Probing entry inhibitors' activity on HIV and development of new fusion inhibitors : integrating evolutionary biology with virology

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2011The general aims of this thesis were: 1) to examine the C2, V3 and C3 envelope regions ofHIV-1 and HIV-2 at the molecular, evolutionary and structural levels; 2) to compare HIV-1and HIV-2 susceptibility to entry inhibitors and assess their potential value in HIV-2therapy; 3) to produce a new fusion inhibitor peptide using evolutionary biology basedstrategies.In the first study (Chapter 2), HIV-1 and HIV-2 were compared at the molecular,evolutionary and structural levels in the C2, V3 and C3 envelope regions. We identifiedsignificant structural and functional constrains to the diversification and evolution of C2,V3 and C3 in the HIV-2 envelope but not in HIV-1. In particular, we found that V3 in HIV-2is less exposed and more conserved than in HIV-1, suggesting fundamental differences inthe biology and infection of these viruses as well as in their susceptibility to entryinhibitors.In the second study (Chapter 3) we measured the baseline susceptibility of HIV-1 and HIV-2primary isolates to different fusion inhibitors and coreceptor antagonists, includingenfuvirtide (T-20) and maraviroc (MVC). MVC inhibited HIV-2 R5 variants at significantlyhigher IC90 concentrations than HIV-1 variants. Moreover, as previously found in HIV-1,susceptibility of HIV-2 R5 variants to MVC was inversely related with CD4+ T cell counts attime of virus isolation. These results suggest that the structure of the envelope complex ofR5 variants changes along the course of infection. More importantly, the results call fornew clinical studies to evaluate the efficacy of MVC in HIV-2 infection and to determine itsbest therapeutic dosage in early and late stage disease. We also provide definitiveevidence demonstrating that T-20 is not useful for HIV-2 therapy.In the final study (Chapter 4), we designed a new HIV fusion inhibitor peptide (P3) basedon the ancestral sequences of the HIV-2 and SIV envelope genes. P3 has an a-helixstructure as demonstrated by circular dichroism. It has broad antiviral activity at thenanomolar range against HIV-1 and HIV-2 primary isolates, including HIV-1 variantsresistant to T-20. Binding ELISA assays and selection of resistant mutants suggest that P3prevents viral fusion by binding to the transmembrane protein in the HR1 region. Thesestudies provide proof of concept that viable antiviral peptides can be constructed usingevolutionary biology strategies. Such strategies should be explored to enhance theproduction of peptide drugs and vaccines.O Vírus da Imunodeficiência Humana do tipo 1 e do tipo 2 (VIH-1 e VIH-2) são os agentes etiológicos do Síndrome de Imunodeficiência Adquirida (SIDA). Embora sejam semelhantes na sua organização estrutural e genómica, estes lentivírus humanos apresentam características antigénicas distintas e partilham uma semelhança genética de apenas 50%. Enquanto o VIH-1 é responsável pela pandemia mundial, a infecção pelo VIH-2 localiza-se sobretudo na África Ocidental, em alguns países europeus como Portugal e França, e na Índia. A infecção pelo VIH-2 tem melhor prognóstico, a progressão para a doença é mais lenta e há melhor controlo imunológico do que na infecção pelo VIH-1. Ao contrário do VIH-1, o arsenal terapêutico actualmente disponível para tratar a infecção por VIH-2 é reduzido. Os fármacos antiretrovirais em uso foram especificamente desenvolvidos para o VIH-1 e, consequentemente, a sua actividade pode ser reduzida ou nula no VIH-2. Este é o caso concreto dos inibidores não nucleosídicos da transcriptase reversa e de alguns inibidores da protease. Neste contexto, os inibidores de entrada poderão ser úteis para tratar a infecção por VIH-2. Contudo, a susceptibilidade dos isolados primários de VIH-2 aos inibidores de entrada é actualmente desconhecida. A susceptibilidade do VIH aos inibidores de entrada é determinada pela qualidade da interacção do vírus com