Estimating the Threshold Surface Density of Gp120-CCR5 Complexes Necessary for HIV-1 Envelope-Mediated Cell-Cell Fusion

Reduced expression of CCR5 on target CD4(+) cells lowers their susceptibility to infection by R5-tropic HIV-1, potentially preventing transmission of infection and delaying disease progression. Binding of the HIV-1 envelope (Env) protein gp120 with CCR5 is essential for the entry of R5 viruses into target cells. The threshold surface density of gp120-CCR5 complexes that enables HIV-1 entry remains poorly estimated. We constructed a mathematical model that mimics Env-mediated cell-cell fusion assays, where target CD4(+)CCR5(+) cells are exposed to effector cells expressing Env in the presence of a coreceptor antagonist and the fraction of target cells fused with effector cells is measured. Our model employs a reaction network-based approach to describe protein interactions that precede viral entry coupled with the ternary complex model to quantify the allosteric interactions of the coreceptor antagonist and predicts the fraction of target cells fused. By fitting model predictions to published data of cell-cell fusion in the presence of the CCR5 antagonist vicriviroc, we estimated the threshold surface density of gp120-CCR5 complexes for cell-cell fusion as similar to 20 mu m(-2). Model predictions with this threshold captured data from independent cell-cell fusion assays in the presence of vicriviroc and rapamycin, a drug that modulates CCR5 expression, as well as assays in the presence of maraviroc, another CCR5 antagonist, using sixteen different Env clones derived from transmitted or early founder viruses. Our estimate of the threshold surface density of gp120-CCR5 complexes necessary for HIV-1 entry thus appears robust and may have implications for optimizing treatment with coreceptor antagonists, understanding the non-pathogenic infection of non-human primates, and designing vaccines that suppress the availability of target CD4(+)CCR5(+) cells.

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

Background: India has the third largest HIV-1 epidemic with 2.4 million infected individuals. Molecular epidemiological analysis has identified the predominance of HIV-1 subtype C (HIV-1C). However, the previous reports have been limited by sample size, and uneven geographical distribution. The introduction of HIV-1C in India remains uncertain due to this lack of structured studies. To fill the gap, we characterised the distribution pattern of HIV-1 subtypes in India based on data collection from nationwide clinical cohorts between 2007 and 2011. We also reconstructed the time to the most recent common ancestor (tMRCA) of the predominant HIV-1C strains. Methodology/Principal Findings: Blood samples were collected from 168 HIV-1 seropositive subjects from 7 different states. HIV-1 subtypes were determined using two or three genes, gag, pol, and env using several methods. Bayesian coalescent-based approach was used to reconstruct the time of introduction and population growth patterns of the Indian HIV-1C. For the first time, a high prevalence (10%) of unique recombinant forms (BC and A1C) was observed when two or three genes were used instead of one gene (p<0.01; p = 0.02, respectively). The tMRCA of Indian HIV-1C was estimated using the three viral genes, ranged from 1967 (gag) to 1974 (env). Pol-gene analysis was considered to provide the most reliable estimate 1971, (95% CI: 1965-1976)]. The population growth pattern revealed an initial slow growth phase in the mid-1970s, an exponential phase through the 1980s, and a stationary phase since the early 1990s. Conclusions/Significance: The Indian HIV-1C epidemic originated around 40 years ago from a single or few genetically related African lineages, and since then largely evolved independently. The effective population size in the country has been broadly stable since the 1990s. The evolving viral epidemic, as indicated by the increase of recombinant strains, warrants a need for continued molecular surveillance to guide efficient disease intervention strategies.

Cell Surface Assembly of HIV gp41 Six-Helix Bundles for Facile, Quantitative Measurements of Hetero-oligomeric Interactions

Helix helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.

