Immunogenicity of personalized dendritic-cell therapy in HIV-1 infected individuals under suppressive antiretroviral treatment:interim analysis from a phase II clinical trial

BACKGROUND: We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. METHODS: PBMCs were obtained from 10 HIV(+) individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient’s HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients’ cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4(+) and CD8(+) T-cells. RESULTS: The protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4(+) and CD8(+) T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4(+) T-cells. The number of candidates that increased in vitro the cytokine levels in CD4(+) and CD8(+) T cells upon stimulation with Gag peptides from baseline to day 15 and from baseline to day 30 and day 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. CONCLUSIONS: MDDC had a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment. Trial registration NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829, posted November 11th, 2016) SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12981-021-00426-z

Analysis of similarity between the sequences of genes of HIV-1 and Human Genome

It is possible that viral genes have cellular origins. They may have been gathered during evolution, and each one may correspond to a distinct origin. The similarity between genetic sequences suggest homology and, therefore, an evolutive relationship. The present study aimed at identifying region with similarity, with significant statistic support, between the human and the HIV-1 genome through the comparative analysis between DNA sequences using the BLAST program. It is also our purpose to develop a methodology which allows the comparison between genetic sequences of diverse organisms, quantifying the similarity. Thus, to carry it through, we use the BLASTn software (2.2.20 version) and a local database of human chromosomes. Python (5.1.30 version) is used for sequence processing and the data analysis is made using the SQL (Structured Query Language). The human genome is compared to sequences from: HIV-1 genes, Tobbaco Mosaic Virus genes (negative non-biologic control), chromosomes motifs of Macaca mulatta (positive biologic control) and random sequences (negative non-biologic control). We find differences between all the HIV-1 genes and the LTR region. Some genes show more similarity with the human genome than others, according to the curve fitting analysis. When analyzing the curve obtained at very low E-values, it is observed that all HIV-1 genes are well represented by a non-linear curve fitting y = a0 . xa1 where a0 and a1 are parameters obtained numerically. As a result, the method is validated using the data from the genome of Macaca mulatta for which a high level of similarity with the human genome is observed. Based on this validation, it is possible to form three distinct groups of genes from HIV-1 according to the a1 value where: the env, gag, nef, pol, rev, tat and vif genes have identical a1 values; the vpr gene a very low a1 value; and the vpu a a1 higher value.Os genes virais podem ter origens celulares ou não. Eles podem ter sido reunidos durante a evolução, sendo que cada um pode corresponder a uma origem distinta. A similaridade entre sequências genéticas sugerem homologia e, portanto, um parentesco evolutivo. O presente estudo tem como objetivo identificar regiões de similaridade, com suporte estatístico confiável, entre o genoma do HIV-1 e o genoma humano a partir da análise comparativa entre sequências de DNA, por meio do programa BLAST. Também é de nosso interesse desenvolver uma metodologia que permita comparar sequências genéticas de diversos organismos, de forma que a similaridade possa ser quantificada. Para realizar as comparações entre as sequências genéticas, é utilizada a ferramenta BLASTn (versão 2.2.20) e um banco de dados local composto pelos cromossomos humanos. Python (versão 5.1.30) é utilizada para o processamento das sequências, e a análise dos dados é feita utilizando a linguagem estruturada de consulta SQL (Structured Query Language). O genoma humano é comparado com: sequências dos genes do HIV-1, sequências de genes do vírus do mosaico do tabaco (controle negativo biológico), trechos de cromossomos da Macaca mulatta (controle positivo biológico) e sequências aleatórias (controle negativo não biológico). O presente estudo demonstra diferenças entre os genes do HIV-1 e a região LTR. Alguns genes demonstram maior similaridade com o genoma humano do que outros, de acordo com a análise da curva do fitting. Ao verificar a curva obtida a partir de E-values muito baixos, observa-se que todos os genes do HIV-1 apresentam curva não linear do tipo y = a0 . xa1, onde a0 e a1 são parâmetros obtidos a partir de ajuste numérico. O método é validado utilizando dados do genoma da Macaca mulatta, pelo qual é observada alta similaridade com o genoma humano. A partir desta validação é possível construir três diferentes grupos de genes do HIV-1 de acordo com o valor de a1, onde: os genes env, gag, nef, pol, rev, tat e vif apresentam valores de a1 idênticos, o gene vpr um valor mais baixo e o gene vpu um valor bem mais alto.TEDEBV UNIFESP: Teses e dissertaçõe

Impact of antiretroviral resistance and virological failure on HIV-1 informational entropy

