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1.
J Infect ; 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31812703

RESUMO

OBJECTIVE: Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. METHODS: Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. RESULTS: All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. CONCLUSION: These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

2.
J Gen Virol ; 100(3): 511-522, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30676308

RESUMO

The growth rate of new HIV infections in the Philippines was the fastest of any countries in the Asia-Pacific region between 2010 and 2016. To date, HIV-1 subtyping results in the Philippines have been determined by characterizing only partial viral genome sequences. It is not known whether recombination occurs in the majority of unsequenced genome regions. Near-full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes from plasma samples collected between 2015 and 2017 from 23 newly diagnosed infected individuals in the Philippines. Phylogenetic analysis showed that the newly characterized sequences were CRF01_AE (14), subtype B (3), CRF01/B recombinants (5) and a CRF01/CRF07/B recombinant (1). All 14 CRF01_AE formed a tight cluster, suggesting that they were derived from a single introduction. The time to the most recent common ancestor (tMRCA) for CRF01_AE in the Philippines was 1995 (1992-1998), about 10-15 years later than that of CRF01_AE in China and Thailand. All five CRF01/B recombinants showed distinct recombination patterns, suggesting ongoing recombination between the two predominant circulating viruses. The identification of partial CRF07_BC sequences in one CRF01/CRF07/B recombinant, not reported previously in the Philippines, indicated that CRF07_BC may have been recently introduced into that country from China, where CRF07_BC is prevalent. Our results show that the major epidemic strains may have shifted to an increased predominance of CRF01_AE and its recombinants, and that other genotypes such as CRF07_BC may have been introduced into the Philippines.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , Recombinação Genética , Adulto , Genoma Viral , Genótipo , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Masculino , Filipinas , Filogenia , Adulto Jovem
3.
Cell ; 175(2): 387-399.e17, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30270043

RESUMO

HIV-1 broadly neutralizing antibodies (bnAbs) are difficult to induce with vaccines but are generated in ∼50% of HIV-1-infected individuals. Understanding the molecular mechanisms of host control of bnAb induction is critical to vaccine design. Here, we performed a transcriptome analysis of blood mononuclear cells from 47 HIV-1-infected individuals who made bnAbs and 46 HIV-1-infected individuals who did not and identified in bnAb individuals upregulation of RAB11FIP5, encoding a Rab effector protein associated with recycling endosomes. Natural killer (NK) cells had the highest differential expression of RAB11FIP5, which was associated with greater dysregulation of NK cell subsets in bnAb subjects. NK cells from bnAb individuals had a more adaptive/dysfunctional phenotype and exhibited impaired degranulation and cytokine production that correlated with RAB11FIP5 transcript levels. Moreover, RAB11FIP5 overexpression modulated the function of NK cells. These data suggest that NK cells and Rab11 recycling endosomal transport are involved in regulation of HIV-1 bnAb development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por HIV/imunologia , Vacinas contra a AIDS/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adulto , Linfócitos B/imunologia , Linhagem Celular , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica/métodos , Anticorpos Anti-HIV/imunologia , Infecções por HIV/fisiopatologia , HIV-1/patogenicidade , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Masculino , Pessoa de Meia-Idade
4.
Nat Commun ; 9(1): 2363, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915222

RESUMO

The envelope glycoprotein (Env) trimer ((gp120/gp41)3) mediates human immunodeficiency virus (HIV-1) entry into cells. The "closed," antibody-resistant Env trimer is driven to more open conformations by binding the host receptor, CD4. Broadly neutralizing antibodies that recognize conserved elements of the closed Env are potentially protective, but are elicited inefficiently. HIV-1 has evolved multiple mechanisms to evade readily elicited antibodies against more open Env conformations. Small-molecule CD4-mimetic compounds (CD4mc) bind the HIV-1 gp120 Env and promote conformational changes similar to those induced by CD4, exposing conserved Env elements to antibodies. Here, we show that a CD4mc synergizes with antibodies elicited by monomeric HIV-1 gp120 to protect monkeys from multiple high-dose intrarectal challenges with a heterologous simian-human immunodeficiency virus (SHIV). The protective immune response persists for at least six months after vaccination. CD4mc should increase the protective efficacy of any HIV-1 Env vaccine that elicits antibodies against CD4-induced conformations of Env.