Stochastic Simulations Suggest that HIV-1 Survives Close to Its Error Threshold

The use of mutagenic drugs to drive HIV-1 past its error threshold presents a novel intervention strategy, as suggested by the quasispecies theory, that may be less susceptible to failure via viral mutation-induced emergence of drug resistance than current strategies. The error threshold of HIV-1, mu(c), however, is not known. Application of the quasispecies theory to determine mu(c) poses significant challenges: Whereas the quasispecies theory considers the asexual reproduction of an infinitely large population of haploid individuals, HIV-1 is diploid, undergoes recombination, and is estimated to have a small effective population size in vivo. We performed population genetics-based stochastic simulations of the within-host evolution of HIV-1 and estimated the structure of the HIV-1 quasispecies and mu(c). We found that with small mutation rates, the quasispecies was dominated by genomes with few mutations. Upon increasing the mutation rate, a sharp error catastrophe occurred where the quasispecies became delocalized in sequence space. Using parameter values that quantitatively captured data of viral diversification in HIV-1 patients, we estimated mu(c) to be 7 x 10(-5) -1 x 10(-4) substitutions/site/replication, similar to 2-6 fold higher than the natural mutation rate of HIV-1, suggesting that HIV-1 survives close to its error threshold and may be readily susceptible to mutagenic drugs. The latter estimate was weakly dependent on the within-host effective population size of HIV-1. With large population sizes and in the absence of recombination, our simulations converged to the quasispecies theory, bridging the gap between quasispecies theory and population genetics-based approaches to describing HIV-1 evolution. Further, mu(c) increased with the recombination rate, rendering HIV-1 less susceptible to error catastrophe, thus elucidating an added benefit of recombination to HIV-1. Our estimate of mu(c) may serve as a quantitative guideline for the use of mutagenic drugs against HIV-1.

Design of an Escherichia coli expressed HIV-1 gp120 fragment Immunogen that binds to b12 and induces broad and potent neutralizing antibodies

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.

Simulations reveal that the HIV-1 gp120-CD4 complex dissociates via complex pathways and is a potential target of the polyamidoamine (PAMAM) dendrimer

The polyamidoamine (PAMAM) dendrimer prevents HIV-1 entry into target cells in vitro. Its mechanism of action, however, remains unclear and precludes the design of potent dendrimers targeting HIV-1 entry. We employed steered molecular dynamics simulations to examine whether the HIV-1 gp120-CD4 complex is a target of PAMAM. Our simulations mimicked single molecule force spectroscopy studies of the unbinding of the gp120-CD4 complex under the influence of a controlled external force. We found that the complex dissociates via complex pathways and defies the standard classification of adhesion molecules as catch and slip bonds. When the force loading rate was large, the complex behaved as a slip bond, weakening gradually. When the loading rate was small, the complex initially strengthened, akin to a catch bond, but eventually dissociated over shorter separations than with large loading rates. PAMAM docked to gp120 and destabilized the gp120-CD4 complex. The rupture force of the complex was lowered by PAMAM. PAMAM disrupted salt bridges and hydrogen bonds across the gp120-CD4 interface and altered the hydration pattern of the hydrophobic cavity in the interface. In addition, intriguingly, PAMAM suppressed the distinction in the dissociation pathways of the complex between the small and large loading rate regimes. Taken together, our simulations reveal that PAMAM targets the gp120-CD4 complex at two levels: it weakens the complex and also alters its dissociation pathway, potentially inhibiting HIV-1 entry.

New solid forms of the anti-HIV drug etravirine: salts, cocrystals, and solubility

Thirteen new solid forms of etravirine were realized in the process of polymorph and cocrystal/salt screening to improve the solubility of this anti-HIV drug. One anhydrous form, five salts (hydrochloride, mesylate, sulfate, besylate, and tosylate), two cocrystals (with adipic acid and 1,3,5-benzenetricarboxylic acid), and five solvates (formic acid, acetic acid, acetonitrile, and 2:1 and 1:1 methanolates) were obtained. The conformational flexibility of etravirine suggests that it can adopt four different conformations, and among these, two are sterically favorable. However, in all 13 solid forms, the active pharmaceutical ingredient (API) was found to adopt just one conformation. Due to the poor aqueous solubility of the API, the solubilities of the salts and cocrystals were measured in a 50% ethanol water mixture at neutral pH. Compared to the salts, the cocrystals were found to be stable and showed an improvement in solubility with time. All the salts were dissociated within an hour, except the tosylate, which showed 50% phase transformation after 1 h of the slurry experiment. A structure property relationship was examined to analyze the solubility behavior of the solid forms.

