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Introdução: O desenvolvimento de antirretrovirais (AVRs) para o vírus HIV em pacientes multiexperimentados é prioridade em saúde pública e, além de supressão virológica máxima, recuperação e estabilização imunológica, os ensaios clínicos randomizados (ECRs) que envolvem esquemas de antirretrovirais utilizados em terapias de resgate devem ser capazes de demonstrar segurança na sua utilização, a curto e longo prazos. Objetivo: Realizar revisão sistemática e caracterizar os achados de segurança da terapia antirretroviral (TARV) para a infecção pelo vírus HIV-1 nos ECRs que utilizaram esquemas otimizados de base (EOB) em pacientes multiexperimentados através, principalmente, da avaliação da qualidade da informação sobre riscos baseada na extensão do CONSORT sobre riscos e da avaliação da apresentação de achados laboratoriais que são proxy de desfechos conhecidamente relevantes. Metodologia: Foram revisadas as bases de dados MEDLINE, EMBASE, LILACS, Colaboração Cochrane, SCOPUS e ISI Web of Science visando identificar publicações entre janeiro/2003 e agosto/2014. Também foram revisadas as bases de dados dos seguintes congressos científicos internacionais: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); e International Congress on Drug Therapy in HIV Infection. Não foram feitas restrições em relação à língua da publicação dos estudos. Os critérios para inclusão dos estudos foram: ECRs com pelo menos 16 semanas de duração e com pelo menos 100 indivíduos que avaliaram segurança da terapia antirretroviral (TARV) em pacientes com infecção pelo HIV- 1 caracterizados como multiexperimentados. Resultados: Vinte e quatro ECRs foram incluídos, sendo 16 estudos originais e 8 extensões. Vinte e três (95,8%) estudos foram exclusivamente financiados pela indústria farmacêutica. Apenas 15 (62,5%) estudos apresentaram eventos adversos (EAs) clínicos graves. Ocorrência de limiar para a apresentação de resultados foi observada em 13 (86,7%) estudos que apresentaram EAs graves, 22 (95,6 %) estudos que apresentaram EAs considerados comuns e em 8 (42,1%) estudos que apresentaram informações sobre desfechos laboratoriais. Seis (25%) ECRs não apresentaram informações sobre os desfechos laboratoriais investigados. Oito (33,3%) estudos não apresentaram informações sobre triglicerídeos, colesterol total, LDL (low density lipoprotein) e hiperglicemia, resultando em ausência de informação sobre os referidos desfechos para os medicamentos maraviroque (MVC) e vincriviroc (VIC). Nove estudos não apresentaram informações sobre marcadores hepáticos, resultando em ausência de informação destes desfechos para o medicamento enfuvirtida (ENF). Dezoito (75%) estudos não apresentaram informações sobre creatinina, resultando em ausência de informação sobre este desfecho para os medicamentos ENF, tipranavir (TPV) e darunavir (DRV). Conclusões: A informação sobre riscos em ECRs envolvendo TARV para pacientes multiexperimentados é altamente seletiva e insuficiente. Além disso, desfechos conhecidamente relevantes não tem sido incluídos a priori nestes estudos. Os ECRs que avaliam novos ARVs ainda apresentam as características dos estudos do início da era TARV, quando, diante de um cenário com grande mortalidade associada ao HIV, o principal objetivo era preservar a vida dos pacientes, em detrimento das questões de segurança da terapia. / Introduction: The development of antiretroviral drugs (ARVs) for treating the HIV virus in treatment-experienced patients is a priority in public health. In addition to virological suppression, recovery and stabilization of the immune system, randomized clinical trials (RCTs) involving antiretroviral regimens used in recovery therapies must be able to demonstrate short and long term safety in their use. Objective: To perform a systematic review and to characterize the findings regarding the safety of cART for HIV-1 virus infections in RCTs that used optimized background regimen in treatmentexperienced patients primarily through assessing the quality of information based on the extent of the CONSORT on risks and evaluating the overall presentation of laboratory findings that are known to proxy relevant outcomes. Methodology: The following databases were researched: MEDLINE, EMBASE, LILACS, The Cochrane Collaboration, SCOPUS and ISI Web of Science. We searched for works published between January/2003 and August/2014. The databases of the following international scientific conferences were also researched: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); and International Congress on Drug Therapy in HIV Infection. There were no restrictions regarding the language of the studies. The criteria for inclusion were the following: RCTs that lasted at least 16 weeks and with at least 100 participants who evaluated the safety of antiretroviral therapies (cART) in treatment-experienced patients with HIV-1. Results: Twenty-four RCTs were included, sixteen original studies and eight extensions. Twenty-three (95.8%) of the studies were funded exclusively by the pharmaceutical industry. Only fifteen (62.5%) of the studies showed serious clinical adverse events (AEs). The occurrence threshold for the presentation of results was observed in thirteen (86.7%) of the studies that showed serious AEs, twenty-two (95.6%) of the studies that showed AEs considered to be common, and eight (42.1%) of the studies which presented information on laboratory outcomes. Six (25%) RCTs have not provided information on laboratory outcomes investigated. Eight (33.3%) studies did not provide information on triglycerides, total cholesterol, LDL (low density lipoprotein) and hyperglycemia, resulting in lack of information on these outcomes for the drugs maraviroc (MVC) and vicriviroc (VIC). Nine studies did not provide information on hepatic markers, resulting in lack of information on these outcomes for the drug enfuvirtide (ENF). Eighteen (75%) studies did not provide information on creatinine, resulting in lack of information on this outcome for the drugs ENF, tipranavir (TPV) and darunavir (DRV). Conclusions: Information on the risks of RCTs involving cART for treatment-experienced patients is highly selective and insufficient. Moreover, known relevant outcomes have not been included a priori in these studies. RCTs evaluating new ARVs have the same characteristics of studies of early ARVs, when, faced with a scenario of high mortality associated with HIV, the main goal was to preserve the lives of patients, at the expense of the safety in therapy.