Assuntos
Vacinas contra a AIDS/imunologia , Guanidinas/farmacologia , Proteína gp120 do Envelope de HIV/imunologia , Indenos/farmacologia , Lentivirus de Primatas/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Guanidinas/química , Células HEK293 , Humanos , Imunidade Heteróloga , Imunização , Indenos/química , Macaca mulatta
5.
Nat Commun ; 9(1): 1928, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29765018

RESUMO

Recombination in HIV-1 is well documented, but its importance in the low-diversity setting of within-host diversification is less understood. Here we develop a novel computational tool (RAPR (Recombination Analysis PRogram)) to enable a detailed view of in vivo viral recombination during early infection, and we apply it to near-full-length HIV-1 genome sequences from longitudinal samples. Recombinant genomes rapidly replace transmitted/founder (T/F) lineages, with a median half-time of 27 days, increasing the genetic complexity of the viral population. We identify recombination hot and cold spots that differ from those observed in inter-subtype recombinants. Furthermore, RAPR analysis of longitudinal samples from an individual with well-characterized neutralizing antibody responses shows that recombination helps carry forward resistance-conferring mutations in the diversifying quasispecies. These findings provide insight into molecular mechanisms by which viral recombination contributes to HIV-1 persistence and immunopathogenesis and have implications for studies of HIV transmission and evolution in vivo.


Assuntos
Evolução Molecular , Infecções por HIV/virologia , HIV-1/genética , Recombinação Genética , Variação Genética , Genótipo , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Estudos Longitudinais , Masculino , Filogenia
6.
Retrovirology ; 14(1): 46, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-29017536

RESUMO

BACKGROUND: Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. RESULTS: Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 and fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. CONCLUSIONS: The rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos T CD8-Positivos/imunologia , Genoma Viral/genética , Infecções por HIV/imunologia , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Técnicas de Cultura de Células , ELISPOT , Epitopos de Linfócito T/imunologia , Aptidão Genética/genética , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Evasão da Resposta Imune/genética , Mutação , Seleção Genética/genética , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
7.
Sci Rep ; 6: 38130, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27909304

RESUMO

A severe bottleneck exists during HIV-1 mucosal transmission. However, viral properties that determine HIV-1 transmissibility are not fully elucidated. We identified multiple transmitted/founder (T/F) viruses in six HIV-1-infected subjects by analyzing whole genome sequences. Comparison of biological phenotypes of different T/F viruses from the same individual allowed us to more precisely identify critical determinants for viral transmissibility since they were transmitted under similar conditions. All T/F viruses used coreceptor CCR5, while no T/F viruses used CXCR4 or GPR15. However, the efficiency for different T/F viruses from the same individual to use CCR5 was significantly variable, and the differences were even more significant for usage of coreceptors FPRL1, CCR3 and APJ. Resistance to IFN-α was also different between T/F viruses in 2 of 3 individuals. The relative fitness between T/F viruses from the same subject was highly variable (2-6%). Importantly, the levels of coreceptor usage efficiency, resistance to IFN-α and viral fitness were not associated with proportions of T/F viruses in each individual during acute infection. Our results show that the modest but significant differences in coreceptor usage efficiency, IFN-α sensitivity and viral fitness each alone may not play a critical role in HIV-1 transmission.


Assuntos
Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Variação Genética , Genoma Viral , Infecções por HIV/transmissão , HIV-1/fisiologia , Humanos , Interferon-alfa/farmacologia , Masculino , Fenótipo , Receptores CCR5/fisiologia , Receptores de HIV/fisiologia , Tropismo Viral/genética , Replicação Viral/efeitos dos fármacos
8.
PLoS One ; 11(12): e0167839, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27973597

RESUMO

A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , Recombinação Genética , Adolescente , Adulto , Evolução Molecular , Feminino , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Paquistão , Filogenia , Análise de Regressão , Análise de Sequência de DNA , Adulto Jovem
9.
PLoS One ; 11(6): e0157340, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27314585

RESUMO

HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.


