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1.
S. Afr. med. j. (Online) ; 109(8): 25-29, 2019. ilus
Article in English | AIM | ID: biblio-1271225

ABSTRACT

Cellular therapy has become a billion-dollar industry and is set to become one of the therapeutic pillars of healthcare in the 21st century. Adult stem cells, which include haematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal/stem cells (MSCs), is one of the major cell types currently under investigation for use in cell therapy. This review focuses on HSPCs and MSCs and discusses their heterogeneous nature and the problems faced in expanding these cells to therapeutic numbers for use in clinical applications


Subject(s)
Cells , Gene Products, gag , Genetic Heterogeneity , Therapeutics
2.
Protein & Cell ; (12): 640-651, 2018.
Article in English | WPRIM | ID: wpr-756931

ABSTRACT

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.


Subject(s)
Humans , Autophagy , Cell Membrane , Metabolism , Gene Products, gag , Genetics , Metabolism , HEK293 Cells , HIV-1 , Metabolism , Lysosomes , Metabolism , MicroRNAs , Genetics , Metabolism , Virus Assembly
3.
Chinese Journal of Virology ; (6): 170-178, 2016.
Article in Chinese | WPRIM | ID: wpr-296201

ABSTRACT

Therapeutic HIV vaccine was considered as a hopeful curative method for AIDS patients. However, there is still no suitable HIV animal model for vaccine study since the difference in the immune system between human and animals. To evaluate the therapeutic effect of combined immunization strategy with multiple vector vaccines in macaque models. Plasmid DNA, recombinant Ad5 and MVA vaccines which expressing SIV gag and env genes were constructed. Sequential and repeated immune strategy were applied to immunize mice with these three vaccines. Cellular immune responses in mice immunized with these three vaccines were measured by ELISPOT test in vitro and CTL assay in vivo. The results were analyzed and compared with different antigen combination, order of vaccines and intervals to choose a suitable immunization strategy for macaque immunization in future. It indicated that strong SIV-Gag/Env-specific cellular immune responses were induced by these three vector vaccines. It laid a foundation for evaluating the therapeutic effect of combined immunization strategy with multiple vector vaccines in SIV infected macaque models.


Subject(s)
Animals , Female , Humans , Mice , AIDS Vaccines , Genetics , Allergy and Immunology , Adenoviridae , Genetics , Metabolism , Antibodies, Viral , Allergy and Immunology , Gene Products, env , Genetics , Allergy and Immunology , Gene Products, gag , Genetics , Allergy and Immunology , Genetic Vectors , Genetics , Metabolism , HIV Infections , Allergy and Immunology , Virology , Immunization , Mice, Inbred BALB C , Simian Immunodeficiency Virus , Genetics , Allergy and Immunology , Vaccines, DNA , Genetics , Allergy and Immunology
4.
J. vet. sci ; J. vet. sci;: 91-97, 2014.
Article in English | WPRIM | ID: wpr-56429

ABSTRACT

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.


Subject(s)
Animals , Cats , Female , Antibodies, Monoclonal/blood , Diagnostic Tests, Routine/veterinary , Gene Products, gag/blood , Leukemia Virus, Feline/immunology , Leukemia, Feline/diagnosis , Mice, Inbred BALB C , Sensitivity and Specificity
5.
Mem. Inst. Oswaldo Cruz ; 108(6): 730-734, set. 2013. tab, graf
Article in English | LILACS | ID: lil-685488

ABSTRACT

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.


Subject(s)
Humans , Antibodies, Viral , Blotting, Western/standards , Central Nervous System/immunology , Human T-lymphotropic virus 1/immunology , Paraparesis, Tropical Spastic/immunology , Antibodies, Viral/blood , Antibodies, Viral/cerebrospinal fluid , Central Nervous System/metabolism , Enzyme-Linked Immunosorbent Assay , Gene Products, env/immunology , Gene Products, gag/immunology , Immunoglobulin G/blood , Immunoglobulin G/cerebrospinal fluid , Paraparesis, Tropical Spastic/blood , Paraparesis, Tropical Spastic/cerebrospinal fluid , Sensitivity and Specificity
6.
Article in Chinese | WPRIM | ID: wpr-246240