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E-GSH; Grx1-roGFP2) and measured subcellular changes in E-GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E-GSH (approximately -310 mV), active viral replication induces substantial oxidative stress (E-GSH more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E-GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about similar to 25 mV in E-GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E-GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Transmissão vertical do hiv e transplante de fígado

"Aborda dois Projetos de Lei na área de saúde pública em tramitação no Congresso Nacional. As proposições referidas são: o Projeto de Lei n° 5522, de 2005, que dispõe sobre a obrigatoriedade da implementação de protocolo terapêutico para a prevenção da transmissão vertical do HIV, e o Projeto de Lei n.º 2.745, de 2003, que dispõe sobre as regras para elaboração da lista nacional de receptores de fígado do Sistema Nacional de Transplante."

Proceedings of the International Workshop responding to HIV and AIDS in the fishery sector in Africa, Lusaka, Zambia, 21-22 February 2006

The purpose of the workshop was to enable professionals and organizations working with fishing communities in response to HIV and AIDS in Africa to share experiences, appraise the efficacy of their approaches and identify actions in research and development that will further improve their impact. The workshop pursued and achieved the following objectives: 1)Review and compare research findings and approaches applied in response to HIV and AIDS in fishing communities and the wider fishery sector. 2)Identify good practice examples for wider application. 3)Identify next steps in development and research to scale up these examples. 4)Initiate a network of practitioners in Africa for capacity building, scaling-up and further development of approaches. The range of papers presented at the conference reveals the diversity of responses to HIV and AIDS in the fishery sector at all levels. The papers discussed a range of issues within this broad remit, from community level impacts of disease to policy implementation, from spatial mapping to theatre as a mode of communication. (Document contains 92 pages)

Adapting integrated agriculture aquaculture for HIV and AIDS-affected households: the case of Malawi

The WorldFish Center in conjunction with World Vision Malawi carried out a project to improve income and nutrition status of households affected by HIV and AIDS with funding from the World Bank. The project was implemented in Southern Malawi particularly in the West of Zomba District from July 2005 to June 2006. Through participatory approaches, the project identified constraints that limit HIV and AIDS affected households’ realisation of the benefits from fish farming and adapted technologies and practices for the affected beneficiaries to boost fish production and utilization. Specifically, the project sought (1) to identify the constraints that limit HIV and AIDS affected households to realise the benefits from fish farming and based on the constraints, (2) to adapt technologies and practices for use by the affected beneficiaries to boost fish production and utilization. (PDF cotains 17 pages)

Diário da Constituinte [gravação de vídeo] : [programa n. 676 2ª ed.]

Debate acerca do marco legal sobre a comercialização, transfusão e captação de sangue no País. Contaminação de pacientes hemofílicos pelo vírus HIV. Convocação, pelo Presidente Ulysses Guimarães, dos constituintes para registro de presença em plenário.

Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents

Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.

In addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.

Vulnerabilidade de enfermeiros no cuidado a pacientes com HIV/Aids: um estudo de representações sociais

Vulnerabilidade e empoderamento apresentam-se como elementos presentes na vida profissional e pessoal dos enfermeiros. Delimitou-se como objeto de estudo as representações sociais elaboradas por enfermeiros que cuidam de pacientes com HIV/Aids acerca de sua vulnerabilidade no contexto do cuidar em enfermagem. O objetivo geral foi analisar as representações sociais construídas por enfermeiros acerca de sua vulnerabilidade no contexto do cuidado que exercem. Trata-se de pesquisa qualitativa, descritiva e exploratória, orientada pelo referencial teórico-metodológico das Representações Sociais em sua abordagem processual. Participaram do estudo trinta enfermeiros de um hospital público municipal do Rio de Janeiro. Como técnicas de coleta de dados foram utilizados o questionário sociodemográfico e a entrevista semiestruturada em profundidade. Como técnica de análise de dados adotou-se a análise de conteúdo temático-categorial proposta por Bardin, sistematizada por Oliveira e operacionalizada pelo software QSR NVivo 9.0. Entre os sujeitos, há predomínio do gênero feminino, da faixa etária de 41 a 45 anos, da realização de pós-graduação lato sensu e de tempo de atuação mínimo de 16 anos em HIV/Aids. Sete categorias emergiram na segmentação do material discursivo: 1) O acesso a informações, a formação profissional e o desenvolvimento da naturalização da aids através da experiência: elementos de vulnerabilidade e de empoderamento; 2) A instituição hospitalar e sua infraestrutura como polo de vulnerabilidade e de empoderamento nas construções simbólicas de enfermeiros que cuidam de pacientes com HIV/Aids; 3) Entre o risco e a prevenção: a vulnerabilidade e o empoderamento no contexto dos acidentes ocupacionais biológicos e as práticas preventivas adotadas por enfermeiros frente ao HIV/Aids no cotidiano hospitalar; 4) Relações interpessoais entre enfermeiro e paciente soropositivo para o HIV enquanto mediadoras da vulnerabilidade e do empoderamento de ambos; 5) As limitações psíquicas enfrentadas por enfermeiros no vivenciar do trabalho junto a portadores do HIV/Aids; 6) A busca pela espiritualidade e pela religiosidade como bases de apoio para a vida profissional contextualizada na aids; e 7) O HIV e a aids no contexto de diferentes modalidades de relacionamento: a presença do risco como elemento organizador da discursividade. Na vida profissional, as representações sociais da vulnerabilidade são compostas pela fragilidade, pelo risco e pela dificuldade. O empoderamento, por sua vez, emerge sustentado por um tripéformado pela proteção, suporte e satisfação como elementosdo bem-estar. Na vida pessoal, o risco possui centralidade nas representações. Já o empoderamento se mostra oriundo do bem-estar e da proteção. Conclui-se que a reconstrução sociocognitiva da vulnerabilidade e do empoderamento permitiu o acesso ao arsenal simbólico do qual o grupo dispõe para a superação do que o ameaça. Vulnerabilidade e empoderamento são, portanto, diversificados e mutáveis em suas bases e produtos e, em movimentos de balanço e contrabalanço, corporificam a díade vulnerabilidade-empoderamento.