Assuntos
Genes pol/genética , Infecções por HIV/genética , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Farmacorresistência Viral/genética , Genótipo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Dados de Sequência Molecular , Mutação , Filogenia , Recombinação Genética
10.
Cell Host Microbe ; 18(3): 354-62, 2015 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-26355218

RESUMO

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.


Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/virologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Seleção Genética , Ligação Viral , Sítios de Ligação , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Dados de Sequência Molecular , Mutação , RNA Viral/genética , Análise de Sequência de DNA
11.
Retrovirology ; 11: 101, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25407514

RESUMO

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.


Assuntos
HIV-1/imunologia , HIV-1/fisiologia , Evasão da Resposta Imune , Mutação de Sentido Incorreto , Linfócitos T/imunologia , Replicação Viral , Aminoácidos/genética , HIV-1/genética , Humanos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
12.
PLoS One ; 9(7): e102734, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25028937

RESUMO

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8+ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4+ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Aptidão Genética/genética , Genoma Viral/genética , HIV-1/genética , Evasão da Resposta Imune/genética , Mutação de Sentido Incorreto/genética , Sequência de Bases , Linfócitos T CD8-Positivos/virologia , Primers do DNA/genética , ELISPOT , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Replicação Viral/genética , Replicação Viral/fisiologia
13.
J Immunol Methods ; 409: 117-30, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24447533

RESUMO

The significant diversity among HIV-1 variants poses serious challenges for vaccine development and for developing sensitive assays for screening, surveillance, diagnosis, and clinical management. Recognizing a need to develop a panel of HIV representing the current genetic and geographic diversity NIH/NIAID contracted the External Quality Assurance Program Oversight Laboratory (EQAPOL) to isolate, characterize and establish panels of HIV-1 strains representing global diverse subtypes and circulating recombinant forms (CRFs), and to make them available to the research community. HIV-positive plasma specimens and previously established isolates were collected through a variety of collaborations with a preference for samples from acutely/recently infected persons. Source specimens were cultured to high-titer/high-volume using well-characterized cryopreserved PBMCs from National y donors. Panel samples were stored as neat culture supernatant or diluted into defibrinated plasma. Characterization for the final expanded virus stocks included viral load, p24 antigen, infectivity (TCID), sterility, coreceptor usage, and near full-length genome sequencing. Viruses are made available to approved, interested laboratories using an online ordering application. The current EQAPOL Viral Diversity panel includes 100 viral specimens representing 6 subtypes (A, B, C, D, F, and G), 2 sub-subtypes (F1 and F2), 7 CRFs (01, 02, 04, 14, 22, 24, and 47), 19 URFs and 3 group O viruses from 22 countries. The EQAPOL Viral Diversity panel is an invaluable collection of well-characterized reagents that are available to the scientific community, including researchers, epidemiologists, and commercial manufacturers of diagnostics and pharmaceuticals to support HIV research, as well as diagnostic and vaccine development.


Assuntos
Vacinas contra a AIDS/uso terapêutico , Infecções por HIV/diagnóstico , HIV-1/patogenicidade , Testes Imunológicos/normas , Ensaio de Proficiência Laboratorial/normas , Leucócitos Mononucleares/virologia , Monitorização Imunológica/normas , Indicadores de Qualidade em Assistência à Saúde/normas , Bancos de Espécimes Biológicos/normas , Biomarcadores/sangue , Sobrevivência Celular , Células Cultivadas , Criopreservação/normas , Citocinas/sangue , Genótipo , Fidelidade a Diretrizes/normas , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Humanos , Cooperação Internacional , Leucócitos Mononucleares/imunologia , Variações Dependentes do Observador , Fenótipo , Guias de Prática Clínica como Assunto/normas , Valor Preditivo dos Testes , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Controle de Qualidade , Reprodutibilidade dos Testes , Manejo de Espécimes/normas , Fatores de Tempo , Resultado do Tratamento
14.
Proc Natl Acad Sci U S A ; 110(17): 6626-33, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23542380

RESUMO

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.


