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Objective:To analyze the immune response in mice after immunization with vaccine of rAd5F35-SIVenvT in combination with rMVA-SIVenvT to evaluate the efficacy of different immunization strategies.Methods:Two recombinant viruses were identified in vitro by PCR and Western blot. The BALB/c mice were immunized with homologous and heterologous immune strategies. The numbers of splenic lymphocytes secreting IFN-γ were measured by ELISPOT assay, meanwhile SIV gp120 antibody titer were measured by ELISA assay. Results:SIVenvT protein was expressed effectively by rAd5F35-SIVenvT and rMVA-SIVenvT in HEK293 cells. The specific immune response reached its peak at 4-week post first immunization, then decreased. SIV Env specific cellular immune response and SIV gp120 specific antibody could be detected at 4-16 weeks post first immunization. The specific cellular response was significant stronger in heterologous immunization group than homologous group at 4 week and 16 week. Furthermore, heterologous immunization induced significant higher titer of SIV gp120 antibody at 4 week than homologous group.Conclusions:Specific immune response induced by rAd5F35-SIVenvT in combination with rMVA-SIVenvT was stronger than homologous vector immunization. The results provided references for further study in nonhuman primates.
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Objective To investigate the changes of CD169 expression on the surface of peripheral blood monocytes and different subsets of monocytes in normal rhesus monkeys after SIVmac239 infection and the possible reasons.Methods Normal rhesus monkeys were infected with SIVmac239 through intravenous injection, and changes in the percentage of peripheral blood monocytes and the expression of CD169 before and after SIVmac239 infection were detected by flow cytometry. The peripheral blood CD14 +monocytes of normal rhesus monkeys sorted by flow cytometry were directly infected by SIVmac239 and stimulated by different cytokines,and changes in the expression of CD169 on the cell surface and the cytokine IFN-α were detected by flow cytometry. Results After SIVmac239 infection, the percentage of CD14 +monocytes of the normal rhesus monkeys was decreased and the expression of CD169 on their surface was increased. Meanwhile,the expression of CD169 on the surface of different subsets of peripheral blood monocytes was significantly increased,and the expression of CD169 on the CD14 +CD16 + +monocytes was increased more obviously. CD169 was not expressed on the surface of peripheral blood CD14 +monocytes of the normal rhesus monkeys after stimulated by the cytokines M-CSF, IL-4 and IL-13. However, CD169 was highly expressed after the monocytes were stimulated by the cytokine IFN-α. The expression of CD169 on the surface of CD14 +monocytes and the intracellular cytokine IFN-α was not significantly changed after the monocytes were directly infected with SIVmac239. Conclusions SIVmac239 infection can lead to the increase of CD169 expression on the surface of peripheral blood monocytes in rhesus monkeys. Its expression is not associated with the direct infection of virus,but is related to the cytokine IFN-α secreted by other cells of the monkeys in vivo.
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Objective SIV30 protein of simian immunodeficiency virus(SIV)was prepared by genetic engineering technique as an antigen diagnostic reagent, to establish an immune comb method for the specific detection of anti SIV IgG in monkey serum. Methods Recombinant expression plasmid of SIV SIV30 gene was constructed by prokaryotic expression vector pGEX-4T-1, and expressed in the competent BL21 cells. The recombinant protein was purified as a diagnostic antigen, and a standardized procedure for the detection of immune comb was established and applied for clinical detection. Results The optimum coating amount of antigen was 0.02 mg/mL. The prepared IC was able to specifically detect the positive serum of SIV. There was no cross reaction between the sera of other viruses. It showed a high specificity of the detection method. Sensitivity analysis showed that the SIV30 protein was able to detect 1:400 times diluted SIV positive sera. The result of stability and repeatability test(the same sample was repeated 3 times) showed that the coefficient of variation(CV)was less than 10%. The serum samples of 10 suspicious monkeys were detected by this method, showing a consistent rate of comb method and ELISA test result of 100%, Kappa =1.000. Conclusions SIV30 protein is expressed in prokaryotic cells. The immune comb is prepared,and is successfullyl applied in clinical examination. It shows that the method has a high sensitivity, strong specificity, good reproducibility and practicability.
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Objective To establish a TaqMan probe-based real-time fluorescent quantitative PCR ( real-time PCR) for the quantitative and rapid detection of viral reservoir in peripheral blood mononuclear cells (PBMCs) isolated from rhesus monkeys with simian immunodeficiency virus (SIV) infection and to evaluate its preliminary application. Methods A pair of primers and one TaqMan probe were designed ac-cording to the conserved sequence of SIVmac239 strain for real-time PCR amplification. A length of 2 090 bp of nucleotide fragment was digested from the plasmid p239SpSp5 containing 5′-end long segments of SIV-mac239 strain by restriction enzymes EcoRⅠand SpeⅠ. The standards used for quantitative detection of SIV DNA in peripheral blood samples were prepared by a 10-fold serial dilution and used for graphing the stand-ard curve. The numbers of SIV DNA ( copies per 106 PBMCs) in rhesus monkeys during acute and chronic phases of SIVmac239 infection were determined and the virological characteristics of SIV DNA at different phages of infection were analyzed. Results A linear positive correlation between cycle threshold ( Ct) val-ues and concentrations (10 copies/μl to 109 copies/μl) of the standards was found. High levels of SIV DNA were monitored in SIV-infected monkeys 14 to 22 days after acute infection. The levels of SIV DNA in the acute phase of infection were about 1 to 2 logs higher than those in the chronic phase of infection. The num-bers of SIV DNA ( copies per 106 PBMCs) were 1 log lower than the SIV viral load in peripheral blood of the same monkey. The ratios of SIV DNA load to SIV RNA load ( DNA/RNA) in chronic phase of infection were higher than those in the acute phase. Conclusion The established TaqMan probe-based real-time fluorescent quantitative PCR was a highly sensitive and specific assay for the detection of SIV DNA with an advantage of wide linear range. It could be used for the quantitative evaluation of latent reservoirs of SIV.
