RESUMO
Human immunodeficiency virus (HIV) is still considered a pandemic, and the detection of p24-HIV protein has an important role in the early diagnosis of HIV in adults and newborns. The accessibility of these trials depends on the price and execution difficulty of the method, which can be reduced using electrochemical methods by using enzymeless approaches, disposable and accurate devices. In this work, graphene quantum dots were acquired by a simple synthesis and employed as an electrochemical signal amplifier and support for the aptamer immobilization through a feasible and stable modification of disposable screen-printed electrodes. The device has been easily assembled and used to detect p24-HIV protein without the interference of similar proteins or sample matrix. Using the best set of experimental conditions, a linear correlation between analytical signal and log of p24-HIV concentration from 0.93 ng mL-1 to 93 µg mL-1 and a limit of detection of 51.7 pg mL-1 were observed. The developed device was applied to p24 determination in spiked human serum and provided distinct levels of signal for positive and negative samples, successfully identifying real samples with the target protein. This sensor is a step towards the development of point-of-care devices and the popularization of electrochemical methods for trials and diagnostics of relevant diseases.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Grafite , Infecções por HIV , Pontos Quânticos , Adulto , Técnicas Eletroquímicas , Eletrodos , Infecções por HIV/diagnóstico , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Recém-Nascido , Limite de DetecçãoRESUMO
Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.
Assuntos
Vacinas contra a AIDS/biossíntese , Proteínas do Vírus da Imunodeficiência Humana/biossíntese , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Lactuca/metabolismo , Animais , Escherichia coli , Feminino , Fenômenos Imunogenéticos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Vacinas Sintéticas/biossínteseRESUMO
We previously reported a naturally occurring BF intersubtype recombinant viral protein U (Vpu) variant with an augmented capacity to enhance viral replication. Structural analysis of this variant revealed that its transmembrane domain and α-helix I in the cytoplasmic domain (CTD) corresponded to subtype B, whereas the α-helix II in the CTD corresponded to subtype F1. In this study, we aimed to evaluate the role of the Vpu cytoplasmic α-helix II domain in viral release enhancement and in the down-modulation of BST-2 and CD4 from the cell surface. In addition, as serine residues in Vpu amino acid positions 61 or 64 have been shown to regulate Vpu intracellular half-life, which in turn could influence the magnitude of viral release, we also studied the impact of these residues on the VpuBF functions, since S61 and S64 are infrequently found among BF recombinant Vpu variants. Our results showed that the exchange of Vpu α-helix II between subtypes (BâF) directly correlated with the enhancement of viral release and, to a lesser extent, with changes in the capacity of the resulting chimera to down-modulate BST-2 and CD4. No differences in viral release and BST-2 down-modulation were observed between VpuBF and VpuBF-E61S. On the other hand, VpuBF-A64S showed a slightly reduced capacity to enhance viral production, but was modestly more efficient than VpuBF in down-modulating BST-2. In summary, our observations clearly indicate that α-helix II is actively involved in Vpu viral-release-promoting activity and that intersubtype recombination between subtypes B and F1 created a protein variant with a higher potential to boost the spread of the recombinant strain that harbours it.
Assuntos
Antígenos CD/metabolismo , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Fatores de Virulência/metabolismo , Liberação de Vírus , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Genótipo , HIV-1/classificação , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Proteólise , Recombinação Genética , Proteínas Virais Reguladoras e Acessórias/genética , Fatores de Virulência/genéticaRESUMO
T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Proteínas do Vírus da Imunodeficiência Humana/farmacologia , Peptídeos/farmacologia , Vacinas de DNA , Vacinas contra a AIDS/administração & dosagem , Algoritmos , Animais , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Sequência Consenso , Reações Cruzadas , Epitopos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Antígenos HLA/imunologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Imunização , Interferon gama/imunologia , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Over the last decade, it has become evident that 14-3-3 proteins are essential for primary cell functions. These proteins are abundant throughout the body, including the central nervous system and interact with other proteins in both cell cycle and apoptotic pathways. Examination of cerebral spinal fluid in humans suggests that 14-3-3s including 14-3-3ε (YWHAE) are up-regulated in several neurological diseases, and loss or duplication of the YWHAE gene leads to Miller-Dieker syndrome. The goal of this review is to examine the utility of 14-3-3s as a marker of human immune deficiency virus (HIV)-dependent neurodegeneration and also as a tool to track disease progression. To that end, we describe mechanisms implicating 14-3-3s in neurological diseases and summarize evidence of its interactions with HIV accessory and co-receptor proteins.
