RESUMO
We have evaluated the homologous and heterologous neutralizing antibody response in a cohort of six Macaca nemestrina infected with the cloned virus SIVsm62d that showed different levels of envelope diversification. Two progressor macaques developed AIDS by 1.5 years post-inoculation and four non-progressors were asymptomatic for 3 years of follow-up. All macaques developed high titers of neutralizing antibodies against homologous SIVsm viruses and intermediate titers against SIVsmB670. Heterologous virus neutralization of SIVmac, SIVmne, and HIV-2 was detected at much lower levels in both progressor macaques; only one of four non-progressors had evidence for broader neutralizing antibody activity. We noted changes in potential N-linked glycosylation (PNG) sites in V1/V2, C2, and V4 that were common to multiple macaques. These results support a model for viral neutralization where heterologous neutralization is, in part, driven by a strong homologous response and may be coupled to changes in PNG sites in envelope.
Assuntos
Anticorpos Antivirais/imunologia , Macaca nemestrina , Doenças dos Macacos/imunologia , Doenças dos Macacos/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Primers do DNA , Glicosilação , Proteína gp160 do Envelope de HIV/genética , HIV-2/genética , Funções Verossimilhança , Glicoproteínas de Membrana/genética , Modelos Genéticos , Dados de Sequência Molecular , Testes de Neutralização/veterinária , Análise de Sequência de DNA/veterinária , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genéticaRESUMO
Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunização , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene env/imunologia , 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 , Proteína gp160 do Envelope de HIV/metabolismo , Imunização Secundária , Dados de Sequência Molecular , Testes de Neutralização , Filogenia , Coelhos , Receptores CCR5/metabolismo , Proteínas Recombinantes/imunologia , Solubilidade , Proteínas do Envelope Viral/genética , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the trp structural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5' end of the trp leader transcript. Deletion of this structure resulted in a dramatic increase in expression of a trpE'-'lacZ translational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5' stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5' stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5' stem-loop increases the affinity of TRAP for trp leader RNA four- to fivefold, suggesting that the 5' structure interacts with TRAP. In vitro transcription results indicate that this 5' structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5' stem-loop was used to demonstrate that formation of the 5' structure is required for proper attenuation control of this operon.