The global HIV epidemic continues to expand, exceeding previous predictions and causing tremen-dous suffering. It has been the leading cause of death in Africa. At the same time, a rapid increase of HIV infection has also been found in China in recent years. Therefore, developing HIV vaccines targeting the prevalent strains in China is one of the most important tasks for Chinese HIV/AIDS control and prevention.
Virus-specific cellular immune responses play an important role in the control of HIV infections (1, 6, 23). Recently some studies indicated that only Gag-specific responses were associated with lowering viremia in an untreated HIV-1 infected cohort and HIV-2 long-term nonprogressors, while Env-and Nef-specific responses are positively correlated with a high viral load (4, 7, 13, 15, 16). Thus Gag would be the preferred antigen in HIV candidate vaccines. DNA vaccines, recombinant-viral-vector based vaccines, and their combinations are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. DNA vaccine is safe and easy to manufacture, and it is quite effective as a priming or initial immunogen in a bimodal vaccine strategy (17, 22); Some of the extraordinary features of the SeV vector are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration (5). An extremely efficient antigen expression system in mammalian cell cultures using recombinant SeV were established in the late 1990's (8, 12, 24). Using this system Kano et al demonstrated the excellent protective efficacy of DNA priming followed by simian immunodeficiency virus (SIV) Gag-expressing Sendai virus boosting against a pathogenic simian-human immunodeficiency virus (SHIV89.6PD) infection in macaques (10). Takeda et al obtained similar results by using a DNA prime/replication defective SIV Gag-expressing SeV boost system (19). In the present study a HIV-1 gag gene was used to construct DNA and replication defective rSeV vaccines. High-frequency CTL responses may be elicited by combining these two vaccines.
Although the discontinuation of the Merck rAd5 Phase Ⅱ proof-of-concept STEP study is undoubtedly a significant setback for the field of HIV vaccine development (18), it does not indicate the failure of the T-cell vaccine concept. In their study a trivalent recombinant adenovirus type 5 vaccine expressing Gag, Pol, and Nef respectively was tested. The issues of lack of vaccine efficacy and differential infection rates between vaccine and placebo groups with previous Ad5 immunity are complex ones and require careful consideration. Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. Though immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens, Toapanta et al found that when HIV-1 Env (gp120) and HIV-1 Gag (p55) DNA plasmids were co-inoculated, there was a reduction in the immune responses elicited to HIV-1 Gag (p55). This anti-HIV-1 Gag immune interference was specific to co-immunizations with HIV-1 Env (gp120) and may involve a yet undefined immunological mechanism (20). Recently Kiepiela et al performed a compre-hensive analysis of the 160 dominant CD8+ T cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. They found only Gag-specific responses were associated with lowering viremia (13). So in our study Gag-expressing vaccines would be investigated first, and only if excellent immune efficacy could be induced would other antigens be included in a further study.
Most worldwide vaccine developments have been focused on inducing protection against prevalent strains in North America and Europe. Developing HIV vaccines targeting the prevalent strains in China is of great importance Thailand subtype B is found to be prevalent in several epidemic regions where paid blood donors are the principally affected population. The HIV-1 Subtype B in these epidemic regions is relatively well conserved, so the prevalent strains isolated from these regions was used as the vaccine strain. The constru-ction of DNA and rSeV vector vaccines containing the gag gene of prevalent Thailand B strains in China was therefore the main objectives of our studies.
The codon-modified consensus gag gene from HIV-1 prevalent strains in Henan province was used to construct a DNA vaccine. Firstly, the gag genes were amplified by nest-PCR using specific primers from the DNA from the peripheral blood mononuclear cells (PBMC) of HIV-1 infected patients in Henan province. Twelve gag sequences were obtained from 20 patients and 6 of them had complete open reading frames (ORF). The consensus gag gene was generated by sequence alignment of the 6 gag genes. The consensus gag sequence had different amino acids in 6 positions compared with reference sequences of subtype B (B.FR.HXB2-LAI-IIIB-BRU and B.TH. BK132). 95 % and 96 % of amino acid sequence homology were found with B.FR.HXB2-LAI-IIIB-BRU and B.TH.BK132 respectively. To increase the expression level of Gag protein in DNA vector the codons of the consensus gag gene were modified according to mammalian condon usage. The codon-modified gag gene was synthesized and inserted into plasmid pUC57 by the Shanghai Sangon Biological Engineering Technology & Service Co., Ltd (Shanghai, China). KpnⅠand XhoⅠrestriction enzyme sites were inserted into the gag gene by PCR and cloned into pcDNA3.1 (+) which was named pcDNA3.1 (+)-gag. The pcDNA3.1 (+)-gag was standardized at 1mg/mL in endotoxin-free Buffer TE.
