[IL15 DNA adjuvant enhances cellular and humoral immune responses induced by DNA and adenoviral vectors encoding HIV-1 subtype B gp160 gene].

To enhance the immunogenicity of DNA and adenoviral vector vaccines expressing HIV-1 subtype B gp160, human interleukin 15 (hIL15) DNA adjuvant (pVR-hIL15) was constructed. BALB/c mice received DNA prime/protein boost immunization with pVR-HIVgp160/Ad5-HIVgp160 alone or combined with pVR-hIL15. Cellular and humoral immune responses were evaluated by IFN-gamma enzyme-linked immunosorbent spot assay and enzyme-linked immunosorbent assay, respectively. Compared with those immunized with vaccines alone, the mice immunized with vaccines combined with pVR-hIL15 had significantly increased specific cellular response and antibody titer (P < 0.05). It suggests that the IL15 DNA adjuvant can enhance the immune responses induced by prime-boost regimen using DNA and adenoviral vector encoding HIV-1 subtype B gp160.

[Molecular epidemiological characteristics of HIV-1 B'/C strains in Beijing].

OBJECTIVE: To analyze the molecular epidemiological characteristic of HIV-1 B'/C strains prevalent in Beijing. METHODS: Plasma samples were collected from 200 newly diagnosed HIV-1 B'/C individuals reported during 2006 to 2010 in Beijing. The gag gene fragments were amplified from RNA template extracted from plasma with reverse transcription (RT) and nested polymerase chain reaction (PCR). And the sequences were analyzed by phylogenetic methods and Entropy analysis. RESULTS: A total of 159 sequences were successfully amplified from the gag genes of which 147 was CRF07_BC and 12 CRF08_BC. There were 3 main sub-clusters in CRF07_BC phylogenetic tree and they were named as sub-cluster IDU-Max (89 sequences), sub-cluster IDU-Min (22 sequences) and sub-cluster MSM (34 sequences) based on transmission.No international reference strain was closely related with these three sub-clusters except for one strain identified in Taiwan. All CRF07_BC recombinant strains were remarkable for their low interpatient diversity in gag genes (3.7%, 3.3% and 2.0% for isolates from IDU-Max, IDU-Min and MSM respectively).When compared with sub-cluster IDU-Max, there were 32 and 41 significantly different sites of nucleotide polymorphism compositions in sub-clusters IDU-Min and MSM. CONCLUSION: This is the first report of describing the existence of three main epidemic sub-clusters in CRF07_BC strains prevalent in Beijing. And IDU-Max sub-cluster is the dominant strain. The CRF07_BC in Beijing are less diverse than other strains and may be derived from a common ancestor.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

OBJECTIVE: To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. METHODS: Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. RESULTS: Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. CONCLUSION: We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

[Molecular-epidemiological characteristics of HIV-1 isolated from newly diagnosed female subjects in Beijing, 2006 - 2010].

OBJECTIVE: To analyze the molecular-epidemiological characteristics of HIV-1 strains prevailing among female people living with HIV in Beijing. METHODS: Gag gene fragments from the 100 newly diagnosed female HIV-1 infections during 2006 to 2010 in Beijing were amplified, sequenced, and phylogenetically analyzed. RESULTS: Eighty-two HIV-1 gag gene fragments were amplified and analyzed. 1 (1.22%), 1 (1.22%), 3 (3.66%), 23 (28.05%), 8 (9.76%), 2 (2.44%), 1 (1.22%), 18 (21.95%), 3 (3.66%), 1 (1.22%), 14 (17.07%), 4 (4.88%) and 3 (3.66%) individuals were infected with HIV-1 subtypes A1, A2, B, B', C, D, G, H, CRF01_AE, CRF02_AG, CRF07_BC, CRF08_BC and B'/C recombinants respectively. CONCLUSION: The subtypes circulating in female HIV infections in Beijing were more diverse than in male and the proportions of B' and rare subtypes were relatively high. Surveillance programs on HIV-1 genetic diversity should be strengthened.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by B cell activation and RT-PCR cloning].

