theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

HIV Reverse Transcriptase Fidelity And Inhibition Are Modulated By Divalent Cations In A Concentration-Dependent Manner In Vitro

Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.

Envelope C2-V3-C3-specific antibodies from HIV-1 infected patients from Angola correlate with neutralization activity in plasma

Poster presented at the 2015 Keystone Symposia Conference X5: HIV Vaccines. Banff, Alberta, Canada, 22-27 March 2015

灵长类动物模型在抗艾滋病毒药物研究中的应用

艾滋病(AIDS)灵长类动物模型广泛用于艾滋病毒(HIV)研究.较为常用的有猴免疫缺陷病毒(SIV)/猕猴模型和猴/人免疫缺陷嵌合病毒(SHIV)/猕猴模型,除了在发病机制和抗HIV疫苗研究中的重要作用外,这2种模型在抗HIV药物研究中同样显示出优越性和实用性,体现在药物疗效的研究、新药的开发、耐药性研究、机体免疫反应在药物治疗作用的研究中.通过这些体内抗病毒活性及机制的研究,可进一步揭示药物的抗HIV疗效,对AIDS的临床用药具有重要的指导作用,将使人们重新考虑抗HIV药物的用药原则和药效评价标准.

Immune Responses to Six Synthetic Peptides of Capsid Protein Immune Responses to Six Synthetic Peptides of Capsid Protein

Many B cell epitopes within p24 of human immunodeficiency virus type 1 (HIV-1) were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera (19.4%), all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals. Cellular & Molecular Immunology. 2005;2(4):289-293.

A transcriptase reversa como alvo terapêutico em doenças retrovirais

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Evolutionary and Functional Analyses of the Interaction between the Myeloid Restriction Factor SAMHD1 and the Lentiviral Vpx Protein.

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.

Mutagenicidade de duas espécies do gěnero Alchornea avaliadas através de ensaios com Salmonella microssomo e teste do micronúcleo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oral syphilis: report of three cases and characterization of the inflammatory cells

Syphilis is a sexually transmitted infectious disease caused by Treponema pallidum. This study reports 3 cases of syphilis and highlights the importance of identifying oral lesions for its final diagnosis. Case 1: a 48-year-old male patient presented with a bleeding ulcer in the lower lip. Overall clinical examination revealed patchy alopecia and skin target lesions. Case 2: a 61-year-old male patient presented with white spots on the lateral tongue and nodules on the dorsum of the tongue. Overall clinical examination showed erythematous target lesions on the abdomen, forearm, palms of the hand, and soles of the feet. Case 3: a 17-year-old male patient presented with an ulcerated lesion on the tongue and lymph node involvement. The following serologic tests were requested: Venereal Disease Research Laboratory, fluorescent treponemal antibody-absorption, anti-HIV-1 and anti-HIV-2, and anti-hepatitis C virus. An incisional biopsy revealed epithelial hyperplasia associated with intense and diffuse mononuclear inflammatory cell infiltration consisting mainly of plasma cells, in a perivascular and perineural distribution. The final diagnosis in the 3 patients was syphilis. Treatment consisted of 1 weekly dose of penicillin (2.4 million units, intramuscular) for 2 or 3 weeks. Immunohistochemical reactions for XIIIa, CD3, CD20, CD68, CD163, S100, CD1a, CD11c, CD83, CD138, and CD208 were performed. Clinicians should be familiarized with oral syphilis lesions in order to be able to diagnose this emerging infectious disease of variable clinical presentation.

Prevalência de infecções sexualmente transmissíveis e de alterações da microbiota vaginal e fatores associados em mulheres que fazem sexo com mulheres

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular evolution of primate immunodeficiency viruses and hepatitis delta virus

Primate immunodeficiency viruses, or lentiviruses (HIV-1, HIV-2, and SIV), and hepatitis delta virus (HDV) are RNA viruses characterized by rapid evolution. Infection by primate immunodeficiency viruses usually results in the development of acquired immunodeficiency syndrome (AIDS) in humans and AIDS-like illnesses in Asian macaques. Similarly, hepatitis delta virus infection causes hepatitis and liver cancer in humans. These viruses are heterogeneous within an infected patient and among individuals. Substitution rates in the virus genomes are high and vary in different lineages and among sites. Methods of phylogenetic analysis were applied to study the evolution of primate lentiviruses and the hepatitis delta virus. The following results have been obtained: (1) The substitution rate varies among sites of primate lentivirus genes according to the two parameter gamma distribution, with the shape parameter $\alpha$ being close to 1. (2) Primate immunodeficiency viruses fall into species-specific lineages. Therefore, viral transmissions across primate species are not as frequent as suggested by previous authors. (3) Primate lentiviruses have acquired or lost their pathogenicity several times in the course of evolution. (4) Evidence was provided for multiple infections of a North American patient by distinct HIV-1 strains of the B subtype. (5) Computer simulations indicate that the probability of committing an error in testing HIV transmission depends on the number of virus sequences and their length, the divergence times among sequences, and the model of nucleotide substitution. (6) For future investigations of HIV-1 transmissions, using longer virus sequences and avoiding the use of distant outgroups is recommended. (7) Hepatitis delta virus strains are usually related according to the geographic region of isolation. (8) Evolution of HDV is characterized by the rate of synonymous substitution being lower than the nonsynonymous substitution rate and the rate of evolution of the noncoding region. (9) There is a strong preference for G and C nucleotides at the third codon positions of the HDV coding region. ^

Monitoring and Switching of First-line Antiretroviral Therapy in sub-Saharan Africa: Collaborative Analysis of Adult Treatment Cohorts.

BACKGROUND HIV-1 viral load (VL) testing is recommended to monitor antiretroviral therapy (ART) but not universally available. We examined monitoring of first-line and switching to second-line ART in sub-Saharan Africa, 2004-2013. METHODS Adult HIV-1 infected patients starting combination ART in 16 countries were included. Switching was defined as a change from a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen to a protease inhibitor (PI)-based regimen, with a change of ≥1 NRTI. Virological and immunological failures were defined per World Health Organization criteria. We calculated cumulative probabilities of switching and hazard ratios with 95% confidence intervals (CI) comparing routine VL monitoring, targeted VL monitoring, CD4 cell monitoring and clinical monitoring, adjusted for programme and individual characteristics. FINDINGS Of 297,825 eligible patients, 10,352 patients (3·5%) switched during 782,412 person-years of follow-up. Compared to CD4 monitoring hazard ratios for switching were 3·15 (95% CI 2·92-3·40) for routine VL, 1·21 (1·13-1·30) for targeted VL and 0·49 (0·43-0·56) for clinical monitoring. Overall 58.0% of patients with confirmed virological and 19·3% of patients with confirmed immunological failure switched within 2 years. Among patients who switched the percentage with evidence of treatment failure based on a single CD4 or VL measurement ranged from 32·1% with clinical to 84.3% with targeted VL monitoring. Median CD4 counts at switching were 215 cells/µl under routine VL monitoring but lower with other monitoring (114-133 cells/µl). INTERPRETATION Overall few patients switched to second-line ART and switching occurred late in the absence of routine viral load monitoring. Switching was more common and occurred earlier with targeted or routine viral load testing.

Age in antiretroviral therapy programmes in South Africa: a retrospective, multicentre, observational cohort study.