The wild-type gag gene was used for construction of the recombinant Sendai virus containing gag (rSeV-gag). The rSeV-gag was constructed and amplified by the DNAVEC Corporation (Tsukuba, Japan) as follows: (ⅰ) EIS sequence and NotⅠ restric-tion enzyme sites were incorporated into the gag gene by PCR, cloned into pBluescript KS (+) and was named pBS-HIVgag. (ⅱ) pBS-HIVgag was digested with NotⅠ and the DNA fragment containing gag was inserted into pSeV/ΔF (a viral genomic RNA-en-coding plasmid) that contains SeV full-length cDNA lacking the F gene. The generated plasmid was named pSeV18+HIVgag/ΔF. (ⅲ) Recombinant virus con-taining the gag gene was recovered by co-transfection of 293T/17 cells with pCAGGS-P(z)/4C-(P protein-expressing plasmid), pCAGGS-NP (NP protein-expressing plasmid), pCAGGS-L(TDK) (L protein-expressing plasmid), pCAGGS-F5R (modified-F protein-expressing plasmid), pCAGGS-T7 (T7 RNA polymerase-expressing plasmid) and pSeV18+HIV gag/ΔF. (iv) Recovered virus was cloned and amplified in LLC-MK2/F/A cells expressing the F protein. Virus yield is expressed in cell infectious units (CIU). The recombinant Sendai virus was titrated as follows: LLC-MK2 cells were seeded into a 6-well plate at a cell density of 2×105 cells/2mL/well and incubated at37℃, 5%CO2 for 72 h until 100% confluence. 10-fold dilutions of the virus stock were prepared in PBS containing 1% BSA. The confluent monolayer was washed once with PBS and then was inoculated in duplicate with 0.1 mL of the virus samples/well. The cells were incubated for 1 h at 37℃, 5% CO2, and the plate was tilted every 15 min. Virus inoculum was removed, the cells were washed with PBS and supplemented with 2 mL of DMEM and incubated at 37℃, 5% CO2for 48h. The cells were washed with PBS and fixed with methanol for 10 min at 4℃. After removing methanol the plate was dried for 10 min at room temperature. 0.5 mL of 1:500 diluted polyclonal rabbit-anti SeV antibodies (prepared by the DNAVEC Corporation) was added into each well. The plate was incubated for 45 min at 37℃ then was washed twice with PBS. 0.5 mL of 1:200 diluted goat anti-rabbit immunoglobulin G (Zhongshan Goldenbridge Biotech-nology Co., LTD, Beijing, China)was added into each well. The plate was incubated for 45 min at 37℃ then was washed twice with PBS. The number of positive cells was counted under the fluorescence microscope. The virus titer was calculated by the formula: mean numbers of positive cells in duplicate wells × dilution multiple×10 (CIU/mL). The titer of generated rSeV-gag was 1.8 × 1010 CIU/mL.
LLC-MK2 cells were seeded into a 6-well plate at a cell density of 2×105 cells/2mL/well and incubated at 37℃, 5%CO2 for 72 h to 100% confluence. The confluent monolayer was washed with PBS, then was inoculated with 0.1 mL of the virus samples/well (MOI of 5, to count the cell number per well, the cells in one well was trypsinized and counted under a microscope). The cells were incubated for 1h at 37℃, 5% CO2, and the plate was tilted every 15 min. Virus inoculum was removed, the monolayer was washed with PBS and supplemented with 2mL of DMEM and incubated at 37℃, 5%CO2 for 48 h. The virus which did not contain the foreign gene (rSeV-control) was used as the control. 48 h later, total RNA was isolated from infected cells using Trizol reagent (Promega, Madison, USA). One-Step RT-PCR was used to test the presence of the gag gene in the recombinant virus (wt269F: 5'-GGCAAGCAGGGAACTAGAAC-3', wt-269R: 5' AGAACCGGTCTACATAGTCTC-3').