To obtain protective human monoclonal antibody from HIV-1 infected person, we adapted a technology for isolating antigen specific monoclonal antibody from human memory B cells through in vitro B cell activation coupled with RT-PCT and expression cloning. Human B cells were purified by negative sorting from PBMCs of HIV-1 infected individuals and memory B cells were further enriched using anti-CD27 microbeads. Two hundred memory B cells per well were cultured in 96-well round-bottom plates Env-specific antibodies in supernatants were with feeder cells in medium containing EBV and CpG. screened by ELISA after 1-2 weeks' culture. Cells from positive wells of Env-specific antibody were harvested and total RNA was isolated. Human VH and Vkappa or Vlambda genes were amplified by RT-PCR and cloned into IgG1 and kappa or lambda expressing vectors. Functional VH and Vkappa or Vlambda were identified by cotransfecting 293T cells with individual heavy chain and light chain clones followed by analysis of culture supernatants by ELISA for Env-specific antibodies. Finally, corresponding mAb was produced by transient transfection of 293T cells with the identified VH and Vkappa/lambda pair and purified by protein A affinity chromatography. Purified monocolonal antibodies were used for HIV-1 specific antibody-dependent cell-mediated cytotoxicity (ADCC) and neutralizing activity assay. Four monocolonal Env-specific antibodies were isolated from one HIV-1 subtype B' infected individual. Two of them showed strong ADCC activity and one showed weak neutralizing activity against HIV-1. Its further studies on their application in therapeutic or prophylactic vaccines against HIV-1 should be grounded.

Genetic diversity of HIV type 1 isolated from newly diagnosed subjects (2006-2007) in Beijing, China.

The purpose of this study was to investigate the genetic diversity of HIV-1 circulating in Beijing and its molecular epidemiological linkages with regard to risk factors of viral transmission. HIV-1 from plasma samples of 280 diagnosed individuals (2006-2007) was characterized. The gene fragments of gag, pol, and env from the infected plasma samples were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), sequenced, and phylogenetically analyzed. From the 280 plasma samples analyzed, a total of 496 sequences were successfully amplified from the gag, pol, and env genes. Nine HIV-1 group M subtypes or CRF including A1, B, B', C, CRF01_AE, CRF02_AG, CRF06_cpx, CRF07_BC, and CRF08_BC, and six new B'/C recombinants were identified. CRF07_BC was found to be the most dominant subtype (32.5%) followed by CRF01_AE (25.0%), B (20.0%), and B' (15.7%). The data from this study indicate the existence of multiple HIV-1 subtypes or CRFs in Beijing and may be proven useful in the development of vaccine candidates in the future.

The prevalence of drug resistance mutations among treatment-naive HIV-infected individuals in Beijing, China.

To investigate the prevalence of HIV-1 genotypic mutations for drug resistance among patients in Beijing, blood samples from 145 newly confirmed (2006-2007), treatment-naive HIV-1-infected individuals were analyzed. Seven subtypes or CRF were subsequently determined and scored by the Stanford HIV Drug Resistance algorithm: CRF01_AE HIV-1 (27.6%), subtype B' (24.1%), CRF07_BC (21.4%), subtype B (20.7%), CRF08_BC (3.4%), subtype C (2.1%), and CRF06_cpx (0.7%). Eleven of the 145 subjects studied were found to harbor the strains resistant to either protease inhibitors (PIs) (3.4%), or nucleoside reverse transcriptase inhibitors (NRTIs) (2.1%), or nonnucleoside reverse transcriptase inhibitors (NNRTIs) (3.4%). Although the prevalence of drug resistance was relatively low among the treatment-naive HIV-1 patients in Beijing in comparison to those in industrialized countries, we will continue monitoring newly infected subjects for any potential alteration of the prevalence pattern to ensure the success of the ongoing scale-up of antiretroviral treatment.

[Anti-adenovirus neutralizing antibodies and Gag-specific cellular immune responses in Macaca fascicularis immunized with Ad5-HIVgag].

OBJECTIVE: To 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. METHODS: The 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. RESULTS: High 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. CONCLUSION: Anti-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.

[Study on vector-specific and foreign gene-specific immune responses induced by rAd5 and rAAV2/1 vaccines].

OBJECTIVE: To compare the foreign gene-specific and vector-specific immune responses in BALB/c mice immunized with rAd5 or rAAV2/1 expressing the same gene. METHODS: BALB/c mice were immunized with rAd5-gag or rAAV2/1-gag once, HIV-1 Gag-specific and vector-specific cellular immune responses were analyzed by Elispot assay, HIV-1 P24-specific IgG and vector-specific IgG were tested by ELISA assay. RESULTS: Mice immunized with rAd5-gag induced potent Gag-specific cellular immune responses and that were significantly higher than Ad5-specfic cellular responses, while rAAV2/1-gag elicited weak Gag-specific and AAV2/1-specific cellular responses. Both P24-specific and Ad5-specific IgG titers induced by rAd5-gag were high and in similar level. Higher level of P24-specific IgG was found in mice inoculated with rAAV2/1-gag than rAd5-gag. And the P24-specific IgG titers were higher than the vector-specific IgG titers in mice immunized with rAAV2/1. CONCLUSION: rAd5 could elicit strong foreign gene-specific cellular and humoral immune responses, weak vector-specific cellular responses and strong vector-specific antibodies, rAAV2/1 could induce potent foreign gene-specific antibodies that were much higher than vector-specific IgG, while both foreign gene-specific and vector-specific cellular responses were very low.

[Immunogenicity of recombinant adeno-asscociated virus type 1 expressing HIV-1 gp120 gene in mice and Rhesus macaques].

To study the immunogenicity of recombinant adeno-asscociated virus type 1 expressing HIV-1 gp120 gene (rAAV2/1-gp120) in BALB/c mice and Rhesus macaques. The gp120 gene derived from Chinese HIV-1 isolates was constructed into rAAV2/1 and rAd5 vectors. Firstly, the immunogenicity of rAAV2/1-gp120 was compared with rAd5-gp120 in BALB/c mice when used once or twice in 3 weeks interval. Then the monkeys were immunized with rAAV2/1-gp120 once. The HIV-1 specific IgG levels and neutralization activity to pseudotyped HIV-1 virus were tested using ELISA and neutralization assay, and the cellular immune responses were analyzed by IFN-gamma enzyme-linked immunospot (ELISPOT) and in vivo CTL assays. Compared with rAd5-gp120 immunized mice, mice immunized with rAAV2/1-gp120 once in duced stronger gp120-specific IgG and were sustained for at least 21 weeks. rAd5-gp120 immunized mice generated stronger cellular immune responses than rAAV2/1-gp120 in spleen and draining lymph node. But only moderate gp120-specific in vivo CTL activity was observed in both rAAV2/1-gp120 and rAd5-gp120 immunized mice. Four of five monkeys vaccinated with rAAV2/1-gp120 generated gp120 specific IgG, the titer ranged from 1:100 to 1:400 with end-point dilution. Gp120 specific IgG could be detected 4 weeks after immunization and reached the peak at 10 weeks after immunization. No neutralization activity against pseudotyped HIV-1 virus expressing NL4-3 Env antigen was detected. In Conclusion, rAAV2/1-gp120 induced high level of HIV-1 specific IgG antibody and moderate cellular immune responses. No neutralizing antibody was elicited. It indicates that the env gene and immunization strategy should be optimized to elicit neutralizing antibody against HIV-1 in further studies.

[Immunogenicity of plasmid DNA and adenoviral vectors encoding HIV-1 subtype B env gene].

OBJECTIVE: To construct DNA and recombinant adenovirus vector vaccines containing an env gene from the prevalent subtype B strain in China and try to use them for therapeutic and prophylactic vaccines. METHODS: The candidate plasmid DNA vaccine pVR-gp160 and recombinant adenovirus vaccine rAdV-gp160 were constructed separately. BALB/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gp120-specific cellular responses and antibody levels were detected by ELISPOT and ELISA respectively. RESULTS: DNA vaccine alone and combined vaccines in a DNA prime/rAdV-gp160 boost vaccination regimen induced high level of Gp120-specific cellular responses. While low level of Gp120-specific antibodies were elicited in all groups. CONCLUSION: DNA and rAdV vaccines could efficiently express Gp160 protein and activate specific cellular responses.

[Genetic analysis of the complete env genes of HIV-1 from paid blood donors in Henan province].

Complete HIV-1 env genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) DNA of 60 HIV-1 positive paid blood donors in Henan province, and the amplified full-length genes were sequenced. Twenty one full-length env genes were obtained, sequence analysis found that 15 of them had intact open reading frame (ORF). Fourteen sequences conformed to subtype B', their average genetic distance with the international reference sequence RL42 was 4.87% +/- 0.31%. One was subtype B, its genetic distance with the international reference sequence HXB2 was 5.43%. The amino acid sequences of these env genes were deduced according to their nucleotide sequences and extensive analysis and comparison of important structural motifs were performed. The results indicated that there was no drastic alteration in the number and position of potential N-linked glycosylation sites among these 15 sequences. And the residues involved in forming the CD4 binding site were highly conserved. Genotype prediction of coreceptor usage based on V3 sequence and net charge suggested that most samples use CCR5 coreceptor. GPGR motif at the tetrapeptide crown in the V3 loop was most common in these samples and it was detected in 40% sequences. The cleavage site of gp120/gp41 was highly conserved, so Gp160 precursor of all isolates would be efficiently cleaved into the Gp120 and Gp41 subunits. The known neutralizing antibody binding sites for 2G12, IgG1b12, 4E10 and 2F5 were also highly conserved, it is expected that most of these isolates will be sensitive to neutralization by these antibodies. Further study to elucidate the correlation of the env genotype to functionally relevant motifs is necessary and that will aid vaccine and novel drug design.

[Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation].

OBJECTIVE: To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies. METHODS: A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full-length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells. RESULTS: HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1:204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells. CONCLUSION: HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.

[Comparison of the immnunogenicity of rAAV2/1 and rAd5 rad5 expressing HIV-1 gag].

OBJECTIVE: To compare the immunogenicity of rAAV2/1 and rAd5 expressing HIV-1 gag in BALB/c mice. METHODS: BALB/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. RESULTS: Mice 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. CONCLUSION: rAd5-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.

[Codon modification of HIV-1 subtype B gag gene and its immunogenicity in mice].

HIV-1 subtype B gag genes were cloned from the infected paid blood donors in Henan, and the consensus sequence based on these prevalent strains was obtained by aligning. The codons of the consensus gag sequence were modified according to mammalian codon usage. Western blot analysis was used to compare the expression level of wild type and codon-modified gag gene. It was found that the expression level of Gag protein was improved largely by codon-modification. Then the mod. gag gene was inserted into the adenovirus vector and the recombinant adenovirus rAdV-mod. gag was constructed. 10(8) PFU or 10(9) PFU rAdV-mod, gag vaccinated mice twicely could elicit high level Gag-specific CTL responses in immunized mice. In conclusion,the codon modification of gag gene is successful. The recombinant adenovirus vaccine harbouring mod. gag can induce robust Gag-specific CTL immune response in mice.

[Immune responses induced by recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys].

OBJECTIVE: To observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys. METHODS: Sixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time. RESULTS: EBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response. CONCLUSION: The recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

[Immunogenicity of a chimeric adenovirus type 5 vector with type 35 fiber containing HIV-1 gag in mice].

OBJECTIVE: To study the immune effect of a chimeric adenovirus type 5 vector with type 35 fiber (rAd5/F35) vaccine in BALB/c mice. METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.

[Construction and immune potency of recombinant adenovirus containing codon-modified HIV-1 gp120].

BACKGROUND: To construct replication-deficient recombinant adenovirus expressing wild and codon-modified HIV-1 gp120. METHODS: The viral codons were changed to the codon usage of highly expressed mammal gene, the resulting modified gp120 gene was synthesized. The wild and modified gp120 genes were cloned into shuttle vector pShuttle-CMV respectively, and then the constructed plasmids containing gp120 gene was cotransformed with the backbone vector pADeasy-1 into E.coli BJ5183. Transfection of the recombinant AdEasy plasmid into 293 cells was performed to obtain recombinant adenoviruses. The mice were immunized with the recombinant adenoviruses. Their immunogenicity was evaluated by testing antibody and CTL levels of immunized mice. RESULTS: Two strains of recombinant adenovirus expressing wild and codon-modified HIV-1 gp120 were obtained. The protein expressing level of the recombinant adenoviruses containing modified genes was much higher than that containing wild genes. The mice immunized with recombinant adenoviruses elicited HIV-1 specific antibody and CTL response. The rAd-mod gp120 group was better than the rAd-wt gp120 group. CONCLUSION: Replication-deficient recombinant adenovirus expressing HIV-1 gp120 can elicit HIV-1 specific humoral and cellular response, the codon-modified recombinant virus was more efficient than the native.

[Immune potency of recombinant adeno-associated virus combined with recombinant adenovirus vaccine containing HIV-1 gp120].

OBJECTIVE: To study the immune effect of recombinant adeno-associated virus (rAAV) combined with recombinant adenovirus (rAdV) vaccine in BALB/c mice. METHODS: The codon-modified HIV-1 gp120 gene was inserted into plasmid of adeno-associated virus and adenovirus vector separately. Then the rAAV and rAdV vaccines were constructed. BALB/c mice were immunized with rAAV and rAdV vaccines in different administration scheme. The IgG antibody was detected by ELISA and CTL response was detected by intracellular cytokine stain assay. RESULTS: Both rAAV and rAdV vaccine could express gp120 gene; the mice primed with rAAV at week 0, 2 and boosted with rAdV at week 5, 14 and 20 elicited the strongest gp120 specific CTL and IgG antibody response. CONCLUSION: The mice primed with rAAV and boosted with rAdV could elicit specific CTL response and IgG antibody.