RESUMO
Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Terapia Genética/métodos , Infecções por HIV/prevenção & controle , HIV-1 , Imunização/métodos , Vacinas de DNA/administração & dosagem , Adenoviridae/genética , Animais , Anticorpos Antivirais/sangue , DNA Viral/administração & dosagem , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , HIV-1/genética , HIV-1/imunologia , Imunização Secundária , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Testes de Neutralização , Vaccinia virus/fisiologia , Proteínas Virais/genética , Fenômenos Fisiológicos ViraisRESUMO
Apoptosis-inducing caspases have been tested for immunomodulatory effect on a gene gun-delivered DNA vaccine which expresses influenza hemagglutinin. Attenuated murine caspase 2 and a chimera of murine caspase 2 prodomain and human caspase 3 strongly enhanced humoral and cell-mediated immune response to hemagglutinin when they were co-administered with an immunogen DNA. In contrast, wild-type caspases did not enhance the DNA-raised immune response. Caspase dose-dependent antibody response curve revealed that the antibody level was in inverse relation to the amount of administered caspase. These findings indicate that bland apoptosis of antigen-harboring cells can elicit enhanced immune responses. Extensive apoptosis interferes with the generation of immune response. Gene gun delivery involving caspases elicited type-2 immune responses that characterized with dominant IL-4 and IgG1 production. ELISPOT assays showed that CD4 T cells were preferentially activated, while CD8 T cell response remained at marginal level. Using attenuated caspases for gene gun DNA vaccination is a useful approach to amplify type-2 immune responses.
Assuntos
Apoptose , Biolística , Caspases/genética , Vacinas contra Influenza/administração & dosagem , Vacinas de DNA/administração & dosagem , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Hemaglutininas/genética , Imunoglobulina G/imunologia , Vacinas contra Influenza/imunologia , Interleucina-4/imunologia , Ativação Linfocitária , CamundongosRESUMO
A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.
Assuntos
Feto/imunologia , Prenhez/imunologia , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Feminino , Transferência Genética Horizontal , Imunidade Materno-Adquirida , Vacinas contra Influenza/imunologia , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , Gravidez , Vacinação , Vacinas de DNA/imunologiaRESUMO
The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Produtos do Gene rev/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Anticorpos Anti-HIV/biossíntese , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp160 do Envelope de HIV/administração & dosagem , HIV-1/imunologia , Interleucina-12/genética , Fragmentos de Peptídeos/administração & dosagem , Vacinas contra a AIDS/imunologia , Administração Cutânea , Animais , Biomarcadores , Biópsia , Dermabrasão , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Produtos do Gene rev/genética , Produtos do Gene rev/imunologia , Antígenos HIV/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/genética , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Células de Langerhans/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas S100/análise , Pele/imunologia , Pele/patologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
We studied the use of a DNA vaccine expressing the matrix (M) gene of the influenza virus A/PR/8/34. Mice were immunized by painting the DNA vaccine three times on the skin after removal of its keratinocytic layers. Immunization by this method produced M-specific antibodies and cytotoxic T lymphocyte (CTL) response, and acquired resistance against influenza virus challenge. This protection was abrogated by the in vivo injection of anti-CD8 or anti-CD4 monoclonal antibody. We further found that simultaneous topical application (t.a.) of GM-CSF expression plasmid (pGM-CSF) or liposomes plus mannan produced stronger immune response competence and enhanced the protective effect against influenza virus challenge. The present study revealed that administering DNA vaccine by topical application can elicit both humoral and cell-mediated immunity (CMI).
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/uso terapêutico , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas de DNA/uso terapêutico , Administração Cutânea , Animais , Anticorpos Antivirais/biossíntese , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Edema/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pele/patologia , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologiaRESUMO
We constructed a recombinant replication defective adenovirus vector containing the env gene (Ad-Bal) derived from macrophage-trophic HIV-1 (HIV-1 Bal). We then immunized mice with this vector using several administration routes and protocols, and examined the immune response. When the Ad-Bal viral vector (over 1 x 10(7) pfu) was injected subcutaneously, both humoral and cell-mediated immunities were induced. However, immune response induced by the Ad-Bal vector alone was weaker than that induced by the recombinant vaccinia viral vector. We then employed the following three immunization protocols: (l) DNA vaccination followed by immunization with the Ad-Bal; (2) vaccination using the Ad-Bal vector followed by DNA vaccination; and (3) DNA vaccination followed by Ad-Bal infection and passive transfer of dendritic cells (DCs) infected with the Ad-Bal. Among the three protocols, the last gave the strongest humoral and cell-mediated immunity. These results suggest that the combination of DNA vaccination, Ad-Bal vector infection and passive transfer of Ad-Bal-infected DCs can induce strong immunity against HIV-1 Bal.
Assuntos
Vacinas contra a AIDS/imunologia , Adenovírus Humanos , Vetores Genéticos , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Células Dendríticas/imunologia , Feminino , Anticorpos Anti-HIV/biossíntese , Humanos , Hipersensibilidade Tardia/imunologia , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linfócitos T Citotóxicos/imunologia , Vacinação/métodos , Replicação ViralRESUMO
Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.
Assuntos
Dependovirus/imunologia , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Vacinas Virais/imunologia , Vacinas contra a AIDS/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Citocinas/biossíntese , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Produtos do Gene rev/imunologia , Produtos do Gene tat/imunologia , Genes env/genética , Genes tat/genética , Anticorpos Anti-HIV/sangue , HIV-1/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Soros Imunes/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Vírus da Influenza A/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Vacinas Sintéticas/imunologia , Vacinas Virais/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.
Assuntos
Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Plasmídeos/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Viral/administração & dosagem , DNA Viral/imunologia , Cães , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Infecções por Orthomyxoviridae/mortalidade , Plasmídeos/administração & dosagem , Plasmídeos/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Proteínas da Matriz Viral/administração & dosagem , Proteínas da Matriz Viral/biossínteseRESUMO
We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.
Assuntos
DNA Viral , HIV-1/genética , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Citocinas/genética , Expressão Gênica , Imunização , Plasmídeos , Células Th1/imunologia , Células Th2/imunologiaRESUMO
From a series of preclinical studies and animal experiments, we have been able to demonstrate that DNA vaccines are a promising tool in strategies for protecting hosts from a variety of infectious diseases. Since the promoter activity of the human cytomegalovirus immediate-early promoter/ enhancer (CMV promoter) is known to be responsive to an elevation in the level of intracellular cAMP, we hypothesized that use of cAMP analogue (8-Bromo adenosine 3'5'-cyclic monophosphate, 8 Br-cAMP) would increase the level of transgene expression supported by the CMV, and enhance the ability of DNA vaccines to evoke an immune response against the transgene product in vivo. To evaluate this hypothesis, immune responses against HIV-1 envelope protein, gp160, an immunogenic HIV-1 component expressed under the control of the CMV promoter, were evaluated in BALB/c mice with or without stimulation by 8 Br-cAMP. DNA vaccine with 8 Br-cAMP was intramuscularly (i.m.) or intranasally (i.n.) administered to BALB/c mice twice on days 0 and 14. Regardless of which route was used, the combination increased the serum IgG antibody (Ab) titer, HIV-1-specific cytotoxic T lymphocyte (CTL) activity and the delayed-type hypersensitivity (DTH) response, compared with the effect of using the vaccine alone. When administered via the i.n. route, the combination also remarkably increased the titer of secretory IgA (sIgA). Moreover, it induced increased production of interferon-gamma with reduction in IL-4 synthesis, and decreased the ratio of serum IgG1/IgG2a. However, these enhancements were not observed when 8 Br-cAMP was coadministered with peptide vaccine or protein antigen. These data suggest that 8 Br-cAMP is able to enhance both humoral and cellular immune responses induced by the DNA vaccine. The induction of T helper type 1 (Th1) immunity against HIV-1 was also enhanced by coadministration of 8 Br-cAMP. A CAT assay study demonstrated that the adjuvant effect of 8 Br-cAMP may be due to the activation of the CMV promoter in the DNA vaccine. The virus challenge experiment in a mouse influenza model also proved our hypothesis.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/uso terapêutico , Terapia Genética/métodos , Proteína gp160 do Envelope de HIV/genética , Hipersensibilidade Tardia/tratamento farmacológico , Linfócitos T Citotóxicos/efeitos dos fármacos , Vacinas de DNA/uso terapêutico , Administração Intranasal , Animais , Terapia Combinada , Citomegalovirus/genética , Relação Dose-Resposta a Droga , Vetores Genéticos/administração & dosagem , Hipersensibilidade Tardia/imunologia , Imunoglobulina G/análise , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Regiões Promotoras Genéticas , Linfócitos T Citotóxicos/imunologiaRESUMO