Objectives: The present study investigated the relationship between genomic variability and resistance of HIV-1 sequences in protease (PR) and reverse transcriptase (RT) regions of the pol gene. In addition, we analysed the resistance among 651 individuals presenting antiretroviral virological failure, from 2009 to 2011, in the state of Sao Paulo, Brazil. Methods: The genomic variability was quantified by using informational entropy methods and the relationship between resistance and replicative fitness, as inferred by the residual viral load and CD4+ T cell count. Results: The number of antiretroviral schemes is related to the number of resistance mutations in the HIV-1 PR (alpha = 0.2511, P = 0.0003, R-2 = 0.8672) and the RT (alpha = 0.7892, P = 0.0001, R-2 = 0.9141). Increased informational entropy rate is related to lower levels of HIV-1 viral loads (alpha = -0.0121, P = 0.0471, R-2 = 0.7923), lower levels of CD4+ T cell counts (alpha = -0.0120, P = 0.0335, R-2 = 0.8221) and a higher number of antiretroviral resistance-related mutations. Conclusions: Less organized HIV genomes as inferred by higher levels of informational entropy relate to less competent host immune systems, lower levels of HIV replication and HIV genetic evolution as a consequence of antiretroviral resistance.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Fed Univ Sao Paulo UNIFESP, Paulista Sch Med, Div Infect Dis, Sao Paulo, SP, BrazilUniv Sao Paulo, Sch Engn, Telecommun & Control Engn Dept, Sao Paulo, SP, BrazilFed Univ Sao Paulo UNIFESP, Paulista Sch Med, Div Infect Dis, Sao Paulo, SP, BrazilFAPESP: 2011/12156-0Web of Scienc

Dolutegravir-associated resistance mutations after first-line treatment failure in Brazil

Abstract Background Since January 2017, the recommended first-line antiretroviral regimen in Brazil is the fixed-dose combination of tenofovir plus lamivudine with dolutegravir (TL + D). According to the literature, integrase resistance-associated mutations (INRAMs) are rarely found upon virologic failure to first-line dolutegravir plus two nucleoside reverse transcriptase inhibitors. We evaluated the HIV antiretroviral genotypic resistance profile of patients referred for genotyping in the public health system who failed first-line TL + D after at least six months of therapy on or before December 31, 2018. Methods HIV Sanger sequences of the pol gene were generated from plasma of patients with confirmed virologic failure to first-line TL + D in the Brazilian public health system before December 31, 2018. Results One hundred thirteen individuals were included in the analysis. Major INRAMs were detected in seven patients (6.19%), four with R263K, one with G118R, one with E138A, and one with G140R. Four patients with major INRAMs also had the K70E and M184V mutations in the RT gene. Sixteen (14.2%) additional individuals presented minor INRAMs, and five (4,42%) patients had both major and minor INRAMS. Thirteen (11.5%) patients also presented mutations in the RT gene selected by tenofovir and lamivudine, including four with both the K70E and M184V mutations and four with only M184V. The integrase mutations L101I and T124A, which are in the in vitro pathway for integrase inhibitor resistance, were found in 48 and 19 patients, respectively. Mutations not related to TL + D, thus probable transmitted resistance mutations (TDR), were present in 28 patients (24.8%): 25 (22.1%) to nucleoside reverse transcriptase inhibitors, 19 (16.8%) to non-nucleoside reverse transcriptase inhibitors, and 6 (5.31%) to protease inhibitors. Conclusions In marked contrast to previous reports, we report a relatively high frequency of INRAMs among selected patients failing first-line TL + D in the public health system in Brazil. Possible reasons for this discrepancy include delays in detecting virologic failure, patients inadvertently on dolutegravir monotherapy, TDR, and/or infecting subtype

High prevalence and incidence of HIV-1 in a counseling and testing center in the city of Itajaí, Brazil

ABSTRACT Itajaí is a port city in southern Brazil with one of the highest incidence and mortality rates from AIDS in the country. The prevalence and incidence of HIV infection were investigated in 1085 of 3196 new HIV-1 infection cases evaluated in the counseling and testing center of Itajaí from January 2002 to August 2008. Recent infections were assessed using the BED(tm), and polregion sequencing was performed in 76 samples. The prevalence ranged from 3.08% to 6.17% among women and from 10.26% to 17.36% among men. A total of 17% of infections were classified as recent, with annual incidence varying from 1.6% to 4.8 per 100 patient/year among women and from 2.05% to 8.5 per 100 patient/year among men. Pol sequences were obtained from 38 randomly recent infections selected individuals: 71% were infected by subtype C, 24% B, 2% D, and 2% F1. Among 38 subjects with established infection, 76% were subtype C, and 24% B. Transmitted drug resistance was detected in 18.4% of recent infection subjects (7.8% to nucleoside analog reverse-transcriptase inhibitors, 5.2% to non-nucleoside reverse-transcriptase inhibitors, and 5.2% protease inhibitors) and 5.2% of subjects with established infection had nucleoside analog reverse-transcriptase inhibitors resistance. The high prevalence and incidence of HIV infection in this region is unprecedented in studies involving cases evaluated in the counseling and testing centers in Brazil

Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes