A specific structure and high richness characterize intestinal microbiota of HIVexposed seronegative individuals

Intestinal microbiota facilitates food breakdown for energy metabolism and influences the im-mune response and maintaining mucosal homeostasis. Overall, HIV infection is associated with intestinal dysbiosis and immune activation, which has been related to seroconversion in HIV-exposed individuals. However, to date, it is unclear whether microbiota dysbiosis is the cause or the effect of immune alterations and disease progression. We characterize the intestinal microbiota and determine its association with immune regulation in HIV-exposed seronegative individuals (HESN), HIV-infected progressors (HIV+), and healthy control (HC) subjects. For this, feces and blood were collected. The microbiota composition of HESN showed a significantly higher alpha and beta diversity compared to HC, but similar to HIV+. A lower Treg percentage was observed in HESN than HC and HIV+, with enrichment of the genus Butyrivibrio being characteristic of this profile. Interestingly, an increase in Succinivibrio and Prevotella and a re-duction in Bacteroides genus were observed in HESN compared to HC, which is typical of HIV-infected individuals. Thus, HESNs have a microbiota profile, similar to that observed in HIV+, most likely because HESN are cohabiting with their HIV+ partners.Intestinal microbiota facilitates food breakdown for energy metabolism and influences the im-mune response and maintaining mucosal homeostasis. Overall, HIV infection is associated with intestinal dysbiosis and immune activation, which has been related to seroconversion in HIV-exposed individuals. However, to date, it is unclear whether microbiota dysbiosis is the cause or the effect of immune alterations and disease progression. We characterize the intestinal microbiota and determine its association with immune regulation in HIV-exposed seronegative individuals (HESN), HIV-infected progressors (HIV+), and healthy control (HC) subjects. For this, feces and blood were collected. The microbiota composition of HESN showed a significantly higher alpha and beta diversity compared to HC, but similar to HIV+. A lower Treg percentage was observed in HESN than HC and HIV+, with enrichment of the genus Butyrivibrio being characteristic of this profile. Interestingly, an increase in Succinivibrio and Prevotella and a re-duction in Bacteroides genus were observed in HESN compared to HC, which is typical of HIV-infected individuals. Thus, HESNs have a microbiota profile, similar to that observed in HIV+, most likely because HESN are cohabiting with their HIV+ partners.https://scienti.minciencias.gov.co/cvlac/EnProdArticulo/query.do?cod_producto=73&cod_rh=0000157775https://scholar.google.com.co/citations?hl=en&user=VLZxl1UAAAAJCOL0112548https://orcid.org/0000-0002-7351-873

Vitamin D modulates the expression of HLA-DR and CD38 after in vitro activation of T-cells

Objective: Vitamin D (VitD) is an anti-inflammatory hormone; however, some evidence shows that VitD may induce the expression of activation markers, such as CD38 and HLA-DR. We explored its effect on the expression of these markers on CD4+ and CD8+ T-cells in vitro, and their potential correlations in vivo. Materials and methods: CD38 and HLA-DR expression was measured by flow cytometry in PHA/IL-2-activated mononuclear cells cultured under VitD precursors: three cholecalciferol (10−11M, 10−9M, 10−7M; n=11) and two calcidiol (40 ng/mL, 80 ng/mL; n=9) concentrations. The correlation between the expression of these markers in freshly isolated blood cells and serum levels of calcidiol was also explored (n=10). Results: Cholecalciferol at 10−7M increased the proportion of CD4+ CD38+ and CD8+ CD38+ cells, and decreased CD8+HLA-DR+ cells. As co-expression, it increased the CD38+HLA-DR− and decreased CD38−HLA-DR+ subpopulations in both CD4+ and CD8+ T-cells, and decreased CD4+CD38−HLA-DR− and CD8+ CD38+HLA-DR+; whereas both calcidiol concentrations decreased the proliferation of CD38−HLA-DR− and CD38−HLA-DR+ subpopulations. Both forms of VitD increased the number of CD38 molecules per cell. In contrast, there was a positive but non-significant correlation between serum calcidiol levels and the expression of CD38 and HLA-DR in CD4+ and CD8+ T-cells. Conclusion: Although no significant correlations were observed in vivo in healthy subjects, VitD treatment in vitro modulated immune activation by increasing the expression of CD38 and decreasing the proliferation of HLA-DR+ and resting cells, which may correlate with improved effector and decreased proliferative capabilities. These results highlight the potential use of VitD as therapeutic strategy in immune disorders