BACKGROUND As access to antiretroviral therapy (ART) expands, increasing numbers of older patients will start treatment and need specialised long-term care. However, the effect of age in ART programmes in resource-constrained settings is poorly understood. The HIV epidemic is ageing rapidly and South Africa has one of the highest HIV population prevalences worldwide. We explored the effect of age on mortality of patients on ART in South Africa and whether this effect is mediated by baseline immunological status. METHODS In this retrospective cohort analysis, we studied HIV-positive patients aged 16-80 years who started ART for the first time in six large South African cohorts of the International Epidemiologic Databases to Evaluate AIDS-Southern Africa collaboration, in KwaZulu-Natal, Gauteng, and Western Cape (two primary care clinics, three hospitals, and a large rural cohort). The primary outcome was mortality. We ascertained patients' vital status through linkage to the National Population Register. We used inverse probability weighting to correct mortality for loss to follow-up. We estimated mortality using Cox's proportional hazards and competing risks regression. We tested the interaction between baseline CD4 cell count and age. FINDINGS Between Jan 1, 2004, and Dec 31, 2013, 84,078 eligible adults started ART. Of these, we followed up 83,566 patients for 174,640 patient-years. 8% (1817 of 23,258) of patients aged 16-29 years died compared with 19% (93 of 492) of patients aged 65 years or older. The age adjusted mortality hazard ratio was 2·52 (95% CI 2·01-3·17) for people aged 65 years or older compared with those 16-29 years of age. In patients starting ART with a CD4 count of less than 50 cells per μL, the adjusted mortality hazard ratio was 2·52 (2·04-3·11) for people aged 50 years or older compared with those 16-39 years old. Mortality was highest in patients with CD4 counts of less than 50 cells per μL, and 15% (1103 of 7295) of all patients aged 50 years or older starting ART were in this group. The proportion of patients aged 50 years or older enrolling in ART increased with successive years, from 6% (290 of 4999) in 2004 to 10% (961 of 9657) in 2012-13, comprising 9% of total enrolment (7295 of 83 566). At the end of the study, 6304 (14%) of 44,909 patients still alive and in care were aged 50 years or older. INTERPRETATION Health services need reorientation towards HIV diagnosis and starting of ART in older individuals. Policies are needed for long-term care of older people with HIV. FUNDING National Institutes of Health (National Institute of Allergy and Infectious Diseases), US Agency for International Development, and South African Centre for Epidemiological Modelling and Analysis.

Perfil clínico y social del paciente adicto hospitalizado

Objetivos: Determinar las características clínicas y morbilidad de los pacientes (pac.) adictos ingresados a un Servicio de Clínica Médica. Material y métodos: Estudio protocolizado, observacional, descriptivo y transversal. Criterio de inclusión: pac. adicto con consumo de sustancias ilícitas. Informe preliminar: Periodo: 24 meses. Datos analizados en Epi info 6.4 Resultados: Se incluyeron 40 pac. Prevalencia: 0.2/1000 egresos. El 82.5% eran hombres. Edad media: 31.5 años (DS±9.65). Permanencia media: 11 días (DS±13.98) vs 7.12 (DS±10.1) del Servicio (p=0.016). Consumían cocaína el 82.5% (IC95%67.22-92.66) marihuana el 77.5% (IC95% 61.65- 89.16), tolueno el 10% (IC95%2.79-23.66), floripondio y hachis el 2.5% (IC95% 0.06-13.16) cada uno. En el 2.5% la vía de administración era endovenosa y el 60% eran poliadictos. Solo el 10% recibió tratamiento para abandonar la adicción (100% tratamiento psicológico y 5.13% farmacológico). De clase social pobre el 75%. El 62.5% de los pac. estaban desocupados, tenían antecedentes judiciales el 10% y el 7.5% había estado en prisión. El 92.5% (IC95% 76.34-97.21) era heterosexual, 2.5% hombres que tenían sexo con hombres y bisexuales 5% (IC95% 0.61-16.92). Solo el 10% tenían secundaria completa. El 80% ingreso por Emergencias y por causa infecciosa el 45%. El 12.5% ingreso por complicaciones de la adicción. Las manifestaciones relacionadas con la adicción fueron: manifestaciones del sistema nervioso central: 12.5% (IC95%4.19-26.80); síndrome de abstinencia y temblor 7.5% cada uno y excitación psicomotriz, delirium, signo de foco neurológico y rigidez 5% cada uno. El 100% presentaba alguna comórbida; tabaquismo 80%, depresión 12.8% Y alcoholismo 57.5%, entre otras. Presentaban enfermedades de transmisión sexual 5 pac. (4 HIV, 2 VHC y 1 pediculus pubis). No hubo mortalidad hospitalaria. Conclusión: El paciente adicto internado se caracteriza por ser joven, pertenecer a un grupo social desprotegido, sin trabajo y sin educación, tener poliadicción, con alta carga de comórbidas y de internación, principalmente por complicaciones infecciosas.

Multiplex detection of four pathogenic retroviruses using molecular beacons

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100,000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.