Assuntos
Proteínas 14-3-3/líquido cefalorraquidiano , Complexo AIDS Demência/líquido cefalorraquidiano , Sistema Nervoso Central/metabolismo , HIV/fisiologia , Doenças Neurodegenerativas/líquido cefalorraquidiano , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Animais , Biomarcadores/líquido cefalorraquidiano , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Cognição , Progressão da Doença , Regulação da Expressão Gênica , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/virologia , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
One of the most studied topics about AIDS disease is the presence of different progression levels in patients infected by HIV. Several studies have shown that this progression is directly associated with host genetics, although viral factors are also known to play a role. Here we explore the contribution of Vpu protein in the evolution of viral population. The sequence variation of Vpu was analyzed during HIV infection in peripheral blood monocyte cells of 12 patients in different clinical stages of HIV-1 infection early and late stages of infections, separated by at least 4 years. The clustering analysis of Vpu sequences showed higher diversity of early alleles, non-random distribution of sequences, and viral evolution strains selection. Forty-two amino acid modifications were found in the multiple alignments of the 57 different alleles found for early stage were 23 modifications were found in the late stage dataset. Interestingly fourteen alteration of early stage were located in conserved site related with Vpu functions alterations while these alterations appear with less frequency in the late stage of infection. Moreover, late stage alleles tend to be similar with the Vpu wild type sequence, suggesting viral selection toward populations harboring more efficient variants during the course of infection. This would contribute to higher infectivity and viral replication actually observed at the aggressive late stages of infection. These data, in conjunction with in vitro experiments, will be important to elucidation of the physiological relevance of Vpu protein in the pathogenic mechanisms of AIDS.
Assuntos
Alelos , Progressão da Doença , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Seleção Genética , Proteínas Virais Reguladoras e Acessórias/genética , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Análise por Conglomerados , Estudos de Coortes , Infecções por HIV/sangue , HIV-1/classificação , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/química , Humanos , Massachusetts , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monócitos/virologia , Alinhamento de Sequência , Proteínas Virais Reguladoras e Acessórias/química , Adulto JovemRESUMO
Existen evidencias que apuntan a la existencia de una asociación inversa entre la ingesta de proteínas vegetales y la presión arterial. En este estudio, los datos de adolescentes VIH +, que acuden a la consulta del Centro de Atención a Pacientes con Enfermedades Infectocontagiosas "Dra. Elsa La Corte" (CAPEI) de la Universidad Central de Venezuela (UCV), fueron analizados para estudiar la relación entre el consumo de proteínas vegetales y la presión arterial tanto sistólica como diastólica, ajustando por índice de masa corporal (IMC) y consumo de energía. Estudio transversal en 43 adolescentes VIH+ con edades entre 15 y 18 años en ambos sexos, que acudieron al CAPEI en el año 2009. Se analizó la media de dos lecturas obtenidas con 5 minutos de intervalo en una visita. Se determinaron peso y altura y se calculó el IMC. Para la determinación de la ingesta de proteínas vegetales se aplicó la técnica de recordatorio de 24 horas. El análisis estadístico se basó en el modelo de regresión lineal. Los resultados muestran una asociación negativa y significativa entre el consumo de proteínas vegetales y la presión arterial sistólica y diastólica, después de ajustar por consumo de energía e IMC, las diferencias de presión arterial sistólica y diastólica asociada con una ingesta de proteínas vegetales de 57,6 kilocalorías por ciento (variación intercuartil) fue de -2,8 mm Hg y -2,4 mm Hg, respectivamente (p <0,05 para ambos). La promoción y planificación de dietas con altos contenidos de proteínas vegetales puede ser de utilidad para prevenir y controlar valores elevados de la presión arterial
Data are available that indicate an independent inverse relationship of dietary vegetable protein to blood pressure (BP). In this investigation data from HIV adolescents attending CAPEI/UCV, were analyzed to study the relationship between dietary vegetable protein and systolic/diastolic pressures, with control for body mass index (BMI) and calorie intake. This was a cross-sectional study with 43 HIV adolescents 15 to 18 years of age. BP was measured 2 times at 1 visit; height and weight were measured, and BMI was calculated; dietary data were obtained from 24-hour dietary recalls. Multivariate regression was applied.The results showed that with control for BMI and calorie intake, estimated average BP differences associated with a vegetable protein intake that was higher by 57,6 %kcal (interquartile range) were -2,8 mm Hg systolic and -2,4 mm Hg diastolic (p <0,05 both). Broad improvement in diets high in vegetable protein can be important in preventing and controlling high blood pressure
