONThe data from this study indicate the existence of multiple HIV-1 subtypes or CRFs in Hubei province and the surveillance of HIV-1 gene variation should be paid more attention to.

Adolescent ; Adult ; Aged ; Child ; China ; epidemiology ; Genotype ; HIV Infections ; epidemiology ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Phylogeny ; Young Adult ; gag Gene Products, Human Immunodeficiency Virus ; genetics