ABSTRACT

<p><b>OBJECTIVE</b>To characterize the Gag-Specific T lymphocyte responses and identify immunodominant region recognized in Chinese HIV-1 recombinant subtype B/C infectors.</p><p><b>METHODS</b>10 antiretroviral treatment (ART) naive HIV-1 recombinant subtype B/C infectors with infected time in 1 year, 25 ART-naive infectors with infected time > 3 years and 10 HIV-1-seronegative healthy individuals were enrolled. HIV-1-specific T lymphocyte responses were analyzed by an IFN-gamma Elispot assay against 123 overlapping peptides spanning HIV-1 Gag protein in the present study.</p><p><b>RESULTS</b>Gag-specific T lymphocyte responses of interferon-gamma secretion were identified in 8(8/10) Chinese HIV-1 recombinant subtype B/ C infectors with infected time in 1 year, the specific T lymphocytes are mainly targeted at five seperated peptides. Responses were identified in 17(68%) infectors with infected time more than 3 years, the specific T lymphocytes are mainly targeted at one peptide in p17 and six in p24. There was obviously positive correlation (P = 0.0318, r = 0.519) between the magnitude of responses and viremia in infectors infected time > 3 years. The magnitude of response in infectors infected in 1 year was significantly higher than group infected time > 3 years (P = 0.021). None of healthy individuals produced positive responses.</p><p><b>CONCLUSIONS</b>HIV-1 recombinant subtype B/C Infectors at different stages of diseases recognize different region of gag.</p>


Subject(s)
Humans , CD4-Positive T-Lymphocytes , Allergy and Immunology , Virology , Gene Products, gag , Genetics , Allergy and Immunology , HIV Infections , Genetics , Allergy and Immunology , Virology , HIV-1 , Classification , Genetics , Allergy and Immunology , Immunodominant Epitopes , Interferon-gamma , Genetics , Allergy and Immunology , Recombination, Genetic
7.
Chinese Journal of Virology ; (6): 339-343, 2009.
Article in Chinese | WPRIM | ID: wpr-297951

ABSTRACT

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.


Subject(s)
Humans , Gene Products, gag , Genetics , Gene Products, pol , Genetics , Human T-lymphotropic virus 1 , Genetics , Molecular Probes , Polymerase Chain Reaction , Methods , Viral Proteins , Genetics
8.
Chinese Journal of Virology ; (6): 190-195, 2008.
Article in Chinese | WPRIM | ID: wpr-334825

ABSTRACT

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.


Subject(s)
Animals , Female , Humans , Mice , Adenoviridae , Genetics , Blotting, Western , Codon , Genetics , Gene Products, gag , Genetics , Allergy and Immunology , Metabolism , HIV-1 , Genetics , Allergy and Immunology , Metabolism , Immunization , Methods , Mice, Inbred BALB C , Mutation , Recombinant Proteins , Allergy and Immunology , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Cytotoxic , Allergy and Immunology
9.
Article in Chinese | WPRIM | ID: wpr-305513

ABSTRACT

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


Subject(s)
Animals , Female , Mice , AIDS Vaccines , Genetics , Allergy and Immunology , Adenoviridae , Genetics , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Gene Products, gag , Genetics , Allergy and Immunology , Metabolism , Genetic Vectors , Genetics , HIV Antibodies , Blood , HIV-1 , Genetics , Allergy and Immunology , Immunization , Methods , Immunoglobulin G , Blood , Mice, Inbred BALB C , Random Allocation , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism
10.
Chin. med. j ; Chin. med. j;(24): 496-502, 2007.
Article in English | WPRIM | ID: wpr-344867

ABSTRACT

<p><b>BACKGROUND</b>Although 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.</p><p><b>METHODS</b>Vector 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.</p><p><b>RESULTS</b>It 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.</p><p><b>CONCLUSIONS</b>The 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.</p>