os receptores celulares. O VIH-1 e VIH-2 são substancialmente diferentes a este nível. Por exemplo, o VIH-2 pode ligar-se ao co-receptor CCR5 independentemente do receptor CD4 e da região V3 do invólucro. Por outro lado, as regiões C2, V3 e C3 do VIH-2 são substancialmente diferentes do VIH-1 a nível antigénico. Colectivamente, estes dados indicam que a estrutura e conformação das glicoproteínas de superfície do VIH-1 e VIH-2 são substancialmente diferentes e sugerem que a susceptibilidade e resistência dos dois tipos de vírus aos inibidores de entrada podem também ser diferentes. Os principais objectivos desta tese foram: 1) analisar as características moleculares, estruturais e evolutivas das regiões C2, V3 e C3 no VIH-1 e VIH-2; 2) comparar a susceptibilidade do VIH-1 e VIH-2 aos inibidores de entrada e avaliar o seu potencial terapêutico na infecção por VIH-2; 3) produzir um novo inibidor de fusão para o VIH-2. Para melhor compreender as potenciais diferenças destes dois vírus na resposta aos inibidores de entrada começámos por analisar as características moleculares, estruturais e evolutivas da região V3 e as regiões circundantes C2 e C3, num número significativo de vírus VIH-1 e VIH-2 isolados em Portugal e noutras regiões do globo, com recurso a diferentes metodologias de biologia evolutiva e computacional (Capitulo 2). Apesar da menor variabilidade das 3 regiões no VIH-2, verificámos que a região C3 está sob forte selecção positiva e encontra-se exposta à superfície sugerindo que, tal como no VIH-1, esta região poderá constituir um domínio neutralizante. No entanto, ao contrário do VIH-1, a maioria das mutações adaptativas no VIH-2 são prejudiciais e levam à extinção das linhagens virais pelo que o efeito final é um forte constrangimento à variabilidade das regiões analisadas. Ao contrário do VIH-1, verificámos que a ansa V3 do VIH-2 se encontra oclusa no complexo glicoproteico do invólucro, numa conformação que parece ser estabilizada por interacções que mantém com alguns resíduos da regiões C2 e C3. Estes resultados são consistentes com o facto de a V3 não ser imunodominante no VIH-2, ficando assim mais protegida da resposta imunitária e das eventuais mutações que dela resultam. A forte conservação da V3, da C2 e da C3 também é consistente com a sua potencialmente importante actividade imunosupressora. Em conclusão, este primeiro estudo permitiu caracterizar algumas das características estruturais e funcionais que distinguem as glicoproteínas do invólucro do VIH-1 e do VIH-2 e que estão associadas às diferentes características biológicas e fenotípicas destes dois vírus. Estes dados podem ter impacto na resposta dos dois vírus aos inibidores de entrada (analisado no Capítulo 3) e no desenvolvimento de novas vacinas. No segundo estudo (Capítulo 3) comparámos a actividade antiviral dos antagonistas dos coreceptores (AMD3100, TAK-779 e maraviroc) e dos inibidores de fusão (T-20 e T-1249) entre um grupo de 20 isolados de VIH-2 (19 isolados primários + um isolado laboratorial) e nove isolados de VIH-1 (sete isolados primários + dois isolados laboratoriais). Verificámos que a sensibilidade ao AMD3100 e ao TAK-779 é semelhante no VIH-1 e o VIH-2. No entanto, o perfil da curva dose-resposta do maraviroc (MVC) obtido para os isolados R5 foi diferente nos dois tipos de vírus. No VIH-2 os valores de IC90 foram significativamente mais elevados do que no VIH-1; por outro lado, os declives da curva dose-resposta foram mais baixos no VIH-2 do que no VIH-1. Colectivamente, estes resultados sugerem que poderão ser necessárias concentrações mais elevadas de MVC para tratar os doentes infectados pelo VIH-2. Adicionalmente, encontrámos uma correlação forte e de sentido inverso entre as susceptibilidade do VIH-2 ao MVC e o número de células T CD4+ dos doentes quando os vírus foram isolados. Vírus isolados em doentes em fase de SIDA foram menos susceptíveis ao MVC do que os vírus isolados em doentes com uma contagem de células T CD4+ superior a 200 células/ul. Ao contrário do VIH-1 