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

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

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 x 10(9) and 0.86 x 10(9), respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 x 10(4) IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.
Animals ; Antibodies, Monoclonal/blood ; Cats ; Diagnostic Tests, Routine/*veterinary ; Female ; Gene Products, gag/*blood ; Leukemia Virus, Feline/immunology/*isolation & purification ; Leukemia, Feline/*diagnosis ; Mice, Inbred BALB C ; Sensitivity and Specificity

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 compare the immunogenicity of rAAV2/1 and rAd5 expressing HIV-1 gag in BALB/c mice.

METHODSBALB/c mice were immunized with rAAV2/1-gag or rAd5-gag once or twice. HIV-1 specific cellular immune responses were analyzed by in vivo CTL and intracellular cytokine staining assays. HIV-1 Gag specific antibodies were tested by ELISA.

RESULTSMice immunized with rAd5-gag once induced stronger Gag specific cellular immune responses and similar level of Gag specific antibody compared with rAAV2/1-gag. Mice immunized with rAd5-gag reached the peak immune responses more rapidly than rAAV2/1-gag. However, mice immunized with rAAV2/1-gag twice elicited better Gag specific IgG.

CONCLUSIONrAd5-gag induced strong HIV-1 specific cellular and antibody responses, and rAAV2/1-gag induced high level of HIV-1 specific IgG and moderate cellular immune responses.

AIDS Vaccines ; administration & dosage ; genetics ; immunology ; Adenoviridae ; genetics ; metabolism ; Animals ; Dependovirus ; genetics ; metabolism ; Female ; Gene Expression ; HIV Antibodies ; blood ; immunology ; HIV Infections ; immunology ; prevention & control ; virology ; HIV-1 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Random Allocation ; gag Gene Products, Human Immunodeficiency Virus ; administration & dosage ; genetics ; immunology

OBJECTIVEHIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately.

METHODSHIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method.

RESULTSpCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1.

CONCLUSIONHIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

AIDS Vaccines ; immunology ; Animals ; Antibodies, Viral ; blood ; Cell Line, Tumor ; Dependovirus ; genetics ; Gene Products, gag ; genetics ; metabolism ; HIV-1 ; genetics ; immunology ; L-Lactate Dehydrogenase ; blood ; Mastocytoma ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, DNA ; immunology

BACKGROUNDAlthough DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed.

METHODSVector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice.

RESULTSIt was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner.

CONCLUSIONSThe 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

AIDS Vaccines ; immunology ; Amino Acid Sequence ; Animals ; Blotting, Western ; CD8-Positive T-Lymphocytes ; immunology ; Enhancer Elements, Genetic ; Female ; Gene Products, gag ; immunology ; HIV Antibodies ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; Simian virus 40 ; genetics ; Vaccination ; Vaccines, DNA ; immunology ; Vaccinia ; immunology

OBJECTIVETo study the immune effect of a chimeric adenovirus type 5 vector with type 35 fiber (rAd5/F35) vaccine in BALB/c mice.

METHODSThe expression of HIV Gag protein was determined using indirect immunofluorescent staining. The rAd5/F35-mod.gag vector was injected intramuscularly to mice. The IgG antibody was detected by ELISA and CTL response was detected by intracellular cytokine stain assay.

RESULTSThe rAd5/F35-mod.gag vector could express HIV Gag protein in vitro and generate strong HIV-specific immune responses in vivo. But anti-Ad5 immunity could limit its immunogenicity in vivo.

CONCLUSIONThe rAd5/F35-mod.gag vector can elicit specific CTL response and IgG antibody in animal model. In mice with high Ad5 vector-specific immunity, Ad5/F35-mod.gag showed lower level of Gag specific CTL and antibody response than in mice without pre-existing adenovirus type 5 immunity. The results indicated that fiber exchange alone does not evade pre-existing Ad5 immunity.

AIDS Vaccines ; genetics ; immunology ; Adenoviridae ; genetics ; Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique, Indirect ; Gene Products, gag ; genetics ; immunology ; metabolism ; Genetic Vectors ; genetics ; HIV Antibodies ; blood ; HIV-1 ; genetics ; immunology ; Immunization ; methods ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.

METHODSThe recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.

RESULTSThere was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.

CONCLUSIONThe recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.

Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cells, Cultured ; Chick Embryo ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; cytology ; metabolism ; ultrastructure ; Fowlpox ; blood ; immunology ; virology ; Fowlpox virus ; genetics ; immunology ; Gene Products, gag ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp120 ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Immunization ; methods ; Interleukin-6 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transfection ; Viral Vaccines ; genetics ; immunology ; metabolism