Calcitriol decreases HIV-1 transfer in vitro from monocyte-derived dendritic cells to CD4 + T cells, and downregulates the expression of DC-SIGN and SIGLEC-1.

Dendritic cells (DCs) promote HIV-1 transmission by acting as Trojan horses, capturing viral particles, facilitating the infection of CD4+ T-cells. Vitamin D (VitD) has shown to decrease T cell activation, reducing susceptibility to HIV-1 infection of CD4+ T-cells in vitro; however, if VitD decreases viral transfer from DCs to CD4+ T-cells is unknown. In this study, we co-cultured HIV-1-pulsed immature and LPS mature monocytes-derived DCs (iDCs and LmDCs, respectively), differentiated in presence or absence of calcitriol (VitD active form), with PHA-activated autologous CD4+ T-cells from 16 healthy donors. In co-cultures of iDCs and LmDCs treated with calcitriol, there was a significant decrease in frequency of infected CD4+ T-cells, evaluated by flow cytometry. However, p24 levels evaluated by ELISA were not significantly reduced in culture supernatants. Moreover, calcitriol-treated iDCs exhibited decreased expression of genes involved in HIV-1 transfer compared to the control. Both, calcitriol-treated iDCs and LmDCs exhibit a similar gene expression profile, probably related to a transcriptional balance achieved after long treatment with calcitriol. Since calcitriol-differentiated DCs express on their surface a lower amount of DC-SIGN and SIGLEC-1 molecules, widely associated with HIV-1 transfer, suggesting that this mechanism contributes to a lower transfer of viral particles by the DCs

Vitamin D treatment of peripheral blood mononuclear cells modulated immune activation and reduced susceptibility to HIV-1 infection of CD4+ T lymphocytes.

IntroductionMucosal immune activation, in the context of sexual transmission of HIV-1 infection, is crucial, as the increased presence of activated T cells enhance susceptibility to infection. In this regard, it has been proposed that immunomodulatory compounds capable of modulating immune activation, such as Vitamin D (VitD) may reduce HIV-1 transmission and might be used as a safe and cost-effective strategy for prevention. Considering this, we examined the in vitro effect of the treatment of peripheral blood mononuclear cells (PBMCs) with the active form of VitD, calcitriol, on cellular activation, function and susceptibility of CD4+ T cells to HIV-1 infection.MethodsWe treated PBMCs from healthy HIV unexposed individuals (Co-HC) and frequently exposed, HIV-1 seronegative individuals (HESNs) from Colombia and from healthy non-exposed individuals from Canada (Ca-HC) with calcitriol and performed in vitro HIV-1 infection assays using X4- and R5-tropic HIV-1 strains respectively. In addition, we evaluated the activation and function of T cells and the expression of viral co-receptors, and select antiviral genes following calcitriol treatment.ResultsCalcitriol reduced the frequency of infected CD4+ T cells and the number of viral particles per cell, for both, X4- and R5-tropic viruses tested in the Co-HC and the Ca-HC, respectively, but not in HESNs. Furthermore, in the Co-HC, calcitriol reduced the frequency of polyclonally activated T cells expressing the activation markers HLA-DR and CD38, and those HLA-DR+CD38-, whereas increased the subpopulation HLA-DR-CD38+. Calcitriol treatment also decreased production of granzyme, IL-2 and MIP-1β by T cells and increased the transcriptional expression of the inhibitor of NF-kB and the antiviral genes cathelicidin (CAMP) and APOBEC3G in PBMCs from Co-HC.ConclusionOur in vitro findings suggest that VitD treatment could reduce HIV-1 transmission through a specific modulation of the activation levels and function of T cells, and the production of antiviral factors. In conclusion, VitD remains as an interesting potential strategy to prevent HIV-1 transmission that should be further explored

The First Chemically-Synthesised, Highly Immunogenic Anti-SARS-CoV-2 Peptides in DNA Genotyped Aotus Monkeys for Human Use

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRβ1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world’s population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides’ potential coverage for the world populations up to 62.9%. These peptides’ 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology’s potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human populationIncluye referencias bibliográfica

The Hydroalcoholic Extract of Uncaria tomentosa (Cat’s Claw) Inhibits the Infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) In Vitro

The coronavirus disease 2019 (COVID-19) has become a serious problem for public health since it was identified in the province of Wuhan (China) and spread around the world producing high mortality rates and economic losses. Nowadays, WHO recognizes traditional, complementary, and alternative medicine for treating COVID-19 symptoms. Therefore, we investigated the antiviral potential of the hydroalcoholic extract of Uncaria tomentosa stem bark from Peru against SARS-CoV-2 in vitro. The antiviral activity of U. tomentosa against SARS-CoV-2 in vitro was assessed in Vero E6 cells using cytopathic effect (CPE) and plaque reduction assay. After 48h of treatment, U. tomentosa showed an inhibition of 92.7 % of SARS-CoV-2 at 25.0 µg/mL (p<0.0001) by plaque reduction assay on Vero E6 cells. In addition, U. tomentosa, induced a reduction of 98.6 % (p=0.02) and 92.7 % (p=0.03) in the CPE caused by SARS-CoV-2 on Vero E6 cells at 25 µg/mL and 12.5 µg/mL, respectively. The EC50 calculated for U. tomentosa extract by plaque reduction assay was 6.6 µg/mL (4.89 – 8.85 µg/mL) for a selectivity index of 4.1. The EC50 calculated for U. tomentosa extract by TCID50 assay was 2.57 µg/mL (1.05 – 3.75 µg/mL) for a selectivity index of 10.54. These results showed that U. tomentosa known as Cat´s claw has antiviral effect against SARS-CoV-2 observed as a reduction in the viral titer and CPE after 48h of treatment on Vero E6 cells. Therefore, we hypothesized that U. tomentosa stem bark, could be promissory to the development of new therapeutic strategies against SARS-CoV-2.The coronavirus disease 2019 (COVID-19) has become a serious problem for public health since it was identified in the province of Wuhan (China) and spread around the world producing high mortality rates and economic losses. Nowadays, WHO recognizes traditional, complementary, and alternative medicine for treating COVID-19 symptoms. Therefore, we investigated the antiviral potential of the hydroalcoholic extract of Uncaria tomentosa stem bark from Peru against SARS-CoV-2 in vitro. The antiviral activity of U. tomentosa against SARS-CoV-2 in vitro was assessed in Vero E6 cells using cytopathic effect (CPE) and plaque reduction assay. After 48h of treatment, U. tomentosa showed an inhibition of 92.7 % of SARS-CoV-2 at 25.0 µg/mL (p<0.0001) by plaque reduction assay on Vero E6 cells. In addition, U. tomentosa, induced a reduction of 98.6 % (p=0.02) and 92.7 % (p=0.03) in the CPE caused by SARS-CoV-2 on Vero E6 cells at 25 µg/mL and 12.5 µg/mL, respectively. The EC50 calculated for U. tomentosa extract by plaque reduction assay was 6.6 µg/mL (4.89 – 8.85 µg/mL) for a selectivity index of 4.1. The EC50 calculated for U. tomentosa extract by TCID50 assay was 2.57 µg/mL (1.05 – 3.75 µg/mL) for a selectivity index of 10.54. These results showed that U. tomentosa known as Cat´s claw has antiviral effect against SARS-CoV-2 observed as a reduction in the viral titer and CPE after 48h of treatment on Vero E6 cells. Therefore, we hypothesized that U. tomentosa stem bark, could be promissory to the development of new therapeutic strategies against SARS-CoV-2.https://scienti.minciencias.gov.co/cvlac/EnProdArticulo/query.do?cod_producto=73&cod_rh=0000157775https://scholar.google.com.co/citations?hl=en&user=VLZxl1UAAAAJCOL0112548https://orcid.org/0000-0002-7351-873

