Surveillance of HIV type 1 drug resistance among naive patients from Venezuela.

We have studied 65 HIV-1-infected untreated patients recruited in Caracas, Venezuela with TCD4 counts > or =350/microl. The reverse transcriptase and protease sequences of the virus were sequenced, aligned with reference HIV-1 group M strains, and analyzed for drug resistance mutations. Most of the viruses were subtype B genotype in both the protease and RT genomic regions. Five of the 62 virus isolates successfully amplified showed evidence of recombination between protease and RT, with their protease region being non-B while their RT region was derived from subtype B. Four strains were found bearing resistance mutations either to NRTIs, NNRTIs, or PIs. The prevalence of HIV-1 isolates bearing resistance mutations was therefore above the 5% threshold of WHO.

Manipulation of the ubiquitin-proteasome system by HIV-1 : role of the accessory protein Vpr

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte.

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetylpepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.

Cytokine profile and clinical metabolic alterations in HIV-1 infected individuals with and without lipodistrophy

The extensive use of Highly Active Antiretroviral Therapy (HAART) has transformed HIV infection into a chronic condition. Thus, metabolic alterations including lipodystrophy and dyslipidemia have been associated with the use of such medications. The objective of the present study was to analyze clinical metabolic alterations and the profile of TNF-α, IFN-γ, IL-2, IL-10, and TNF-α type II soluble receptor in serum of HIV-1 individuals with and without lipodystrophy. Eighty-four adults were evaluated, 42 males and 42 females, mean age 37 years, and HAART time of at least 15 months. Two groups were formed, G1: 42 individuals with lipodystrophy, and G2: 42 without lipodistropy. From the HAART used, stavudine was more associated with the lipodystrophy group and zidovudine with the non-lipodystrophy group. CD4 and CD8 values, viral load, glucose, albumin, and lipids were not different between groups, except for triglycerides, which were high in the lipodystrophy group, and HDL, whose concentration was reduced in G1. TNF-α, TNF-RII, and IL-10 profiles were high and had positive correlation; IL-2 and IFN-γ had reduced levels in the lipodystrophy group. High TNF-α and its receptor levels seem to be associated with lipodystrophy development in individuals under HAART therapy.

Frequency of subtype B and F1 dual infection in HIV-1 positive, Brazilian men who have sex with men

Background: Because various HIV vaccination studies are in progress, it is important to understand how often inter- and intra-subtype co/superinfection occurs in different HIV-infected high-risk groups. This knowledge would aid in the development of future prevention programs. In this cross-sectional study, we report the frequency of subtype B and F1 co-infection in a clinical group of 41 recently HIV-1 infected men who have sex with men (MSM) in Sao Paulo, Brazil. Methodology: Proviral HIV-1 DNA was isolated from subject's peripheral blood polymorphonuclear leukocytes that were obtained at the time of enrollment. Each subject was known to be infected with a subtype B virus as determined in a previous study. A small fragment of the integrase gene (nucleotide 4255-4478 of HXB2) was amplified by nested polymerase chain reaction (PCR) using subclade F1 specific primers. The PCR results were further confirmed by phylogenetic analysis. Viral load (VL) data were extrapolated from the medical records of each patient. Results: For the 41 samples from MSM who were recently infected with subtype B virus, it was possible to detect subclade F1 proviral DNA in five patients, which represents a co-infection rate of 12.2%. In subjects with dual infection, the median VL was 5.3 x 10(4) copies/ML, whereas in MSM that were infected with only subtype B virus the median VL was 3.8 x 10(4) copies/ML (p > 0.8). Conclusions: This study indicated that subtype B and F1 co-infection occurs frequently within the HIV-positive MSM population as suggested by large number of BF1 recombinant viruses reported in Brazil. This finding will help us track the epidemic and provide support for the development of immunization strategies against the HIV.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirao Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm(3) [standard deviation (SD) = 99] and a mean viral load of 5.09 log(10) copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno([clinical20%]) algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naive HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

An in-depth analysis of the HIV-1/AIDS dynamics by comprehensive mathematical modeling

This work presents major results from a novel dynamic model intended to deterministically represent the complex relation between HIV-1 and the human immune system. The novel structure of the model extends previous work by representing different host anatomic compartments under a more in-depth cellular and molecular immunological phenomenology. Recently identified mechanisms related to HIV-1 infection as well as other well known relevant mechanisms typically ignored in mathematical models of HIV-1 pathogenesis and immunology, such as cell-cell transmission, are also addressed. (C) 2011 Elsevier Ltd. All rights reserved.

Incidence of HIV-1 drug resistance among antiretroviral treatment-naive individuals starting modern therapy combinations

Estimates of drug resistance incidence to modern first-line combination antiretroviral therapies against human immunodeficiency virus (HIV) type 1 are complicated by limited availability of genotypic drug resistance tests (GRTs) and uncertain timing of resistance emergence.