O cuidar de enfermeiras na Estratégia Saúde da Família diante da vulnerabilidade feminina para HIV e o contexto familiar

Este estudo tem como objeto de pesquisa as ações de cuidar da enfermeira na Estratégia de Saúde da Família (ESF) diante da vulnerabilidade feminina para o Vírus da Imunodeficiência Humana (HIV) considerando o contexto familiar. Discutir a vulnerabilidade para o HIV, ainda constitui um desafio social, principalmente considerando a mesma, a partir das relações de gênero existente em nossa sociedade no que diz respeito ao papel social e sexual de homens e mulheres no interior de suas famílias. Esta pesquisa tem como objetivos: descrever a percepção sobre o HIV no contexto familiar para a enfermeira da ESF; compreender a percepção da enfermeira da ESF sobre a vulnerabilidade feminina para o HIV no contexto familiar e analisar as ações de cuidar da enfermeira da ESF acerca da vulnerabilidade feminina. Trata-se de uma pesquisa exploratória com abordagem qualitativa, a qual teve como sujeitos da pesquisa onze enfermeiras que foram selecionadas e atuavam na ESF no ano 2012, na Área Programática 2.2 do município do Rio de Janeiro. A coleta de dados foi realizada através de entrevistas semi-estruturadas. A técnica de análise do conteúdo foi baseada em Bardin. Emergiram três categorias: a) Percepções das enfermeiras em relação ao HIV e o contexto de familiar; b) Percepções das enfermeiras em relação à vulnerabilidade feminina para o HIV; c) Ações de cuidar das enfermeiras relacionadas à vulnerabilidade feminina para o HIV considerando o contexto familiar. Constatamos que o HIV é para as enfermeiras, o determinante de uma doença grave, de difícil acompanhamento, que não tem cura, de caráter complexo e também como um agravo que impõe limites em relação a sobrevida. As enfermeiras pouco valorizam as questões de gênero e o contexto social sobre a condição de vulnerabilidade das mulheres, responsabilizando-as por sua contaminação. A prevenção do HIV é realizada em grande parte nas atividades de educação em saúde desenvolvidas pelas enfermeiras da ESF, entretanto ela não é abordada considerando especificamente cada contexto familiar e social da mulher. Os valores pessoais ainda interferem nas ações das enfermeiras, e o HIV é apontado como um agravo possuidor de estigmas tanto sociais quanto culturais. Considerando a ESF uma ação governamental que tem por objetivo a autonomia do sujeito, as mudanças de paradigmas em relação à saúde dos indivíduos, e importante aliada na minimização dos problemas de saúde pública como a vulnerabilidade para o HIV é necessário que as enfermeiras estejam mais sensibilizadas e capacitadas (educação permanente), nas questões sociais (gênero) especificas da população feminina, para que suas ações possam minimizar a vulnerabilidade ao HIV nessa população.