Genetic vaccination against experimental infection with myotropic parasite strains of Trypanosoma cruzi.

In earlier studies, we reported that a heterologous prime-boost regimen using recombinant plasmid DNA followed by replication-defective adenovirus vector, both containing Trypanosoma cruzi genes encoding trans-sialidase (TS) and amastigote surface protein (ASP) 2, provided protective immunity against experimental infection with a reticulotropic strain of this human protozoan parasite. Herein, we tested the outcome of genetic vaccination of F1 (CB10XBALB/c) mice challenged with myotropic parasite strains (Brazil and Colombian). Initially, we determined that the coadministration during priming of a DNA plasmid containing the murine IL-12 gene improved the immune response and was essential for protective immunity elicited by the heterologous prime-boost regimen in susceptible male mice against acute lethal infections with these parasites. The prophylactic or therapeutic vaccination of resistant female mice led to a drastic reduction in the number of inflammatory infiltrates in cardiac and skeletal muscles during the chronic phase of infection with either strain. Analysis of the electrocardiographic parameters showed that prophylactic vaccination reduced the frequencies of sinus arrhythmia and atrioventricular block. Our results confirmed that prophylactic vaccination using the TS and ASP-2 genes benefits the host against acute and chronic pathologies caused by T. cruzi and should be further evaluated for the development of a veterinary or human vaccine against Chagas disease.

Detection of distinct human immunodeficiency virus type 1 circulating recombinant forms in northeast Brazil.

The extraordinary genetic diversity and immune evasion of human immunodeficiency virus (HIV) pose significant challenges for vaccine development and antiretroviral therapy efficacy. The objective of this study was to characterize the molecular profile of HIV-1 epidemic in Salvador, Bahia, Brazil, determining the genetic subtypes and the presence of antiretroviral resistance mutations. HIV-1 pol DNA sequences from 57 individuals infected with HIV were obtained by PCR, followed by sequencing. The subtypes were determined by phylogenetic analyses and the intersubtype recombination was investigated by bootscanning. The pol subtypes were compared with gag and env subtypes. Antiretroviral susceptibility was evaluated through the Stanford HIV resistance Database. The subtypes frequencies were: 77.2% of subtype B, 1.8% of subtype F1, and 21.0% of BF recombinant forms. Two intergenic and three intragenic BF recombinant patterns were observed. Six (10.5%) viruses were related to CRF28/CRF29, two were related to CRF12 (3.5%), and one (1.8%) was CRF39. Fourteen (24.6%) strains carried one or more mutations associated with at least intermediate resistance: 24.6% had resistance to nucleoside reverse transcriptase inhibitors, 21.0% to non-nucleoside reverse transcriptase inhibitors, and 7% to protease inhibitors. The substitutions I54V (7.0%), M184V (14.0%), and K103N (10.5%) were the most frequent within each class of drugs. The results show a high diversity of BF genotypes and a lower prevalence of major reverse transcriptase and protease drug resistance mutations in Salvador, compared with other regions of Brazil. These findings may contribute to improve treatment strategies of patients infected with HIV-1 from this Brazilian region.

Lack of high-level resistance mutations in HIV type 1 BF recombinant strains circulating in northeast Brazil.

Abstract The genetic variability and the prevalence of drug resistance-associated mutations (DRAM) of HIV-1 isolates from 50 women and 8 children from Feira de Santana, Bahia, Brazil were investigated. DNA samples were obtained and pol sequences were generated by PCR and direct sequencing. Phylogenetic analysis showed that 39 (67.2%) samples were subtype B, four (6.9%) F, one (1.7%) C, and 14 (24.1%) BF recombinants. Four different BF recombination patterns were detected. Twelve (20.7%) samples shared the same breakpoint within the reverse transcriptase (RT) sequence. Fifty-five (94.8%) isolates showed several resistance-associated mutations in the RT and the protease (PR) genes. Ten (17.2%) isolates presented mutations associated with a high level of resistance: nine (15.5%) to nucleoside RT inhibitors (NRTI), four (6.9%) to nonnucleoside RT inhibitors (NNRTI), and three (5.2%) to PR inhibitors (PIs). Subtype B-infected patients had, on average, 0.5 high-level DRAM per sequence while no mutations were observed in BF recombinants, although the two groups were under ARV for a similar period of time. Our data indicate the predominance of the subtype B, followed by BF recombinants in this population, and the dissemination of a recombinant strain in Bahia, which could be related to adaptive advantages of these variants over the predominant subtype B.