Infants born to HIV-1 positive mothers are at risk of acquiring the infection through pro-longed breastfeeding due to the presence of HIV-1 cell-free RNA and cell-associated DNA in breast milk. However, there is limited focus on vaccines to prevent mother-to-child transmission (MTCT) via breastfeeding. Mycobacterium tuberculosis (M. tuberculosis) the causative agent of Tuberculosis (TB) is the most common cause of AIDS-related deaths. Most infants in Africa receive Bacillus Calmette-Guérin (BCG) at birth or soon after birth and it is the only licensed vaccine for TB. The development of a dual platform vaccine against HIV-1 and TB would be a logical effort to combat these two deadly diseases. Thus, rBCG expressing an HIV-1 derived immunogen may induce HIV-1 responses at birth and these responses can be boosted at adolescence, by a heterologous vector such as modified vaccinia Ankara (MVA) or Chimpanzee adenovirus serotype 63 (ChAdV63). In the first study, I assessed BCG-based vaccines derived from BCG Danish SSI-1331 (BCG₁₃₃₁), expressing an HIV-1 immunogen HIVconsv either by an episomal plasmid (BCG.HIVconsv401^epi) or integrated into the BCG chromosome (BCG.HIVconsv401^int) in a prime-boost regimen. BALB/c mice were immunised with the different prime-boost regimens. rBCG alone was unable to induce detectable HIV-1-specific T-cell responses, however, when used in a prime-boost strategy, elevated HIV-1-specific T-cell responses were observed. In the second study, I aimed to construct marker-less mycobacterium-vectored HIV-1 vaccines using the operator-repressor titration (ORTA®) system as an alternative system for antibiotic resistance gene free vaccines. This rBCG vaccine would express the HIVconsv immunogen. I first constructed plasmids carrying the lac operator lacO and tetracycline operator tetO to enable use in ORT Escherichia coli (E.coli) and Mycobacterium strains, respectively. The ORT system was successful in E. coli and not in mycobacterium. I also constructed plasmids carrying mycobacterium essential genes that would allow for genetic manipulation in Mycobacterium and the use of ORT in mycobacterium. Although the plasmid construction was successful, in the end, genetic manipulations in Mycobacterium and the production of an ORT based BCG (ORT-VAC) was not successful. Finally, I evaluated the immunogenicity of conventional DNA plasmid pTH.HIVconsv compared to Semliki Forest virus replicon DREP.HIVconsv in rhesus macaques. Immunisations were done in a prime-boost strategy with heterologous vectors MVA or ChAdV63 delivering the same immunogen, HIVconsv. It was found that DREP.HIVconsv which was at least 20-fold lower dose than pTH.HIVconsv was capable of inducing comparable T-cell responses and in some experiments, the responses were superior to the conventional DNA plasmid pTH.HIVconsv.