Subject(s)
Animals , Female , Mice , AIDS Vaccines , Allergy and Immunology , Amino Acid Sequence , Blotting, Western , CD8-Positive T-Lymphocytes , Allergy and Immunology , Enhancer Elements, Genetic , Gene Products, gag , Allergy and Immunology , HIV Antibodies , Blood , Immunoglobulin G , Blood , Mice, Inbred BALB C , Molecular Sequence Data , Plasmids , Simian virus 40 , Genetics , Vaccination , Vaccines, DNA , Allergy and Immunology , Vaccinia , Allergy and Immunology
11.
Article in Korean | WPRIM | ID: wpr-61962

ABSTRACT

Xenotransplantation, as a potential solution to the shortage of human organs, is associated with a number of concerns including immunologic rejection and xenogenic infection. While the pigs are considered the most suitable organ source for xenotransplantation, there is a potential public health risk due to zoonosis. Among the known porcine zoonotic microbes, Porcine Endogenous Retrovirus (PERV) is the most considerable virus. PERV belongs to the Gammaretrovirus and has been divided into three groups (A, B, and C). To characterize the gag of PERVs, we isolated the genomic DNAs from three pig breeds (Birkshire, Duroc, and Yorkshire) and two types of SPF miniature pigs. About 1.5 kb fragments covering full length of gag were amplified and cloned into T-vector. A total of 38 clones were obtained and sequenced. Nucleotide sequences were analyzed and phylogenetic trees were constructed from the nucleotide and deduced amino acids. PERV-A, -B and -C were present in the proportion of 47, 19 and 34%, respectively. Regardless of origin or subgroups, gag clones showed highly homology in nucleotide and deduced amino acid sequences. Deduced amino acids sequence alignments showed typical conserve sequences, Cys-His box and processing sites. Among analyzed clones, about 28% of isolates had the correct open reading frame. To test the functional expression of Gag protein, gag was subcloned into expression vector and confirmed its expression in HeLa cell. This research provides the fundamental information about molecular characteristics of gag gene and functional Gag protein related xenotropic PERVs.


Subject(s)
Humans , Amino Acid Sequence , Amino Acids , Base Sequence , Clone Cells , DNA , Endogenous Retroviruses , Gammaretrovirus , Gene Products, gag , Genes, gag , HeLa Cells , Korea , Open Reading Frames , Public Health , Sequence Alignment , Swine , Transplantation, Heterologous
12.
Infection and Chemotherapy ; : 374-382, 2006.
Article in Korean | WPRIM | ID: wpr-721398

ABSTRACT

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.


Subject(s)
Humans , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , Enzyme-Linked Immunospot Assay , Gene Products, gag , Haplotypes , HIV , HIV Infections , HIV-1 , Immunity, Cellular , Interferon-gamma , RNA , T-Lymphocytes, Cytotoxic
13.
Infection and Chemotherapy ; : 374-382, 2006.
Article in Korean | WPRIM | ID: wpr-721903

ABSTRACT

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.


Subject(s)
Humans , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , Enzyme-Linked Immunospot Assay , Gene Products, gag , Haplotypes , HIV , HIV Infections , HIV-1 , Immunity, Cellular , Interferon-gamma , RNA , T-Lymphocytes, Cytotoxic
14.
Zhonghua Bing Li Xue Za Zhi ; (12): 220-223, 2005.
Article in Chinese | WPRIM | ID: wpr-265145

ABSTRACT

<p><b>OBJECTIVE</b>To assess the infectivity of porcine endogenous retrovirus (PERV) via in vitro infection of human embryonic kidney cell line HEK-293.</p><p><b>METHODS</b>PERV particles were detected by immunoelectron microscopy. PERV DNA and mRNA were studied in HEK-293 24 hours after the infection using polymerase chain reaction and reverse transcriptase-PCR respectively. The PERV types were also analyzed. PERV-gag protein was observed by confocal microscopy.</p><p><b>RESULTS</b>Retroviral particles were round under electron microscope. PERV-gag pol gene and gag protein were detected and expressed in the infected HEK-293 cells. The types of PERV were PERV-A and PERV-B. PERV-gag protein was also identified in the cytoplasm of infected cells by confocal microscopy.</p><p><b>CONCLUSIONS</b>PERV is able to infect HEK-293 cell line in vitro; types of PERV-gag protein is also expressed as a result. Further studies are thus necessary in order to evaluate the possibility of xenozoonoses in pig-to-human xenotransplantation.</p>