Lower prevalence of human immunodeficiency virus type 1 Brazilian subtype B found in northeastern Brazil with slower progression to AIDS.

Besides being extremely useful in measuring the level of HIV-1 diversity and prevalence in populations, the molecular analysis of genomic sequences provides crucial surveillance support and aids in the development of new therapies and effective vaccines. The present study focused on gag and env DNA and amino acid sequences that were generated from samples taken from 61 infected patients in the City of Salvador, Bahia, located in northeastern Brazil. In order to determine selective pressure and predict coreceptor usage, Bioinformatics tools were employed in phylogeny reconstruction. Fifty-six (91.8%) viruses were classified as belonging to subtype B, three (4.9%) from F1, and two (3.3%) from BF1 recombinants. Based on the characterization of the V3 region, the subtype B strains were represented by eight (18.2%) Brazilian variants (B'-GWGR), 20 (46.5%) European/EUA B variants (GPGR), and 15 (34.9%) GXGX variants. The mean time elapsed since diagnosis was 13 years among subtype B' and 9 years in subtype B. The mean dN/dS ratios from the GWGR, GPGR, and GXGX groups, when compared to an HXB2 reference, were 0.72, 0.77, and 0.67, respectively. Seventy-six percent of the viruses studied were predicted to use the CCR5 coreceptor for cell entry (R5 viruses), while 24% were predicted to use the CXCR4 or were classified as dual tropic viruses. The prevalence of subtypes B' and recombinant B/F1 was shown to be lower than findings from previous studies performed both in Brazil (B') and in Bahia (B/F1). The association between subtype B' and a lengthy period of time since diagnosis can be correlated with a slower disease progression in infected patients, when compared with those infected with subtype B.

Diversidade molecular do vírus da imunodeficiencia humana tipo 1 no estado da Bahia / Molecular diversity of human immunodeficiency virus type 1 in the state of Bahia

A análise de sequências genômicas é uma forma poderosa para se estudar a diversidade do HIV-1 e sua expansão nas populações, além de ser crucial para dar suporte à vigilância epidemiológica e ao desenvolvimento de terapias e vacinas eficazes. Neste estudo foram geradas sequências dos genes gag e env do HIV-1 de 61 pacientes infectados em Salvador e do pol de 58 pacientes de Feira de Santana, Bahia, Brasil. A bioinformática foi utilizada para subtipar, genotipar as mutações de resistência, determinar a pressão seletiva e predizer o uso de coreceptor. Em Salvador, 92,6% das amostras foram do subtipo B e 7,4% foram recombinantes BF1. Em Feira de Santana, 67,2% foram do subtipo B, 6,9% F1, 1,7% C e 24,1% BF1. Onze (19,0%) destes isolados apresentaram mutações de resistência. Pacientes infectados pelo B tinham em média 0,4 mutações de resistência e nenhuma mutação deste tipo foi observada em BF1. Com base na caracterização da alça V3 do gene env em 43 amostras de Salvador, foram encontradas 18,2% de variantes brasileiras (B'-GWGR), 46,5% de GPGR e 34,9% de GXGX. O tempo médio de diagnóstico positivo foi de 13 anos para o subtipo B' e 9 anos para o subtipo B (GPGR + GXGX). Setenta e seis por cento destes vírus fazem uso do coreceptor CCR5, enquanto 24% foram classificados como capazes de usar o CXCR4. Encontramos uma associação entre o subtipo B' e um maior tempo em anos de diagnóstico positivo para HIV-1. As prevalências de subtipo B' e de recombinante B/F1 em Salvador foram menores do que as encontradas em estudos anteriores na Bahia e no Brasil, por outro lado, em Feira de Santana foi encontrada uma alta prevalência de B/F1.