Variants in the CYP7B1 gene region do not affect natural resistance to HIV-1 infection

Ajuts: Estrategia de Sostenibilidad 2014-2015 de la Universidad de Antioquia, NIH‑NIDCR R01 DE018925‑04, HIVACAT program i CUTHIVAC 241904Abstract.BACKGROUND: The genetic bases of natural resistance to HIV-1 infection remain largely unknown. Recently, two genome-wide association studies suggested a role for variants within or in the vicinity of the CYP7B1 gene in modulating HIV susceptibility. CYP7B1 is an appealing candidate for this due to its contribution to antiviral immune responses. We analyzed the frequency of two previously described CYP7B1 variants (rs6996198 and rs10808739) in three independent cohorts of HIV-1 infected subjects and HIV-1 exposed seronegative individuals (HESN). FINDINGS:rs6996198 and rs10808739 were genotyped in three case/control cohorts of sexually-exposed HESN and HIV-1-infected individuals from Italy, Peru and Colombia. Comparison of the allele and genotype frequencies of the two SNPs under different models showed that the only significant difference was seen for rs6996198 in the Peruvian sample (nominal p = 0.048, dominant model). For this variant, a random-effect meta-analysis yielded non-significant results (dominant model, p = 0.78) and revealed substantial heterogeneity among cohorts. No significant effect of the rs10808739 allelic status on HIV-1 infection susceptibility (additive model, p = 0.30) emerged from the meta-analysis.CONCLUSIONS: Although our study had limited power to detect association due to the small sample size, comparisons among the three cohorts revealed very similar allelic and genotypic frequencies in HESN and HIV-1 positive subjects. Overall, these data indicate that the two GWAS-defined variants in the CYP7B1 region do not strongly influence HIV-1 infection susceptibility

A 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 confers protections against HIV-1 infection.