O HIV é dividido em HIV-1 e 2. O HIV-1 é classificado em três grupos, o M (main) responsável pela maioria das infecções, O (outlier) e N(new). Na atualidade, nove subtipos puros e 43 formas recombinantes do grupo M são reconhecidos incluindo sub subtipos. O subtipo C é o mais prevalente, representando aproximadamente 56% das infecções no mundo. Não foram descritas as conseqüências exatas destes subtipos, algumas evidências indicam que alguns subtipos podem ter vantagem na transmissão viral, enquanto outros na replicação, além de influenciar nas vias de mutação, aparecimento de resistência mais rapidamente e fracasso do tratamento. No Brasil os subtipos B e C são os mais freqüentes e acima de 50% dos indivíduos recentemente infectados pertencem aos subtipos não-B. O Rio Grande do Sul (RS) exibe a prevalência mais alta do subtipo C e há uma forma recombinante específica C e B – CRF-31. Entre julho de 2002 e janeiro de 2003 foi realizada a genotipagem e subtipagem do vírus HIV-1 em 178 pacientes, consecutivamente, atendidos no ambulatório de HIV/AIDS do Hospital de Clínicas de Porto Alegre (HCPA) desde 1983, os quais assinaram o termo de consentimento livre e informado. Após revisão dos resultados destes testes restaram 161 pacientes devido a perda de dados relacionados a nome e prontuário (não foi possível relacionar as 17 amostras a um nome e/ou prontuário). Em busca das variáveis demográficas e epidemiológicas realizou-se revisão de prontuário e busca ativa através de telefonema aos pacientes sem seguimento por pelo menos 2 anos. Consultou-se também o obituário de HIV/AIDS. O objetivo principal deste estudo é avaliar os padrões variáveis do HIV-1, prevalência nos indivíduos assistidos no Hospital de Clínicas de Porto Alegre e relacionar estes subtipos com características demográficas e epidemiológicas. Dos 161 pacientes, 90 eram do sexo masculino e 71 do sexo feminino. O subtipo B foi o mais prevalente em nossa amostra sendo encontrado em 53% dos pacientes. Até 1990 o subtipo B era encontrado na grande maioria dos pacientes com HIV, contribuindo com 75% das infecções. A partir de 2000 o subtipo C passou a representar a maioria das infecções, ficando com 33,3% dos casos, porém este aumento não foi estatisticamente significativo. Dos 161 pacientes com subtipo identificado, 19 não se conhecia a categoria de exposição. Revelando entre o subtipo B, a grande maioria dos pacientes nas categorias de exposição sexual, subdivisão homossexual e bissexual, o subtipo C foi relacionado com categorias de exposição heterossexual e uso de drogas injetáveis, quando se analisou separadamente o subtipo C com as categorias de exposição, não se demonstrou relação com categoria específica. Já o subtipo B analisado separadamente mostrou grande associação com a categoria de exposição MSM e bissexual. Em relação a procedência dos pacientes, houve associação estatisticamente significativa entre o subtipo B e a cidade de Porto Alegre, onde o mesmo foi encontrado na maioria dos pacientes. O subtipo C foi encontrado na maioria dos pacientes procedentes de regiões metropolitanas e do interior do estado. O conhecimento da prevalência dos subtipos de acordo com a procedência dos pacientes pode ajudar as autoridades competentes a dirigir esforços na prevenção da infecção pelo HIV.

Genetic variability within both the HIV-1 coreceptor, CCR5, and its ligand, CCL3L, has been shown to contribute towards differences between individuals in their susceptibility to HIV-1 infection and rate of disease progression. In this study we investigated the extent of genetic variation within the CCR5 gene as well as CCL3L, CCL3La and CCL3Lb gene copy number distribution in two healthy HIV-1 uninfected South African populations, South African Africans (SAA) and South African Caucasians (SAC). The impact of variations within these genes on the expression of CCR5 and CCL3 was subsequently assessed. Furthermore, CCR5 genetic variability, CCL3L gene copy number distribution and the expression of CCR5 and CCL3, was assessed in a similar way in a small cohort of HIV-1 infected long term nonprogressors (LTNPs). Genotyping of the CCR5 gene in SAA (n=41) and SAC (n=46) HIV-1 uninfected individuals revealed a high degree of genetic variation between the two population groups, both in terms of single nucleotide polymorphism (SNP) profiles and CCR5 haplotype distribution. Seven complex putative haplotypes spanning the length of the sequenced region were identified with only one of the identified haplotypes, SAA/C-HHC, common to both study populations. The effect of genetic variability on promoter activity of four different CCR5 promoter regions for three CCR5 haplotypes,SAA-HHA, SAA/C-HHC and SAC-HHE, were evaluated. Results showed variability in (i) promoter activity between different promoter regions tested, (ii) results obtained with different cells used for analysis, and (iii) the haplotype being analysed, thereby highlighting that both the cellular environment as well as genetic variability within the promoter region, have the capacity to influence the efficiency of a promoter and consequently CCR5 expression levels. Haplotype-specific promoter analysis demonstrated the SAA-HHA haplotype to have the strongest promoter activity in THP-1 and K562 cells for both P1A (downstream) and P2 (upstream) promoter regions, while in the other cell lines tested (Jurkat and U937), HHA demonstrated intermediate promoter strength.Differences seen between the haplotypes tested in this study and other published studies may be attributable to additional SNPs being tested in the promoter constructs used in this study. The two population groups differed significantly with regards to cell activation levels, as measured by HLA-DR expression, in CD4+ T cell (P=0.002) and CD56+ NK cell subsets (P<0.001). CCR5 expression, determined both as the number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, was found to differ between SAA and SAC individuals across all peripheral blood cell types. SAA individuals had larger proportions of CCR5-expressing natural killer (NK) cell subsets (P<0.01) but lower CCR5 molecules per cell density on CCR5+CD8+ T cell and CCR5+ NK cell subsets (CD56+, CD16+CD56+ and CD56dim) (all P<0.05) compared to SAC individuals. These differences were maintained even after CCR3D32 heterozygous SAC individuals were included in the analyses. Furthermore, the previously described haplotypes, HHA and HHC, associated with differences in CCR5 expression on different cell subsets between individuals within the same population group. SAA individuals with the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells compared to SAA individuals that lacked the haplotype (P=0.001). SAC individuals with the HHC haplotype had significantly higher density on NK (CD56+) and CD16+CD56+ NK cell subsets (P=0.030 and P=0.024, respectively) compared to SAC individuals without this haplotype. The latter observation suggests that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals, thus highlighting the potential role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression. Despite significant differences in CCL3La (CCL3L chemokine coding) and CCL3Lb (nonchemokine coding) copy number between SAA and SAC populations, no difference in CCL3 production by peripheral blood mononuclear cells (PBMCs) was noted between the two study populations. Assuming equal contribution of CCL3 and each copy of CCL3La to CCL3 production,we found that SAC individuals produced higher levels of CCL3 per functional copy of CCL3La compared to SAA individuals (P<0.001). Although, when SAA and SAC individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, there was no difference in production per functional copy between the two groups (P=0.974). We also determined CCL3La and CCL3Lb gene copy number for a previously established cohort of HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants and found that differences previously seen in cord blood mononuclear cell (CBMC) CCL3 production between IP and EU infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number. The potential role of differences in CCR5 genotype, CCR5 expression, CCL3 genotypes and CCL3 production levels in the control of HIV-1 infection was then examined by comparing a small group (10 SAA and 4 SAC) of LTNPs to the respective background population. No polymorphisms in the CCR5 open reading frame were detected in these LTNP individuals. However, the HHA haplotype frequency was significantly higher in SAC LTNP individuals compared to SAC control individuals (P=0.010). Interestingly, CCR5 density on CD4+ T cells and monocytes was significantly lower in SAA LTNP individuals (P=0.025 and P=0.022, respectively) with a trend towards a similar relationship in CD8+ T cells (P=0.058), while the proportions of CCR5-expressing CD8+ T cells were elevated compared to SAA controls (P=0.043). This latter finding reflects the increased immune activation in these individuals compared to uninfected individuals, as evidenced by increased proportions of HLA-DR-expressing T cells (CD8+ and CD4+, P<0.0001). In addition,PHA-induced CCL3 production by PBMCs was significantly lower in LTNP (SAA and SAC combined) compared to control individuals (P=0.004). SAA LTNP individuals had higher proportions of CD8+ T cells (P<0.0001) and lower proportions of natural killer cells (CD56+,P=0.002) compared to control SAA individuals. Thus, CCL3 production differences may be partially explained by differences in the distribution of immune cell subsets between the two study groups.Furthermore, PBMCs of LTNP individuals with low viral loads (<400 copies/ml) produced CCL3 at lower levels than those from individuals with higher viral loads, irrespective of whether or not the cells were stimulated (P=0.005 and P=0.035, respectively). In summary, this study demonstrates that: (i) two ethnically divergent populations show marked differences in CCR5 genetic variability, cell activation and CCR5 expression which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression, (ii) both CCR5 genotypic differences and differences in baseline cellular activation levels appear to be contributing towards the observed differences in CCR5 protein expression, and (iii) the two study populations do not differ with respect to CCL3 production by PBMC cultures which suggests that either the two copy per diploid genome gene, CCL3, may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy number. In addition, a pilot study conducted in a small group of LTNP individuals demonstrates that two major determinants of HIV-1 disease progression, CCR5 and CCL3, are both expressed at lower levels in LTNPs individuals compared to healthy uninfected controls and has identified CCR5 haplotypes which are potentially associated with disease progression.