Genomic analysis of sequences is a powerful tool to perform studies in HIV-1 diversity and expansion in the populations and it is crucial to give support to epidemiological surveillance, new therapies and vaccines development. In this study, sequences were generated from gag and env genes from 61 HIV-1 infected patients sampled in Salvador and from pol gene in 58 infected patients sampled in Feira de Santana, Bahia, Brazil. Bioinformatic tools were used to subtype, genotype the resistance mutations, determine the selective pressure and to predict coreceptor use. In Salvador, 92.6% of the samples were subtype B and 7.4% were recombinant BF1. In Feira de Santana, 67.2% of the samples were subtype B, 6.9% were F1, 1.7% was C and 24,1% were recombinant BF1. Eleven (19.0%) of these isolates presented resistance mutations. HIV-1 subtype B infected individuals had, in average, 0.4 resistance mutations by sequence and no mutation was observed in BF1. With regard to V3 loop of gene env, it was found 18.2% of Brazilian variants (B'-GWGR), 46.5% of GPGR and 34.9% of GXGX. The env sequences showed through to negative pressure. The time, in average, since the diagnosis was 13 years among subtype B' and 9 years among subtype B isolates. Seventy-six percent of these viruses use the CCR5 coreceptor, while 24% were able to use CXCR4. We have found an association between subtype B’ and a longer period of time since the HIV positive diagnosis. The prevalence of B’ and B/F1 in Salvador were smaller than the found in previous studies in Bahia state and in Brazil, on the other hand, in Feira de Santana it was found a higher prevalence of B/F1.

Diversidade genética do vírus da imunodeficiência humana tipo 1 (HIV-1) em mulheres infectadas de uma cidade do nordeste do Brasil / Genetic diversity of human immunodeficiency virus type-1 (HIV-1) in infected women from a northeast city of Brazil

OBJETIVO: descrever a diversidade genética dos isolados de HIV-1 de mulheres soropositivas acompanhadas em um centro de referência. MÉTODOS: estudo transversal, no qual foram incluídas 96 mulheres com dois testes sorológicos ELISA e um teste confirmatório Western Blot. Das amostras de sangue periférico, foram determinadas a carga viral pelo kit b-DNA e a contagem de linfócitos T CD4 e T CD8 pela citometria de fluxo excalibur. A extração e purificação do DNA pró-viral foi realizada pela reação em cadeia da polimerase (PCR), utilizando o kit QIAamp Blood (Qiagen Inc., Chatsworth, CA, USA). O sequenciamento da região pol foi realizado em 52 isolados com o (3100 Genetic Analyzer, Applied Biosystems Inc., Foster City, CA) e a genotipagem foi investigada pela ferramenta Rega (Rega Subtyping Tool). O padrão de resistência aos antirretrovirais (ARV) foi inferido pelo algoritmo do banco de dados Stanford HIV Resistance. Os estágios clínicos das participantes foram definidos como A, B ou C segundo os critérios do Center for Diseases Control (CDC). Para a análise estatística dos dados, foram utilizados os testes do χ2 para as variáveis categóricas e o teste t de Student para as variáveis numéricas. RESULTADOS: a média de idade da amostra, o tempo médio de doença e de tratamento foram: 33,7; 3,8 e 2,5 anos, respectivamente. A média da carga viral foi log10 2,3 cópias/mL; a dos linfócitos T CD4 e T CD8 foi 494,9 células/µL e 1126,4 células/µL. Sobre o estágio clínico, 30 mulheres estavam no estádio A, 47 no B e 19 no C. O sequenciamento dos 52 isolados encontrou 33 do subtipo B, quatro do F, um do C e 14 do recombinante BF. A análise da resistência aos ARV mostrou 39 (75,0 por cento) isolados susceptíveis, 13 (25,0 por cento) resistentes aos inibidores da transcriptase reversa (INTR) e três (5,7 por cento) aos inibidores da protease (IP). CONCLUSÕES: Houve grande diversidade do HIV-1 e elevado percentual de isolados resistentes aos ARV na amostra estudada.

