RESUMO
Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.
Assuntos
Proteína gp120 do Envelope de HIV/biossíntese , Proteína gp41 do Envelope de HIV/biossíntese , Infecções por HIV/imunologia , Planticorpos/imunologia , Animais , Cloroplastos/imunologia , Epitopos de Linfócito T/imunologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/administração & dosagem , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Imunização , Camundongos , Nicotiana/citologia , Nicotiana/imunologiaRESUMO
HIV-infected PM show restricted replication as compared with MDM. We aimed to determine at what point in the viral replication cycle this restriction occurs in PM as compared with MDM. We performed Alu-LTR PCR for proviral DNA to detect differences in HIV integration, real-time RT-PCR to measure env and gag mRNA levels, and Western blot analysis to detect differences in viral protein expression. PM and MDM were infected with HIV-1 BaL, and DNA was extracted after 24 h and at 6 days p.i. for real-time PCR studies. At 6 and 12 days p.i., cells were lysed for Western blot analyses. We found no difference in viral integration between PM and MDM but significantly lower levels of viral protein gp120 in PM than in MDM. Real-time RT-PCR analyses revealed 24-fold less env mRNA and tenfold less gag mRNA in PM. These results suggest that HIV-1 restriction in PM occurs at the level of transcription. This study is significant, as it advances our understanding of HIV-1 infection in PM and its contribution to decreased in utero vertical transmission.
Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Macrófagos/virologia , Placenta/virologia , Provírus/metabolismo , Transcrição Gênica , Replicação Viral/fisiologia , Células Cultivadas , DNA Viral/genética , DNA Viral/metabolismo , Feminino , Proteína do Núcleo p24 do HIV/biossíntese , Proteína do Núcleo p24 do HIV/genética , Proteína gp120 do Envelope de HIV/biossíntese , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Placenta/metabolismo , Placenta/patologia , Gravidez , Provírus/genética , RNA Viral/biossíntese , Fatores de Tempo , Integração Viral/fisiologiaRESUMO
The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.
Assuntos
Eritropoetina/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Animais , Células COS , Eritropoetina/biossíntese , Expressão Gênica , Vetores Genéticos , Proteína gp120 do Envelope de HIV/biossíntese , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/genética , TransfecçãoRESUMO
The V3 loop of the gp120 envelope glycoprotein contains the principal neutralizing determinant of HIV-1. Many serological studies have been carried out to assess the reactivity of HIV-1 infected individuals against V3 loop synthetic peptides from different HIV-1 subtypes. V3 directed serology has also been used to demonstrate the association between ELISA reactivity and progression to AIDS in HIV patients, and to study the reactivity against the V3 region in sera from vaccinated animals and human volunteers. The advantage of the use of bovine serum albumin (BSA) conjugated V3 peptides over free V3 peptides for ELISA is described. 15 meric V3 peptides representing several HIV-1 isolates were synthesized, chemically coupled to BSA, and used to coat ELISA microplates. Conjugated peptides were compared with free peptides for the detection of anti V3 antibodies in the sera from rabbits immunized with V3 containing chimeric proteins and from HIV-1 infected individuals. No differences in reactivity against free or BSA-peptide were found for most rabbit sera, however human plasma recognized preferentially the BSA conjugated peptides. Although technically more complex, ELISA with BSA coupled V3 peptides is more sensitive and appropriate for serological studies of HIV-1 infected persons.