The mechanism of immune activation induced by a plasmid-encoding GM-CSF (pGM-CSF), administered in combination with a DNA vaccine encoding the envelope of HIV, was studied. Injecting pGM-CSF i.m. into mice 3 days before DNA vaccination primarily induced a Th2 response. Simultaneous administration of the DNA vaccine plus pGM-CSF activated both a Th1 and a Th2 response. When the plasmid was injected 3 days after DNA vaccination, enhancement of Th1 immunity predominated. These results suggest that the timing of cytokine expression determines the phenotype of the resultant Th response. After 3 days of pGM-CSF injection, the increased percentages of CD11c+, CD8+ cells were observed in the regional lymph nodes. In addition, many infiltrated cells, including S-100 protein-positive cells, were found in the pGM-CSF-injected tissue. The importance of these S-100+ cells or both CD8+ and CD11c+ cells, especially that of dendritic cells (DCs), was also studied. DCs derived from bone marrow and cultured in RPMI 1640 medium containing IL-4 and GM-CSF were incubated with DNA vaccine and then transferred into naive mice. Mice receiving DCs showed strong HIV-1-specific Th2 immune responses. Our results suggest that DCs play important roles in the activation or modification of the Th2-type immune response induced by DNA vaccination.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , HIV-1/imunologia , Plasmídeos/administração & dosagem , Células Th1/imunologia , Células Th2/imunologia , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Movimento Celular/imunologia , Células Cultivadas , Citocinas/administração & dosagem , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/transplante , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/genética , Esquemas de Imunização , Injeções Intramusculares , Interleucina-4/administração & dosagem , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos/imunologia , Células Th1/metabolismo , Células Th1/virologia , Células Th2/metabolismo , Células Th2/virologia , Vacinas de DNA/imunologiaAssuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Ativação Linfocitária , Linfócitos T/transplante , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Células Cultivadas , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , HIV-1/genética , Imunoglobulina A Secretora/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia , Vacinação , Vacinas de DNA/administração & dosagemRESUMO
An effective vaccine for human immunodeficiency virus (HIV) is needed to stimulate the immune response of the genital mucus to prevent mucosal transmission of the virus. We have developed a macromolecular multicomponent peptide vaccine candidate, VC1. Both rectal and vaginal immunization of VC1 mixed with cholera toxin (CT) induced HIV-1-specific IgA antibody in mouse fecal extract solution and vaginal wash. These antibody productions were enhanced by the combination with IL-4 or GM-CSF expressing plasmids. Either fecal extract or vaginal wash solution from immunized mice inhibited production of HIV-1IIIB p24 protein. The mononuclear cells from spleen, intestinal lymph nodes, or Peyer's patches from VC1- and CT-immunized mice released IFN-gamma or IL-4, when these cells were co-cultured with VC1 antigen. In addition, the regional lymphoid cells from rectal and vaginal region of mice immunized with VC1 and CT also elicited a substantial level of HIV-1-specific cytotoxic T cell (CTL) response. This CTL response was enhanced by the addition of IL-12 expressing plasmid. Our results clearly demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunities specific for HIV-1.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Peptídeos/imunologia , Reto/imunologia , Vacinas Sintéticas/imunologia , Vagina/imunologia , Vacinas contra a AIDS/administração & dosagem , Administração Intravaginal , Administração Retal , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Biopolímeros/imunologia , Feminino , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Humanos , Imunidade nas Mucosas/imunologia , Imunoglobulina A Secretora/biossíntese , Imunoglobulina A Secretora/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.
Assuntos
Baculoviridae/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1 , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Proteínas Recombinantes/metabolismo , Spodoptera/virologiaRESUMO
In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.
Assuntos
Eritrócitos/enzimologia , Predisposição Genética para Doença/genética , Genótipo , Intoxicação por Chumbo/genética , Doenças Profissionais/genética , Sintase do Porfobilinogênio/genética , Adulto , Alelos , Humanos , Intoxicação por Chumbo/enzimologia , Masculino , Doenças Profissionais/induzido quimicamente , Doenças Profissionais/enzimologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Fatores de Risco , Análise de Sequência de DNARESUMO
Cytokines play important roles in regulating immune response. This study evaluated the adjuvant effect of an expression plasmid encoding RANTES (regulated on activation normal T-cell expressed and secreted) chemokine on the immunity induced by a DNA vaccine. This vaccine consists of expression plasmids encoding the env and rev genes of human immunodeficiency virus type 1 (HIV-1). DNA vaccination with RANTES plasmid induced significantly higher titers of serum HIV-1-specific IgG and IgG2a antibodies than DNA vaccination alone on both intramuscular and intranasal immunization. This combination also increased HIV-1-specific cytotoxic T lymphocyte activity and delayed-type hypersensitivity. Intranasal immunization induced a higher titer of fecal secretory IgA antibody than intramuscular immunization. These results demonstrate that coadministration of RANTES plasmid dominantly induced HIV-1-specific cell-mediated immunity.