Infection of cells by enveloped viruses is a multi-step process requiring both the binding of viral glycoproteins to specific cellular receptors/coreceptors and less specific interactions with accessory molecules whose main function is to locate the virus closer to its receptor(s) [1]. Among such attachment factors, a key role has been attributed to the TIM (T-cell immunoglobulin and mucin domain containing) family receptors, cell surface glycoproteins that control both innate and acquired immune responses during allergy, asthma, tolerance, autoimmunity, as well as viral infections [2]. In the human genome, three genes (HAVCR1, HAVCR2 and TIMD4) encode TIM proteins (TIM-1, TIM-3, and TIM-4, respectively). Structurally, all TIM proteins have a conserved ectodomain consisting of an immunoglobulin (IgV)-like domain and an heavily glycosylated mucin-like domain, anchored to the cell through a transmembrane domain followed by a cytoplasmic tail [3]. TIM-1, in particular, is mostly expressed by hepatocytes and lymphoid cells, preferentially Th2 cells, and is a key T-cell costimulatory molecule that controls T cell activation [4,5]. Human TIM-1, originally identified as the receptor for hepatitis A virus (HAV) [6], promotes the entry of a wide variety of enveloped viruses in host cells [7]. Virus internalization occurs when TIM binds phosphatidylserine (PtdSer) on the viral envelope; this process seems to be independent of viral glycoprotein interaction with cellular receptors [7,8]. A recent analysis of the role of TIM-1 domains indicated that, whereas the IgV domain is essential for virus binding and internalization, the mucin-like domain also plays a key role in enhancing viral entry [8]. Specifically, the use of deletion mutants indicated that a stalk of adequate length is necessary to form an extended structure that places the IgV domain within the appropriate distance from the host cell membrane, thus allowing optimal interactions with the virus [8]. Interestingly, the HAVCR1 gene is highly polymorphic in human populations. In particular, natural selection has maintained high nucleotide diversity in exon 4, which encodes a portion of the mucin-like domain [9]. The selective pressure acting on this region is believed to be virus-mediated, suggesting that polymorphisms in exon 4 modulate viral infection susceptibility and/or diseases severity. In fact, an 18-bp insertion/deletion polymorphism in the exon, causing a six amino acid insertion/deletion variant (157ins/delMTTTVP), was associated with the risk to develop acute liver failure following HAV infection [10]; the same variant was found to modulate AIDS progression in HIV-1 infected subjects [11]. Notably, in both studies the deleted (short) allele of 157ins/delMTTTVP exerted a protective effect. In the HAV study, having one or two copies of the long form of TIM1 was associated with a greater risk to develop severe liver failure, indicating that the protective effect of the short allele is recessive [10]. These data are in line with the observation that the length of the mucin-like domain is critical for enhancing enveloped virus entry [8]. In fact, TIM-1 molecules with a short mucin-like domain (157delMTTTVP) bind HAV less efficiently than those with a long domain (157insMTTTVP) [10]. Herein we assessed whether the HAVCR1 (Hepatitis A virus cellular receptor 1) 18-bp insertion/deletion polymorphism modulates susceptibility to HIV-1 infection in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals. Results indicated that homozygosity for the short allele is associated with natural protection from infection and lower rate of HIV-1 replication in CD4þ T lymphocytesWe investigated whether a 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 (T-cell immunoglobulin and mucin domain 1), modulates susceptibility to HIV-1 infection. The polymorphism was genotyped in three case/control cohorts of HIV-1 exposed seronegative individuals (HESN) and HIV-1 infected subjects from Italy, Peru, and Colombia; data from a Thai population were retrieved from the literature. Across all cohorts, homozygosity for the short TIM-1 allele was more common in HESNs than in HIV-1 infected subjects. A metaanalysis of the four association analyses yielded a p value of 0.005. In vitro infection assays of CD4þ T lymphocytes indicated that homozygosity for the short allele is associated with lower rate of HIV-1 replication. These results suggest that the deletion allele protects from HIV-1 infection with a recessive effect

Variants in the CYP7B1 gene region do not affect natural resistance to HIV-1 infection

Background: The genetic bases of natural resistance to HIV-1 infection remain largely unknown. Recently, two genome-wide association studies suggested a role for variants within or in the vicinity of the CYP7B1 gene in modulating HIV susceptibility. CYP7B1 is an appealing candidate for this due to its contribution to antiviral immune responses. We analyzed the frequency of two previously described CYP7B1 variants (rs6996198 and rs10808739) in three independent cohorts of HIV-1 infected subjects and HIV-1 exposed seronegative individuals (HESN). Findings: rs6996198 and rs10808739 were genotyped in three case/control cohorts of sexually-exposed HESN and HIV-1-infected individuals from Italy, Peru and Colombia. Comparison of the allele and genotype frequencies of the two SNPs under different models showed that the only significant difference was seen for rs6996198 in the Peruvian sample (nominal p = 0.048, dominant model). For this variant, a random-effect meta-analysis yielded non-significant results (dominant model, p = 0.78) and revealed substantial heterogeneity among cohorts. No significant effect of the rs10808739 allelic status on HIV-1 infection susceptibility (additive model, p = 0.30) emerged from the meta-analysis. Conclusions: Although our study had limited power to detect association due to the small sample size, comparisons among the three cohorts revealed very similar allelic and genotypic frequencies in HESN and HIV-1 positive subjects. Overall, these data indicate that the two GWAS-defined variants in the CYP7B1 region do not strongly influence HIV-1 infection susceptibility