Subject(s)
Animals , Humans , Cell Line , DNA, Viral , Embryo, Mammalian , Endogenous Retroviruses , Virulence , Gene Amplification , Gene Products, gag , Genetics , Genes, gag , Kidney , Metabolism , Virology , RNA, Messenger , Genetics , Swine
15.
Article in Chinese | WPRIM | ID: wpr-333025

ABSTRACT

<p><b>OBJECTIVE</b>To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>There 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.</p><p><b>CONCLUSION</b>The 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.</p>


Subject(s)
Animals , Chick Embryo , Mice , Antibodies, Viral , Blood , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Cell Biology , Metabolism , Fowlpox , Blood , Allergy and Immunology , Virology , Fowlpox virus , Genetics , Allergy and 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, Inbred BALB C , Microscopy, Electron , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Transfection , Viral Vaccines , Genetics , Allergy and Immunology , Metabolism
16.
Rev. argent. microbiol ; Rev. argent. microbiol;36(4): 151-157, Oct.-Dec. 2004. graf, tab
Article in English | LILACS | ID: lil-634474

ABSTRACT

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.


El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.


Subject(s)
Humans , Antigen-Antibody Reactions , Enzyme-Linked Immunosorbent Assay/methods , Gene Products, gag/immunology , HIV Antibodies/immunology , HIV Antigens/immunology , /immunology , HIV-1 , Molecular Mimicry , Peptide Fragments/immunology , Viral Proteins/immunology , Amino Acid Sequence , Amino Acid Substitution , Circular Dichroism , gag Gene Products, Human Immunodeficiency Virus , Gene Products, gag/chemistry , HIV Antibodies/isolation & purification , HIV Antigens/chemistry , /chemistry , HIV Infections/blood , HIV Infections/immunology , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/immunology , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Peptide Fragments/chemical synthesis , Solutions , Viral Proteins/chemistry
17.
Article in Chinese | WPRIM | ID: wpr-279562

ABSTRACT

<p><b>OBJECTIVE</b>HIV-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.</p><p><b>METHODS</b>HIV-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.</p><p><b>RESULTS</b>pCI-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.</p><p><b>CONCLUSION</b>HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.</p>


Subject(s)
Animals , Mice , AIDS Vaccines , Allergy and Immunology , Antibodies, Viral , Blood , Cell Line, Tumor , Dependovirus , Genetics , Gene Products, gag , Genetics , Metabolism , HIV-1 , Genetics , Allergy and Immunology , L-Lactate Dehydrogenase , Blood , Mastocytoma , Metabolism , Pathology , Mice, Inbred BALB C , Recombination, Genetic , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Transfection , Vaccines, DNA , Allergy and Immunology
18.
Chinese Journal of Pediatrics ; (12): 586-589, 2003.
Article in Chinese | WPRIM | ID: wpr-276954

ABSTRACT

<p><b>OBJECTIVE</b>To estimate prevalence of HIV/AIDS among children and the transmission routes in a highly endemic villages of AIDS.</p><p><b>METHODS</b>Totally 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.</p><p><b>RESULTS</b>Thirty-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.</p><p><b>CONCLUSION</b>Mother-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.</p>


Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Acquired Immunodeficiency Syndrome , Diagnosis , Epidemiology , Antibodies, Viral , Blood , China , Epidemiology , Gene Products, gag , Genetics , HIV Antigens , Genetics , HIV Infections , Diagnosis , Epidemiology , HIV-1 , Genetics , Allergy and Immunology , Infectious Disease Transmission, Vertical , Polymerase Chain Reaction , Prevalence , Viral Proteins , gag Gene Products, Human Immunodeficiency Virus
19.
Arq. bras. endocrinol. metab ; Arq. bras. endocrinol. metab;44(5): 417-24, out. 2000. ilus, tab
Article in Portuguese | LILACS | ID: lil-276100