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The Human Immunodeficiency Virus -1 (HIV-1) has been the source of substantial human misery since it was first discovered in the early 1980s. Despite remarkable progress that has been made towards understanding HIV- 1, there is no cure, no vaccine and life-prolonging therapies are beyond the reach of millions in need. Our research sought to both gain insight into potential therapeutic targets as well as the preclinical testing of drug candidates. We demonstrate that a RhoA derived peptide is an effective inhibitor of HIV-1 entry. Although this peptide inhibits HIV-1 due to its poly-anionic nature, it nevertheless demonstrates that endogenous host proteins may be repurposed for novel therapeutic uses. We also characterized the mechanism and effectiveness of Basant, a polyherbal topical microbicide candidate for the prevention of HIV-1 transmission. Our data demonstrate that it inhibits the entry of both CCR5 and CXCR4 tropic HIV-1 at non-toxic concentrations. Finally, data is presented on the characterization of a novel HIV-1 protein expressed from an alternative reading frame of the HIV-1 polymerase gene. We demonstrate that this protein is localized to the nucleolus and is likely expressed from a novel HIV-1 transcript. This work lays the foundation for further studies to target this protein for drug development. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from May 9, 2011 - May 9, 2012

Chemokines are important mediators of leukocyte migration. Chemokines bind to G protein–coupled receptors (GPCR) and cause conformational changes that trigger intracellular signaling pathways involved in inflammation, injury healing, cancer, metastasis, and HIV infections. No direct structural information about any chemokine receptor is available, but the structure of chemokines has been well studied. Structural studies of chemokines coupled with cell-biological investigations may lead to a better understanding of the mechanisms of chemokine-receptor interactions. In this Ph.D. project, I studied the structural and functional relationship between chemokines and chemokine receptors using NMR, X-ray crystallography, and mutagenesis approaches, coupled with several different cell-biology assays. We found that the conserved “chemokine fold” can support different dimerization types in the chemokines family, although changing the dimers from CC- to CXC-type fold is not readily accomplished. I also used an engineered covalently-bound dimer of the MIP-1β mutant, MIP-1β-A10C, to study the relationship between dimerization of chemokines and their interaction with the CCR5 receptor. My results suggest that MIP-1β dimer neither bind nor activate the CCR5 receptor. I also studied the biophysical properties of one N-terminal awkward mutant of P2-RANTES, which was originally selected by others from a phage display using CCR5-expressing cells. Although the NMR and X-ray crystal studies revealed that the wild type RANTES is a tight homodimer, analytical ultracentrifugation reveals that P2-RANTES is a monomer in solution, the 1.7 Å resolution X-ray crystal structure of P2-RANTES was found to be a packed tetramer. The mutated N-terminal residues play a very important role in the tetramerization in the X-ray crystal structure. Finally I used the HIV-1 env mediated cell-cell fusion assay to study the combination of chemokines or chemokine variants with anti-HIV peptides C37 or/and T-20. A surprisingly synergistic effect was found between P2-RANTES and C37 or T-20. This combination stratagem may lead to further useful drug combinations or drug delivery for more potent anti-HIV treatments.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 142-166). Also available online through Digital Dissertations.

The great adaptability of the Human Immunodeficiency Virus type 1 (HIV-1) and the exclusive use of zidovudine (3$ sp prime$azido-3$ sp prime$deoxythymidine or AZT) in the treatment of AIDS has motivated our search for HIV-1 strains resistant to this drug. / Our first attempt at obtaining such strains was made by using an in vitro approach in which a lymphoid cell line, chronically infected with HIV-1, was exposed to various drug concentrations. Although this system has never generated such mutants, we found that the progeny virus population, present in the culture fluids of these chronically infected drug-treated cells lost infectivity. These results suggest a potential post-integrational role for zidovudine, possibly acting at the level of viral assembly or budding. / However, we were successful in isolating zidovudine-resistant HIV-1 strains from the blood of patients undergoing AZT treatment. Our work shows that a minimum of 27 weeks of treatment is necessary for the appearance of the resistant phenotype and that the majority (75%) of patients treated with AZT for more than one year will generate such resistant strains. No correlation between the clinical status of the patients and the occurrence of resistant variants can be drawn from our work. However, in vitro experiments have shown that the resistant isolates are more cytopathic, although less infectious, than the sensitive strains. Reverse transcriptase enzymes from both AZT-resistant and -sensitive strains were virtually identical when their respective enzymatic activities or their affinity for the substrate or the inhibitor were compared. Finally, some zidovudine-resistant isolates demonstrate cross-resistance to other nucleoside analogs with potential clinical applications.