Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity.

One of the critical issues that should be addressed in the development of a BCG-based HIV vaccine is genetic plasmid stability. Therefore, to address this issue we have considered using integrative vectors and the auxotrophic mutant of BCG complemented with a plasmid carrying a wild-type complementing gene. In this study, we have constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVAint, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector uses an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate the vaccine BCG.HIVA2auxo.int. Presence of the HIVA gene sequence and protein expression was confirmed. We demonstrated that the in vitro stability of the integrative plasmid p2auxo.HIVAint was increased 4-fold, as compared with the BCG strain harboring the episomal plasmid, and was genetically and phenotypically characterized. The BCG.HIVA2auxo.int vaccine in combination with modified vaccinia virus Ankara (MVA).HIVA was found to be safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. We have engineered a more stable and immunogenic BCG-vectored vaccine using the prototype immunogen HIVA. Thus, the use of integrative expression vectors and the antibiotic-free plasmid selection system based on "double" auxotrophic complementation are likely to improve the mycobacterial vaccine stability in vivo and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.

HIV-1/HAART-Related Lipodystrophy Syndrome (HALS) Is Associated with Decreased Circulating sTWEAK Levels.

BACKGROUND AND OBJECTIVES: Obesity and HIV-1/HAART-associated lipodystrophy syndrome (HALS) share clinical, pathological and mechanistic features. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that plays an important role in obesity and related diseases. We sought to explore the relationship between HALS and circulating levels of soluble (s) TWEAK and its scavenger receptor sCD163. METHODS: This was a cross-sectional multicenter study of 120 HIV-1-infected patients treated with a stable HAART regimen; 56 with overt HALS and 64 without HALS. Epidemiological and clinical variables were determined. Serum levels of sTWEAK and sCD163 levels were measured by ELISA. Results were analyzed with Student's t-test, Mann-Whitney U and χ2 test. Pearson and Spearman correlation were used to estimate the strength of association between variables. RESULTS: Circulating sTWEAK was significantly decreased in HALS patients compared with non-HALS patients (2.81±0.2 vs. 2.94±0.28 pg/mL, p = 0.018). No changes were observed in sCD163 levels in the studied cohorts. On multivariate analysis, a lower log sTWEAK concentration was independently associated with the presence of HALS (OR 0.027, 95% CI 0.001-0.521, p = 0.027). CONCLUSIONS: HALS is associated with decreased sTWEAK levels.

Pharmacogenetics of efficacy and safety of HCV treatment in HCV-HIV coinfected patients: significant associations with IL28B and SOCS3 gene variants.

BACKGROUND AND AIMS: This was a safety and efficacy pharmacogenetic study of a previously performed randomized trial which compared the effectiveness of treatment of hepatitis C virus infection with pegylated interferon alpha (pegIFNα) 2a vs. 2b, both with ribavirin, for 48 weeks, in HCV-HIV coinfected patients. METHODS: The study groups were made of 99 patients (efficacy pharmacogenetic substudy) and of 114 patients (safety pharmacogenetic substudy). Polymorphisms in the following candidate genes IL28B, IL6, IL10, TNFα, IFNγ, CCL5, MxA, OAS1, SOCS3, CTLA4 and ITPA were assessed. Genotyping was carried out using Sequenom iPLEX-Gold, a single-base extension polymerase chain reaction. Efficacy end-points assessed were: rapid, early and sustained virological response (RVR, EVR and SVR, respectively). Safety end-points assessed were: anemia, neutropenia, thrombocytopenia, flu-like syndrome, gastrointestinal disturbances and depression. Chi square test, Student's T test, Mann-Whitney U test and logistic regression were used for statistic analyses. RESULTS: As efficacy is concerned, IL28B and CTLA4 gene polymorphisms were associated with RVR (p<0.05 for both comparisons). Nevertheless, only polymorphism in the IL28B gene was associated with SVR (p = 0.004). In the multivariate analysis, the only gene independently associated with SVR was IL28B (OR 2.61, 95%CI 1.2-5.6, p = 0.01). With respect to safety, there were no significant associations between flu-like syndrome or depression and the genetic variants studied. Gastrointestinal disturbances were associated with ITPA gene polymorphism (p = 0.04). Anemia was associated with OAS1 and CTLA4 gene polymorphisms (p = 0.049 and p = 0.045, respectively), neutropenia and thromobocytopenia were associated with SOCS3 gene polymorphism (p = 0.02 and p = 0.002, respectively). In the multivariate analysis, the associations of the SOCS3 gene polymorphism with neutropenia (OR 0.26, 95%CI 0.09-0.75, p = 0.01) and thrombocytopenia (OR 0.07, 95%CI 0.008-0.57, p = 0.01) remained significant. CONCLUSIONS: In HCV-HIV coinfected patients treated with PegIFNα and ribavirin, SVR is associated with IL28B rs8099917 polymorphism. HCV treatment-induced neutropenia and thrombocytopenia are associated with SOCS3 rs4969170 polymorphism.

Pharmacogenetics of the lipodystrophy syndrome associated with HIV infection and combination antiretroviral therapy.