Social Meets Molecular: Combining Phylogenetic and Latent Class Analyses to Understand HIV-1 Transmission in Switzerland

Switzerland has a complex human immunodeficiency virus (HIV) epidemic involving several populations. We examined transmission of HIV type 1 (HIV-1) in a national cohort study. Latent class analysis was used to identify socioeconomic and behavioral groups among 6,027 patients enrolled in the Swiss HIV Cohort Study between 2000 and 2011. Phylogenetic analysis of sequence data, available for 4,013 patients, was used to identify transmission clusters. Concordance between sociobehavioral groups and transmission clusters was assessed in correlation and multiple correspondence analyses. A total of 2,696 patients were infected with subtype B, 203 with subtype C, 196 with subtype A, and 733 with recombinant subtypes (mainly CRF02_AG and CRF01_AE). Latent class analysis identified 8 patient groups. Most transmission clusters of subtype B were shared between groups of gay men (groups 1-3) or between the heterosexual groups "heterosexual people of lower socioeconomic position" (group 4) and "injection drug users" (group 8). Clusters linking homosexual and heterosexual groups were associated with "older heterosexual and gay people on welfare" (group 5). "Migrant women in heterosexual partnerships" (group 6) and "heterosexual migrants on welfare" (group 7) shared non-B clusters with groups 4 and 5. Combining approaches from social and molecular epidemiology can provide insights into HIV-1 transmission and inform the design of prevention strategies.

Correlates of latent and productive HIV type-1 infection in tonsillar CD4+ T cells

Correlates of virus load and characteristics of virus-producing cells in tonsillar tissue were investigated. Our results suggest that when less than 1:100 tonsillar CD4+ T cells from individuals infected with HIV type-1 (HIV-1) contain replication competent provirus, the level of CD4+ T cells in tonsils is comparable to that observed in uninfected individuals. Virus load at or above this level was associated with low CD4 cell numbers in tonsillar tissue. Only a few percent of all infected T cells in tonsillar tissue were active virus producers, with minor differences observed between individuals. Plasma viremia was found to correlate with infectious virus load in tonsillar tissue. With less than 1:1,000 of CD4 cells in lymphoid tissues being involved in active virus production, direct cytopathic effect by HIV-1 on infected CD4 cells is unlikely to fully explain the immunodeficiency seen in AIDS.

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and β-chemokine production

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4+ T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-γ, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Δnef, up to 2% of all CD4+T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles

The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactive vpr, vpu, and nef coding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 107 colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+ cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1 stage of the cell cycle by density-dependent inhibition of growth. Human CD34+ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.

A vaccine for HIV type 1: The antibody perspective

Antibodies that bind well to the envelope spikes of immunodeficiency viruses such as HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) can offer protection or benefit if present at appropriate concentrations before viral exposure. The challenge in antibody-based HIV-1 vaccine design is to elicit such antibodies to the viruses involved in transmission in humans (primary viruses). At least two major obstacles exist. The first is that very little of the envelope spike surface of primary viruses appears accessible for antibody binding (low antigenicity), probably because of oligomerization of the constituent proteins and a high degree of glycosylation of one of the proteins. The second is that the mature oligomer constituting the spikes appears to stimulate only weak antibody responses (low immunogenicity). Viral variation is another possible obstacle that appears to present fewer problems than anticipated. Vaccine design should focus on presentation of an intact mature oligomer, increasing the immunogenicity of the oligomer and learning from the antibodies available that potently neutralize primary viruses.

Prevalence of Human T-Cell Lymphotropic Virus (HTLV-1/2) in Individuals from Public Health Centers in Mozambique

The prevalence of human T-cell lymphotropic viruses types 1 and 2 (HTLV-1/2) in Mozambique is not known. The present study examined blood samples from 208, 226, and 318 individuals from Northern, Central, and Southern Mozambique, respectively, of all socioeconomic and demographic strata attending public health centers in Mozambique for HTLV-1/2-specific antibodies. Serum samples were assessed for HIV- and HTLV-1/2-specific antibodies by using enzyme immunoassays, and infections with HTLV-1 and -2 were confirmed by using Western blot. An overall HTLV-1/2 prevalence of 2.3% (2.9% in female and 1.1% in male subjects) was observed, and the prevalence of infection increased with age. Regional variation in the prevalence of HIV and HTLV-1/2 was observed; 32.2%, 65.5%, and 44% of individuals tested HIV positive in Northern, Central, and Southern Mozambique, respectively, and 2.4%, 3.9%, and 0.9% tested HTLV-1/2 positive in the same regions. HTLV-1 infection was confirmed in these individuals. No association between HTLV-1 infection and socio-demographic variables or HIV status was detected, although the low number of HTLV-1-positive cases did not allow robust statistical analyses. The results obtained suggest different risk factors and epidemiologic correlates of HIV and HTLV-1 transmission in Mozambique. Furthermore, our results suggested that North and Central Mozambique should be considered endemic regions for HTLV-1 infection. As no cases of HTLV-2 were detected, HTLV-2 appears to have not been introduced into Mozambique.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.