PURPOSE: to describe the genetic diversity of HIV-1 isolates from serum positive women followed up at a reference center. METHODS: transversal study, including 96 women with two ELISA serological tests and a Western Blot confirmatory test. The viral charge was determined by the b-DNA kit, and the counting of T CD4 and T CD8 lymphocytes, by the Excalibur flow cytometry, from the samples of peripheral blood. The extraction and purification of pro-viral DNA was performed by the polymerase (PCR) chain reaction, using the QIAamp Blood kit (Qiagen Inc., Chatsworth, CA, U.S.A.). Sequencing of the pol region was done in 52 isolates with the 3100 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA), and the genotyping was assessed by the Rega Subtyping Tool. The resistance pattern to anti-retrovirals (ARV) was inferred by the algorithm from the Stanford HIV Resistance data bank. Participants' clinical stages were defined as A, B or C, according to the criteria established by the Center for Diseases Control (CDC). For statistical analysis, the χ2 test was used for the categorical variables and the Student's t test, for the numerical variables. RESULTS: The average age of the sample, the disease and treatment average duration were respectively: 33.7 years old, 3.8 and 2.5 years. The viral charge average was log10 2.3 copies/mL; the T CD4 e T CD8 lymphocytes, 494.9 cells/µL and 1126.4 cells/µL. Concerning the clinical stage, 30 women were in stage A, 47 in B and 19 in C. Sequencing from the 52 isolates found 33 of B subtype, 4 of F, 1 of C and 14 of BF recombinant. The analysis of resistance to ARV has shown 39 (75.0 percent) susceptible isolates, 13 (25.0 percent) resistant to reversal transcriptase inhibitors (RTIN), and 3 (5.7 percent) resistant to protease inhibitor (PI). CONCLUSIONS: There has been a large variety of HIV-1 and a high percentage of isolates resistant to ARV in the studied sample.

[Genetic diversity of human immunodeficiency virus type-1 (HIV-1) in infected women from a northeast city of Brazil]. / Diversidade genética do vírus da imunodeficiência humana tipo 1 (HIV-1) em mulheres infectadas de uma cidade do nordeste do Brasil.

PURPOSE: to describe the genetic diversity of HIV-1 isolates from serum positive women followed up at a reference center. METHODS: transversal study, including 96 women with two ELISA serological tests and a Western Blot confirmatory test. The viral charge was determined by the b-DNA kit, and the counting of T CD4 and T CD8 lymphocytes, by the Excalibur flow cytometry, from the samples of peripheral blood. The extraction and purification of pro-viral DNA was performed by the polymerase (PCR) chain reaction, using the QIAamp Blood kit (Qiagen Inc., Chatsworth, CA, U.S.A.). Sequencing of the pol region was done in 52 isolates with the 3100 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA), and the genotyping was assessed by the Rega Subtyping Tool. The resistance pattern to anti-retrovirals (ARV) was inferred by the algorithm from the Stanford HIV Resistance data bank. Participants' clinical stages were defined as A, B or C, according to the criteria established by the Center for Diseases Control (CDC). For statistical analysis, the chi(2) test was used for the categorical variables and the Student's t test, for the numerical variables. RESULTS: The average age of the sample, the disease and treatment average duration were respectively: 33.7 years old, 3.8 and 2.5 years. The viral charge average was log(10) 2.3 copies/mL; the T CD4 e T CD8 lymphocytes, 494.9 cells/microL and 1126.4 cells/microL. Concerning the clinical stage, 30 women were in stage A, 47 in B and 19 in C. Sequencing from the 52 isolates found 33 of B subtype, 4 of F, 1 of C and 14 of BF recombinant. The analysis of resistance to ARV has shown 39 (75.0%) susceptible isolates, 13 (25.0%) resistant to reversal transcriptase inhibitors (RTIN), and 3 (5.7%) resistant to protease inhibitor (PI). CONCLUSIONS: There has been a large variety of HIV-1 and a high percentage of isolates resistant to ARV in the studied sample.