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Objective To study the mutations of Env sequence of SIVmac239 after infection of Chinese rhesus monkeys, and compare the differences and characteristics of Gp120 sequences of enterotropic and neurotropic SIV strains. Methods Six strains of simian immunodeficiency virus were analyzed in this study: four separated from peripheral blood mononuclear cells of SIVmac239-infected monkeys and two neurotropic SIVmac251 strains.Isolated and cultured monoclonal virus was obtained by limiting dilution assay.Gp120 sequences were amplified after the RNA extraction and phylogenetic analysis was processed thereafter.So did the Gp120 amino acid sequence and N-glycosylation sites analysis of the enterotropic and neurotropic strains.Results SIVmac239 had different mutations in four rhesus monkeys.The diversity in amino acid sequences of the enterotropic and neurotropic strains concentrated in the V1 and V4 regions of Gp120.The enterotropic strains had an addition of glycosylation site in V4 but the glycosylation site changes of neurotropic strains were located in the conservative regions of C1, C2 and C3.Conclusions The addition of one glycosylation site in V4 region of GP120 and loss of one glycosylation site in C1 region are associated with enhanced enterotropism and neurotropism.The differences between the enterotropic and neurotropic strains are not dipicted in Gp120 V3 region which is closely related with the tropism of strains.
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Objective To study the effect of repeated rectal exposure of low -dose simian immunodeficiency virus on the systemic cellular immunity in monkeys .Methods Eight 3-to 4-year old rhesus macaques ( Macaca mulatta) (male:female 1:1) were used in this study.The monkeys were inoculated with 10 TCID50 SIVmac239 virus through rectum twice a week for consecutive 6 weeks to establish a multiple rectal exposure model of SIVmac 239 virus infection.Then, plasma viral load, CD4+ T cell count, T cell subsets and IFN-γsecretion of the experiment monkeys were determined . Results Low-dose SIVmac239 virus induced some changes in the immune system through the rectal mucosa , but didn’t induce typical infection.Repeated rectal mucosal low-dose virus exposure can activate the cellular immune system . Conclusions This study defines the effect of repeated low -dose simian immunodeficiency virus exposure on the systemic cellular immunity, and provided basic information for HIV-1 vaccine research.
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The envelope protein(Env) of lentiviruses such as HIV,SIV,FIV and EIAV is larger than that of other retroviruses.The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus,demonstrating that envelope is crucial for vaccine.We compared Env variation of the four kinds of lentiviruses.Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor,and the relationship of HIV and EIAV was the furthest.EIAV had the shortest Env length and the least number of potential N-linked glycosylation sites(PNGS) as well as glycosylation density compared to various immunodeficiency viruses.However,HIV had the longest Env length and the most PNGS.Moreover,the alignment of HIV and SIV showed that PNGS were primarily distributed within extracellular membrane protein gp120 rather than transmembrane gp41.It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env.There are low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.
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Objective To compare the bio-medical parameters in SIV infected Chinese rhesus monkeys with diverse disease progression,by which the pathogenesis of simian AIDS were to be investigated.Methods Sixteen Chinese rhesus monkeys were inoculated intravenously with SIVmac239 and followed-up for 18 months.Based on their progression patterns and plasma viral loads,animals were divided into 3 groups,including 1 rapid progressor( RP),13 normal progressors(NP),and 2 elite controllor(EC).Their parameters of haematology,virology,immunology and pathology were examined and compared. Results Compared with other animals,RM449(RP) showed higher viral load,unresponsive humoral immunity,and higher level of auto-antibodies against lymph node,thymus,and spleen.Additionally,its effector memory CD4 count was lower,with the transformation progress being blocked-like from naive/central memory subsets to effector memory subset,as the flow-cytometry assay showed.Notable decrease in its peripheral B cell was also observed,especially to the sub-population of tissue-like memory B cells and activated memory B cells.Pathological examination showed the depletion of lymphoid tissue,atrophy of spleen and loss of thymus.Moreover,most of these parameters of RM450 and RM453 (EC) changed opposite to that of RP.Conclusion The hallmarks of RM449 were higher viraemia and lower SIV specific IgG level,which may due to the disturbance of T cells and B cells development and differentiation.Moreover,destructions of organs of the immune system may contribute to the disturbance.Our study suggest that the change of micro-environments of thymus induced by SIV infection,which is necessary in T cell and B cell development and differentiation,may contribute at least partially to the AIDS pathogenesis.
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Human immunodeficiency virus (HIV)is a kind of widespread human pathogen in the world. The emergence of HIVs resulted from multiple transmissions and diversification of SIV from nonhuman primates to humans. In this paper, the findings on the transmission of immunodeficiency virus from Chimpanzees to humans and the adaptive evolution in the new host are reviewed, and the significance for science and public health is discussed.
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Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus (SIV) infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR). The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEM×174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA (b-DNA) assays or real-time PCR assays based on TaqMan probes.