ABSTRACT

As doenças auto-imunes da tireóide säo os resultados da quebra da auto-tolerância causada principalmente por fatores genéticos e ambientais. O retrovírus HTLV-1 tem sido implicado como um importante fator ambiental desencadeador destas doenças. Objetivando encontrar evidências da participaçäo deste retrovírus, 47 tecidos tireoidianos parafinados de 45 pacientes com doença de Gravese dois com tireoidite de Hashimoto, juntamente com tecidos tireoidianos normais de seis tireóidesobtidos após necropsia e linfonodo de pacientes com infecçäo pelo HTLV-1, utilizados como controles negativo e positivo, respectivamente, foram estudados,. Com a intençäo de detectar as proteínas p19 (gag) E GP21 (env) do HTLV-1, os tecidos foram submetidos a imuno-histoquímica, utilizando-se um novo método de recuperaçäo antigênica e anticorpos monoclonais anti-19 e anti-gp21. Como resultado obtivemos a mediana do percentual de positividade no sexo feminino de 30 por cento e 41 por cento no masculino de 27 por cento e 52 por cento e total de 30 por cento e 41 por cento, para as proteínas p19 e gp21, respectivamente. Nos tecidos tireoidianos normais, cinco foram positivos para as duas proteínas e apresentaram mediana do percentual de positividade de 17 por cento para a p19 e 39 por cento para a gp21. A detecçäo das proteínas p19 e gp21 no tecido tireoidiano de pacientes com doenças auto-imune da tireóide e em tecidos normais talvez indique a expressäo genética de sequências do HTLV-1 integradas ao genoma dos indivíduos estudados, ou talvez possa representar mimetismo molecular, ou reaçäo cruzada, causada pela presença de antígenos comuns entre as proteínas P19 e gp21 do HTLV-1 e as células epiteliais foliculares da tireóide.


Subject(s)
Humans , Male , Female , Gene Products, env/adverse effects , Gene Products, gag/adverse effects , HTLV-I Infections/etiology , Thyroiditis, Autoimmune/complications , Graves Disease/complications , Immunohistochemistry
20.
Rev. chil. pediatr ; 71(2): 84-8, mar.-abr. 2000. ilus
Article in Spanish | LILACS | ID: lil-268224

ABSTRACT

El virus de inmunodeficiencia humana (VIH), causante del síndrome de inmunodeficiencia adquirida (SIDA), ha constituido desde su emergencia hace 2 décadas, un enorme desafío a la investigación biomédica. Utilizando una amplia gama de recursos para interferir y evadir la respuesta inmune normal, infecta las células CD4+, ingresa a ellas a través de sus receptores de superficie, y expresa una alta frecuencia de mutación lo que le permite cambiar repetidamente sus determinantes antígenos. Los VIH tipo 1 y 2 corresponden a lentivirus, los cuales, junto a los oncornavirus y espumavirus integran la familia de retrovirus RNA humanos. En este artíulo se revisan los conocimientos actuales de la biología molecular del virus, se comentan modernas técnicas de diagnóstico como la reacción de transcriptasa reversa y polimerasa en cadena, usadas actualmente para medir la replicación del virus en la sangre del huésped infectado, y se discuten las actuales líneas de tratamiento exploradas, las que básicamente se dirigen a blancos moleculares susceptibles de intervención farmacológica que no afecten la funcionalidad celular, como son la interacción virus-receptor celular, , transcripción reversa del RNA viral, integración del provirus, procesamiento proteolítico del precursor gag-pol, y la regulación transcripcional virus específica por los productos tat y rev. Finalmente se comentan aspectos relacionados a la investigación en el campo de la inmunización VIH, desafío pendiente para la primera década de este nuevo siglo


Subject(s)
Humans , HIV/genetics , Acquired Immunodeficiency Syndrome/virology , Anti-HIV Agents/pharmacology , Gene Products, gag/genetics , HIV Protease Inhibitors/therapeutic use , HIV-1/genetics , HIV/drug effects , HIV/pathogenicity , Proviruses/genetics , Reverse Transcriptase Inhibitors/pharmacology , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/drug therapy , Virus Replication
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