Assuntos
Humanos , Masculino , Adolescente , Feminino , Pressão Arterial , Proteínas de Vegetais Comestíveis/análise , Proteínas de Vegetais Comestíveis/efeitos adversos , Proteínas de Vegetais Comestíveis/uso terapêutico , Proteínas do Vírus da Imunodeficiência Humana/análiseRESUMO
BACKGROUND: Multiple HIV-1 intersubtype recombinants have been identified in human populations. Previous studies from our lab group have shown that the epidemic in Argentina is characterized by the high prevalence of a circulating recombinant form, CRF12_BF, and many related BF recombinant forms. In these genomic structures a recombination breakpoint frequently involved the vpu coding region. Due to the scarce knowledge of Vpu participation in the virion release process and its impact on pathogenesis and of the functional capacities of intersubtype recombinant Vpu proteins, the aim of this work was to perform a comparative analysis on virion release capacity and relative replication capacity among viral variants harboring either a BF recombinant Vpu or a subtype B Vpu. RESULTS: Our results showed that BF recombinant Vpu was associated to an increased viral particles production when compared to WT B variant in tetherin-expressing cell lines. This observation was tested in the context of a competition assay between the above mentioned variants. The results showed that the replication of the BF Vpu-harboring variant was more efficient in cell cultures than subtype B, reaching a higher frequency in the viral population in a short period of time. CONCLUSION: This study showed that as a result of intersubtype recombination, a structurally re-organized HIV-1 Vpu has an improved in vitro capacity of enhancing viral replication, and provides evidence of the changes occurring in this protein function that could play an important role in the successful spread of intersubtype recombinant variants.
Assuntos
HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Recombinação Genética , Proteínas Virais Reguladoras e Acessórias/genética , Fatores de Virulência/genética , Liberação de Vírus , Replicação Viral , Linhagem Celular , HIV-1/crescimento & desenvolvimento , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Humanos , Carga Viral , Proteínas Virais Reguladoras e Acessórias/fisiologia , Fatores de Virulência/fisiologiaRESUMO
BACKGROUND: Here, we investigated the phylogenetic relationships of the HIV-1 subtype F1 circulating in Angola with subtype F1 strains sampled worldwide and reconstructed the evolutionary history of this subtype in Central Africa. METHODS: Forty-six HIV-1-positive samples were collected in Angola in 2006 and subtyped at the env-gp41 region. Partial env-gp120 and pol-RT sequences and near full-length genomes from those env-gp41 subtype F1 samples were further generated. Phylogenetic analyses of partial and full-length subtype F1 strains isolated worldwide were carried out. The onset date of the subtype F1 epidemic in Central Africa was estimated using a Bayesian Markov chain Monte Carlo approach. RESULTS: Nine Angolan samples were classified as subtype F1 based on the analysis of the env-gp41 region. All nine Angolan sequences were also classified as subtype F1 in both env-gp120 and pol-RT genomic regions, and near full-length genome analysis of four of these samples confirmed their classification as "pure" subtype F1. Phylogenetic analyses of subtype F1 strains isolated worldwide revealed that isolates from the Democratic Republic of Congo (DRC) were the earliest branching lineages within the subtype F1 phylogeny. Most strains from Angola segregated in a monophyletic group together with Romanian sequences; whereas South American F1 sequences emerged as an independent cluster. The origin of the subtype F1 epidemic in Central African was estimated at 1958 (1934-1971). CONCLUSION: "Pure" subtype F1 strains are common in Angola and seem to be the result of a single founder event. Subtype F1 sequences from Angola are closely related to those described in Romania, and only distantly related to the subtype F1 lineage circulating in South America. Original diversification of subtype F1 probably occurred within the DRC around the late 1950s.
Assuntos
Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Filogenia , Angola , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Dados de Sequência Molecular , Romênia , Fatores de TempoRESUMO
Studies examining positive selection on accessory proteins of HIV are rare, although these proteins play an important role in pathogenesis in vivo. Moreover, despite the biological relevance of analyses of molecular adaptation after viral transmission between species, the issue is still poorly studied. Here we present evidence that accessory proteins are subjected to positive selective forces exclusively in HIV. This scenario suggests that accessory protein genes are under adaptive evolution in HIV clades, while in SIVcpz such a phenomenon could not be detected. As a result, we show that comparative studies are critical to carry out functional investigation of positively selected protein sites, as they might help to achieve a better comprehension of the biology of HIV pathogenesis.