não encontrámos qualquer correlação entre a carga da V3 e a susceptibilidade dos isolados R5 de VIH-2 ao MVC. De um modo geral, os nossos resultados sugerem que são necessários ensaios clínicos para avaliar a efectividade do MVC na infecção pelo VIH-2, determinar a dose terapêutica mais adequada e esclarecer se é necessário fazer um ajuste de dose de acordo com a fase da doença. Adicionalmente, e uma vez que isolados VIH-2 X4 e populações duplas/mistas são totalmente ou parcialmente resistentes ao MVC, é de extrema importância o desenvolvimento de um ensaio de tropismo (genotípico e/ou fenotípico) para o VIH-2 de modo a determinar o tropismo antes do início da terapia com MVC. Sem o conhecimento prévio do tropismo viral, o tratamento com MVC poderá seleccionar espécies X4 minoritárias que estão associadas a maior resistência à neutralização e uma progressão mais rápida da doença. No que diz respeito aos inibidores de fusão, verificámos que o T-20 tem actividade reduzida no VIH-2, confirmando estudos anteriores realizados com dois isolados laboratoriais. Por outro lado, observámos uma elevada susceptibilidade deste vírus ao T- 1249, indicando que os inibidores de fusão são potencialmente eficazes na infecção pelo VIH-2. Assim, o desenvolvimento de um novo inibidor de fusão do VIH-2 foi o objectivo do último estudo desta tese (Capítulo 4). No Capítulo 4, desenvolvemos novos péptidos inibidores de fusão a partir da reconstrução de sequências ancestrais da glicoproteína gp36 do invólucro de VIH-2 e de Vírus de Imunodeficiência dos Símios (VIS). Com esta abordagem inovadora pretendemos incorporar a história evolutiva dos vírus na sequência dos péptidos e desta forma melhorar a tolerância destas moléculas aos polimorfismos naturais da sua região alvo bem como às mutações de resistência seleccionadas na sua presença. Obteve-se um péptido ancestral (P3) constituído por 34 aminoácidos, cuja sequência corresponde às posições homólogas 628 – 661 da proteína Env do isolado VIH-1 HXB2 (ou 623 – 656 do isolado VIH-2 ROD). A sequência do P3 difere em 21 aminoácidos da sequência consenso de VIH-1, 14 aminoácidos da sequência do T-20 e 6 aminoácidos da sequência consenso de VIH-2. Ao contrário da natureza não-estruturada do T-20, o P3 tem uma conformação típica em hélice-a, o que lhe poderá conferir maior a estabilidade contra a degradação proteolítica, bem como maior afinidade para a região alvo. Por outro lado, o P3 foi facilmente solúvel em soluções aquosas o que é uma vantagem num futuro desenvolvimento de uma fórmula farmacêutica. O P3 demonstrou ter uma forte actividade antiviral contra isolados primários e laboratoriais de VIH-1 e VIH-2 (IC50 médio, 11 nM para o HIV-1 e 63.8 nM para o HIV-2), incluindo variantes resistentes ao T-20 (IC50, 0.15 – 11.8 nM). Através da passagem consecutiva de vírus em cultura na presença do péptido, foi seleccionada uma mutação de resistência na região HR1 da gp41 (VIH-1), a qual é responsável pela redução da susceptibilidade do VIH-1 ao P3 em 120x. Nas mesmas condições, e após 60 dias em cultura, não foi possível seleccionar mutações de resistência ao P3 no VIH-2. Estes resultado, em conjugação com a sua forte ligação à glicoproteína transmembranar de um isolado de VIH-2, indicam que, tal como outros péptidos baseados na região HR2 (T-20, T- 1249), o P3 inibe a entrada do VIH pela interacção com a região HR1 da gp41 e sugerem que a barreira genética para a resistência ao P3 é significativamente superior no VIH-2 do que no VIH-1. Neste estudo demonstrámos ainda que o P3 é significativamente menos antigénico do que o T-20 nos doentes infectados pelo VIH-1 o que poderá traduzir-se numa maior duração da eficácia clínica do P3 em comparação com o T-20. Os resultados obtidos com o P3 demonstram pela primeira vez que é possível desenvolver péptidos com actividade antiviral significativa utilizando metodologias de biologia evolutiva, pelo que esta abordagem poderá ser explorada no futuro para a produção de medicamentos peptídicos e, eventualmente, de vacinas