LLC-MK2 cells were maintained in DMEM and supplemented with 10% fetal bovine serum (FBS). Cells were transfected with pcDNA3.1(+)-gag or infected with rSeV-gag at a MOI of 5. Proteins of transfected or infected LLC-MK2 cells were extracted 48 h later using TRIzol reagent. The protein samples were subjected to SDS-PAGE and electroblotted onto nitrocellulose blotting membranes. Blots were blocked with 5% fat free milk in phosphate buffered saline (PBS) containing 0.05% Tween 20 and probed with mouse anti-HIV-1 P24 monoclonal antibody(NIH AIDS Research and Reference Reagent program, Germantown, USA)and peroxidase-conjugated goat anti-mouse immunoglobulin G (Zhongshan Goldenbridge Biotechnology Co., LTD, Beijing, China). Proteins were visualized by staining with 3, 3'-Diamino-benzidine.
Four to six-week-old Balb/c female mice were purchased from Institute of Experimental Animal Sciences, Chinese Academy of Medical Science (Beijing, China). To compare the immunogenicity of the DNA and rSeV-gag vaccines applied in single or combined vaccines, mice were inoculated with these vaccines intramuscularly either in single or combined modality at week 0 and week 3. The immunization dose for the DNA vaccine was 100μg per animal and for the rSeV vaccine was 1.8 ×107 CIU per animal. Balb/c mice were randomly divided into four groups of 16. Inoculation was conducted according to the schedule in Table 1. The splenocytes and sera of immunized mice were collected at 1, 5 and 9 weeks post immunization and the cellular and humoral immune responses were analyzed.
Table 1. Immunization schedule of DNA and rSeV vaccines
Freshly isolated splenic lymphocytes (2×106 cells) were suspended in 10% FBS RPMI1640 medium and incubated with H-2d-restricted CTL epitope peptides, Gag197-205(AMQMLKETI), Gag239-247(TTSTLQEQI) and Gag291-300(EPFRDYVDRF) for antigen-specific stimulation or without peptides for mock stimulation. The three peptides were pooled together and the final concentration for each peptide was 10µg/mL. Cells were cultured for 16h at 37℃. For the final 12 h, brefeldin A (Sigma, Saint Louis, USA) was added at 5μg/mL. After stimulation, the cells were stained (60 min, 25℃) for surface markers with 5 μL of R-PE-conjugated rat anti-mouse CD8a (Ly-2) monoclonal antibody (BD Pharmingen, San Diego, USA). Then the cells were sequentially fixed with 4% paraformal-dehyde and permeabilized with 0.3 % saponion for 15 min respectively, and stained for 60 min with 1 μL of FITC-conjugated rat anti-mouse IFN-γ monoclonal antibody (BD Pharmingen, San Diego, USA). Stained samples were collected by a Coulter EPICS Altra flow cytometer (Beckman, Fullerton, USA) and analyzed using the CellQuest software package. Gating was performed on mononuclear cells and then on CD8+ subpopulations. 50 000 of CD8+ T cells were collected in total. From the ratio of CD8+IFN-γ+ cells to CD8+ cells, the frequency of CD8+ IFN-γ+ cells in the total CD8+T cells was calculated. Gag-specific T-cell frequencies were calculated by subtracting the CD8+IFN-γ+-cell frequencies after mock stimulation from those after Gag-specific peptides stimulation.
Specific antibodies were detected by the enzyme-linked immunosorbant assay (ELISA). 96-well microtiter plates were coated with 200ng/well of HIV P24 protein prepared by our laboratory and incubated overnight at 4℃. The wells were blocked with PBS containing 5% fat free milk for 1h at 37℃. They were then treated with 100 μL of serially diluted mice sera and incu-bated for an additional 1h at 37℃. The plates were washed five times with PBS containing 0.05% Tween-20 and incubated for 1h with 1:20 000 diluted goat anti-mouse IgG/HRP. The plates were then washed five times, developed with tetramethylbenzidine, stopped with 2 mol/L H2SO4, and analyzed at A450nm/630nm.