Assuntos
Quimiocina CCL5/imunologia , HIV-1/imunologia , Vacinas de DNA/química , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Especificidade de Anticorpos , Feminino , Histiócitos/química , Histiócitos/citologia , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Hipersensibilidade Tardia/virologia , Imunidade Celular/imunologia , Linfócitos/química , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/citologiaRESUMO
HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.
Assuntos
Encéfalo/virologia , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Fragmentos de Peptídeos/genética , Adulto , Encéfalo/patologia , Evolução Molecular , Feminino , Genes Virais , Infecções por HIV/patologia , HIV-1/classificação , Humanos , Masculino , Filogenia , Análise de Sequência de DNARESUMO
Cytokines are powerful regulators of the immune response. In this study, an HIV-1 envelope DNA vaccine and interleukin 15 (IL-15) expression plasmid were intranasally administered to mice. A significant increase in the HIV-1-specific DTH response and CTL activity, and decrease in the serum IgG/IgG2a ratio was observed in the group which received DNA vaccine and IL-15 expression plasmid compared to DNA vaccination alone. Restimulated immune lymphoid cells from mice which received both agents showed enhanced production of interferon-gamma (IFN-gamma) and reduced secretion of IL-4. However, administration of DNA vaccine with IL-15 and IL-2 or IL-12 expression plasmids did not alter the effect of IL-15 expression plasmid on the DNA vaccine. These results indicate that intranasal administration of DNA vaccine and IL-15 expression plasmid is capable of enhancing the T helper type 1 (Th1) dependent HIV-1-specific cell-mediated immunity, and that the IL-15 and IL-2 or IL-12 expression plasmids may not have a synergistic effect on the immune response induced by DNA vaccine in vivo.
Assuntos
Adjuvantes Imunológicos/genética , DNA Viral/imunologia , HIV-1/genética , HIV-1/imunologia , Interleucina-15/genética , Plasmídeos/imunologia , Vacinas de DNA/imunologia , Administração Intranasal , Sequência de Aminoácidos , Animais , Citocinas/biossíntese , Sinergismo Farmacológico , Fezes/química , Feminino , Anticorpos Anti-HIV/biossíntese , Hipersensibilidade Tardia/imunologia , Imunidade Celular/genética , Imunidade Celular/imunologia , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Injeções Intramusculares , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-15/biossíntese , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/farmacologiaRESUMO
CD8+ cell-secreted CC-chemokines, MIP-1alpha, and MIP-beta have recently been identified as factors which suppress HIV. In this study we co-inoculated MIP-1alpha expression plasmid with a DNA vaccine constructed from HIV-1 pCMV160IIIB and pcREV, and evaluated the effect of the adjuvant on HIV-specific immune responses following intramuscular and intranasal immunization. The levels of both cytotoxic T lymphocyte (CTL) activity and DTH showed that HIV-specific cell-mediated immunity (CMI) was significantly enhanced by co-inoculation of the MIP-1alpha expression plasmid with the DNA vaccine compared with inoculation of the DNA vaccine alone. The HIV-specific serum IgG1/IgG2a ratio was significantly lowered when the plasmid was co-inoculated in both intramuscular and intranasal routes, suggesting a strong elicitation of the T helper (Th) 1-type response. When the MIP-1alpha expression plasmid was inoculated intramuscularly with the DNA vaccine, an infiltration of mononuclear cells was observed at the injection site. After intranasal administration, the level of mucosal secretory IgA antibody was markedly enhanced. These findings demonstrate that MIP-1alpha expression plasmid inoculated together with DNA vaccine acts as a strong adjuvant for eliciting Th1-derived immunity.
Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos , HIV-1/imunologia , Proteínas Inflamatórias de Macrófagos/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/farmacologia , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Produtos do Gene rev/genética , Produtos do Gene rev/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Imunidade Celular , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos , Vacinas de DNA/farmacologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
DNA vaccine against human immunodeficiency virus type-1 (HIV-1) can induce substantial levels of HIV-1-specific humoral and cell-mediated immunity. To develop more potent HIV-1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin-2 (IL-2) expression plasmid on an HIV-1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV-1-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL-2 and interferon-gamma and reduced secretion of IL-4. The level of total antibody to HIV-1 antigen was not greatly changed by coadministration of the DNA vaccine and IL-2 expression plasmid. An analysis of serum HIV-1-specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid-vaccine combination. These results demonstrate that the IL-2 expression plasmid strongly enhances the HIV-1-specific immune response via activation of T helper type-1 cells.