INTRODUCTION: Antiretroviral drugs have been associated with several toxicities that limit their success. Of the chronic toxicities, the lipodystrophy syndrome is of special concern due to the metabolic alterations that can accompany it. Why some patients treated with a particular antiretroviral regimen develop lipodystrophy, while others do not, is a medical mystery, but it has been suggested that individuals may (or may not) have a genetically conditioned predisposition. Pharmacogenetics is the science that studies how the genetic composition of individuals can give rise to interindividual variations in response to drugs and drug toxicity. AREAS COVERED: This article reviews the published investigations on the association between host genetic determinants in treated HIV-infected patients and the presence of lipodystrophy. Studies were identified through a PubMed database search. Case-control and longitudinal studies into pharmacogenetic association were selected. Areas covered include the data on the genetic variants of mitochondrial parameters, cytokines, adipokines, proteins involved in adipocyte biology and proteins involved in stavudine metabolism. EXPERT OPINION: Most studies provide inconsistent data due to partial genetic evaluation, different assessment of lipodystrophy and low number of patients evaluated. The pharmacogenetics of lipodystrophy in HIV-infected patients treated with antiretroviral drugs still belongs in the research laboratory.

Effect of TNF-alpha genetic variants and CCR5 Delta 32 on the vulnerability to HIV-1 infection and disease progression in Caucasian Spaniards.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is thought to be involved in the various immunogenetic events that influence HIV-1 infection. METHODS: We aimed to determine whether carriage of the TNF-alpha-238G>A, -308G>A and -863 C>A gene promoter single nucleotide polymorphisms (SNP) and the CCR5 Delta 32 variant allele influence the risk of HIV-1 infection and disease progression in Caucasian Spaniards. The study group consisted of 423 individuals. Of these, 239 were uninfected (36 heavily exposed but uninfected [EU] and 203 healthy controls [HC]) and 184 were HIV-1-infected (109 typical progressors [TP] and 75 long-term nonprogressors [LTNP] of over 16 years' duration). TNF-alpha SNP and the CCR5 Delta 32 allele were assessed using PCR-RFLP and automatic sequencing analysis methods on white blood cell DNA. Genotype and allele frequencies were compared using the chi 2 test and the Fisher exact test. Haplotypes were compared by logistic regression analysis. RESULTS: The distribution of TNF-alpha-238G>A, -308G>A and -863 C>A genetic variants was non-significantly different in HIV-1-infected patients compared with uninfected individuals: -238G>A, p = 0.7 and p = 0.3; -308G>A, p = 0.05 and p = 0.07; -863 C>A, p = 0.7 and p = 0.4, for genotype and allele comparisons, respectively. Haplotype analyses, however, indicated that carriers of the haplotype H3 were significantly more common among uninfected subjects (p = 0.04). Among the infected patients, the distribution of the three TNF-alpha genetic variants assessed was non-significantly different between TP and LTNP: -238G>A, p = 0.35 and p = 0.7; -308G>A, p = 0.7 and p = 0.6: -863 C>A, p = 0.2 and p = 0.2, for genotype and allele comparisons, respectively. Haplotype analyses also indicated non-significant associations. Subanalyses in the LTNP subset indicated that the TNF-alpha-238A variant allele was significantly overrepresented in patients who spontaneously controlled plasma viremia compared with those who had a detectable plasma viral load (genotype comparisons, p = 0.02; allele comparisons, p = 0.03). The CCR5 Delta 32 distribution was non-significantly different in HIV-1-infected patients with respect to the uninfected population (p = 0.15 and p = 0.2 for genotype and allele comparisons, respectively) and in LTNP vs TP (p = 0.4 and p = 0.5 for genotype and allele comparisons, respectively). CONCLUSIONS: In our cohort of Caucasian Spaniards, TNF-alpha genetic variants could be involved in the vulnerability to HIV-1 infection. TNF-alpha genetic variants were unrelated to disease progression in infected subjects. The -238G>A SNP may modulate the control of viremia in LTNP. Carriage of the CCR5 Delta 32 variant allele had no effect on the risk of infection and disease progression.

Similar HIV-1 evolution and immunological responses at 10 years despite several therapeutic strategies and host HLA Types.

Two sexual partners infected with related HIV-1 viruses and enrolled in different therapeutic strategies including structured treatment interruptions (STI) provided us an opportunity to compare long term (10 years) viral genetic evolution for closely related isolates in different hosts. HLA loci were molecularly typed and different genetic markers were studied. The viral genetic evolution was studied by sequencing pol and env genes. The HIV-specific CD4+ and CD8+ T cell responses were assessed by the lymphoproliferative response (LPR) and an ELISPOT assay, respectively. HLA class I loci of patients A and B were different and both of them were heterozygous for CCR5delta32 gene. During the two STI studies, viral load of both patients rebounded after treatment interruption and both developed a transitory strong helper and CTL responses. After definitive interruption of therapy, viral load remained below 5,000 copies/ml without therapy during the two years of follow-up. Two patients infected with related viruses showed a similar dynamics of viral evolution and CD4 T cells, despite hosts having a different HLA type and being treated with several therapeutic protocols, after 10 years of infection. These results suggest that, in this case, an effective immunological response to STI depended more on the virus than on the characteristics of the host.