Assuntos
Células Dendríticas/imunologia , HIV-1/genética , Fenótipo , Proteínas do Envelope Viral/metabolismo , Vírion/patogenicidade , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/imunologia , Infecções por HIV/transmissão , HIV-1/imunologia , Humanos , Modelos Lineares , Dados de Sequência Molecular , Análise de Sequência de DNA
15.
Retrovirology ; 9: 89, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23110705

RESUMO

BACKGROUND: A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. RESULTS: The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. CONCLUSIONS: These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.


Assuntos
Aptidão Genética , Genoma Viral , HIV-1/genética , Evasão da Resposta Imune/genética , Mutação , Replicação Viral/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Efeito Fundador , HIV-1/imunologia , HIV-1/fisiologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Dados de Sequência Molecular , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
16.
PLoS Pathog ; 8(5): e1002686, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22693444

RESUMO

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4ß7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4ß7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4ß7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4ß7, CD4 or CCR5 more efficiently.


Assuntos
Antígenos CD4/metabolismo , Infecções por HIV/transmissão , HIV-1/patogenicidade , Integrinas/metabolismo , Receptores CCR5/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Clonagem Molecular , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Integrinas/imunologia , Membrana Mucosa/virologia , Testes de Neutralização , Tropismo Viral , Internalização do Vírus , Replicação Viral
17.
J Virol Methods ; 167(2): 146-51, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20362002

RESUMO

Characterization of multiple sites in a single gene that are important in biological phenotypes is challenging due to the difficulty to generate many mutants representing all or a majority of combinations of mutations in the gene. Using the HIV-1 env and pol genes as templates, four random libraries were generated representing different combinations of mutations introduced by up to 36 mutagenesis primers in a single assay. Over 86% of the clones contained mutations and the mutants tended to have single or fewer mutations in the libraries. When protein size was used as a screening marker, all identified clones contained at least 2 mutations and up to 12 mutations were detected in a single clone. Nearly all mutant clones in each library contained unique mutations, indicating that mutants in the library were generated at random. Closely related mutations which were overlapped by neighboring mutagenesis primers were often introduced in this system. Analysis of the env library showed that some potential N-linked glycosylation sites did not increase the Env molecular mass significantly, suggesting they were not used for glycosylation or only limited carbohydrate moieties were added at these sites. This novel method can serve as a powerful tool to study the biological phenotypes of genes whose functions are determined by multiple sites.


Assuntos
Primers do DNA/genética , Biblioteca Gênica , HIV-1/genética , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/genética , Reação em Cadeia da Polimerase/métodos , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
18.
Virology ; 394(1): 91-8, 2009 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-19744690

RESUMO

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vírus da Imunodeficiência Símia/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Reações Cruzadas , Anticorpos Anti-HIV/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Ressonância de Plasmônio de Superfície
19.
J Clin Microbiol ; 43(1): 321-5, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15634989

RESUMO

Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans. Adherence of M. pneumoniae to host cells requires several adhesin proteins, such as P1, P30, and P116. A major limitation in developing a specific diagnostic test for M. pneumoniae is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in Escherichia coli. In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in E. coli. On screening these recombinant proteins with sera from M. pneumoniae-infected patients, only the P1-C1 protein was found to be immunogenic. This protein can be used as an antigen for immunodiagnosis of M. pneumoniae infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/sangue , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/diagnóstico , Proteínas Recombinantes/imunologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Pneumonia por Mycoplasma/microbiologia
20.
FEMS Immunol Med Microbiol ; 34(1): 33-43, 2002 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-12208604

RESUMO

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.


Assuntos
Vacinas Antimaláricas/farmacologia , Malária/imunologia , Malária/prevenção & controle , Plasmodium cynomolgi/genética , Plasmodium cynomolgi/imunologia , Vacinas de DNA/farmacologia , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/genética , Sequência de Bases , DNA de Protozoário/genética , Eritrócitos/parasitologia , Feminino , Imunidade Celular , Imunização , Macaca mulatta , Malária/parasitologia , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Parasitemia/imunologia , Parasitemia/prevenção & controle , Plasmídeos/genética , Plasmodium cynomolgi/crescimento & desenvolvimento , Vacinas de DNA/genética , Vacinas de DNA/imunologia
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