Emerging Challenges and Opportunities in Infectious Disease Epidemiology.

Much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the 20th century epidemiologic transition of countries such as the United States and United Kingdom. After decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. Here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. We offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. We next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. These areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners

Epidemiologic synergy - the contribution of heterosexual HIV transmission to the spread of HIV among men who have sex with men (MSM) in South Africa

Background: Could heterosexual HIV transmission be a driver of HIV infections that occur in men who have sex with men (MSM)? Noting the disproportionately high HIV prevalence among MSM across a variety of settings, this subpopulation is often considered as sources of new infections, overlooking the possibility of HIV transmission from the heterosexual – general – population to MSM. Objective: To assess the relative contribution of heterosexual transmission of HIV for onwards transmission of HIV from one man to another. Method: An agent based model of heterosexual transmission of HIV in South Africa was extended to simulate the HIV epidemic among MSM from 1990 to 2012. The model included gay men (who only have sex with men), bisexual men (who have partners of both sexes) in addition to men who have sex with women. HIV prevalence and sexual behaviour data collected among MSM in South Africa served as calibration data. Results: The model estimated that 28.7% (IQR: 27.4-28.9%) of MSM were HIV positive in 2010. By simulating a counterfactual HIV epidemic in South Africa, where HIV only spreads via male-male sex, we observe a decline in HIV incidence occurring in MSM by 56% over the period of 1990-2010, relative to the historical reality of HIV spreading via heterosexual and male-male sex. Analogously, HIV prevalence among MSM in 2010 under the counterfactual scenario reached only 10.0% (IQR 2.8- 17.4%), substantially less than HIV prevalence estimates from samples of MSM in South Africa. Conclusion: Roughly half of the HIV infections among MSM in South Africa can be attributed to the high levels of HIV prevalence in the general population. Scale up of interventions to target high risk behaviours with male partners should dispel possible misconceptions of bisexually active or heterosexual MSM as lower risk partners, relative to those MSM in gay communities

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle

Integrated analysis of epidemiological and phylogenetic data to elucidate viral transmission dynamics

While infectious disease outbreaks are often summarised by population averages such as the reproductive number, variation between individuals in terms of onwards transmissions modulates the degree of unpredictability of an epidemic, and it needs to be accounted for in models of infection control. This heterogeneity among individuals can be quantified by the dispersion parameter k of the offspring distribution, a distribution that defines the number of secondary infections per infected individual. I have developed an inference framework to estimate k and other epidemiological parameters by fitting stochastic transmission models to both incidence time series and the pathogen phylogeny. Applying the framework to simulated data, I found that more accurate, less biased and more precise estimates of the reproductive number and k were obtained by combining epidemiologic and phylogenetic analyses. Accurately estimating k was necessary for unbiased estimates of the reproductive number, but it did not affect the accurate estimation of epidemic start date and the probability of sampling an infection. I further demonstrated that inference was possible in the presence of phylogenetic uncertainty by sampling from the posterior distribution of phylogenies. In addition to methodological contributions, I found that the inclusion of sequences in statistical inference for polio improved the precision of parameter estimates. Based on sequences collected from patients during a poliovirus outbreak, the estimated values of k were high regardless of the data used. On the other hand, the k estimates were low when a transmission model was fit to environmental sequences collected in Pakistan, which is still endemic for wild poliovirus. Furthermore, analysis of environmental sequences was informative of seasonality parameters whereas inference from incidence time series alone was not. This type of analysis using environmental sequences would be useful as polio eradication draws to a close as the number of symptomatic cases approaches zero.Open Acces

Blocking Zika virus vertical transmission.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity

Bayesian methods for source attribution using HIV deep sequence data

The advent of pathogen deep-sequencing technology provides new opportunities for infec- tious disease surveillance, especially for fast-evolving viruses like human immunodeficiency virus (HIV). In particular, multiple reads per host contain detailed information on viral within- host diversity. This information allows the reconstruction of partial directed transmission networks, where estimates of who is source and who is recipient are directly available from the phylogenetic ordering of the viruses of any two individuals. This is a new approach for phylodynamics, and the topic of my thesis. In this thesis, I present updates to the bioinformatics pipeline used by the Phylogenetics And Networks for Generalised Epidemics in Africa consortium for processing HIV deep sequence data and running the phyloscanner program. I then present a semi-parametric Bayesian Poisson model for inferring infectious disease transmission flows and the sources of infection at the population level. The framework is computationally scalable in high- dimensional flow spaces thanks to Hilbert Space Gaussian process approximations, allows for sampling bias adjustments, and estimation of gender- and age-specific transmission flows at a finer resolution than previously possible. In this sense, the methods that I developed enable us to overcome some problems which have been unable to be solved by conventional phylodynamic approaches. We apply the approach to densely sampled, population-based HIV deep-sequence data from Rakai, Uganda. I focus on characterising age-specific transmission dynamics, and examining the sources of HIV infections in adolescent and young women in particular.Open Acces

Sequential Bottlenecks Drive Viral Evolution in Early Acute Hepatitis C Virus Infection

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection