Current HIV Research - Volume 6, Issue 3, 2008

Techniques for the quantification of HIV-1 load in semen include culture and nucleic acid amplification techniques. The latter tend to be used in the reproductive, public health and research settings due to speed, throughput, sensitivity and capacity to eliminate and control for contamination or inhibitory substances from semen. Commerciallyavailable assays such as nucleic acid sequence-based amplification and reverse transcriptase polymerase chain reaction are equivalent in yielding more reliable and reproducible results than in-house, non-commercial assays, and should be used for the determination of HIV-1 load in semen. Sensitivity is increased when silica extraction methods are used.

The perception of HAART failure in achieving broadest immune reconstitution has further strengthened the rationale to widely explore new adjuvant immunotherapy. Most work has been performed on IL-2, given its potential to correct HIV-driven immune defects, possibly translating in enhanced immune competency. This is a literature review report reviewing different trials on IL-2 immunotherapy in HIV/AIDS in the past ten years through the Cochrane and NIH review database. IL-2 can benefit severely compromised patients, either HAART-naive or lacking HAART-driven immune rescue. Furthermore, by sparing HAART-related toxicity, IL-2 is indicated within treatment interruptions or immunization protocols. Important clinical insights stem from the IL-2-mediated immune reconstitution, with a rise in long-term peripheral T-cell turnover, survival and functional markers. Furthermore, IL-2 immunotherapy proved to interfere with cytokine networks with specific regulatory functions over T-cell homeostasis and function. Despite the plethora of immunological findings exploring the intriguing hypothesis that IL-2 might contribute to amend the skewed T-cell immunophenotype and cytokine milieu in HIV/AIDS, major question on the actual clinical impact remain unanswered. This review is meant to thoroughly explore the possibility that the immunological advantages described during IL-2 immunotherapy might translate into actual clinical benefits in the treatment of HIV/AIDS disease.

Conserved in all primate lentivirus genomes, Nef promotes viral replication and infectivity, influences the trafficking of a large number of surface receptors and interferes with TCR signalling, consequently modulating T-cell activation. In vivo observations with Long Term Non-Progressors harbouring a Nef-defective HIV and vaccination studies with Nef-deleted SIV in Rhesus macaques have shown a prominent role for Nef in lentiviral pathogenesis. Here we review the functions of Nef involved in viral replication and infectivity and speculate on a possible role for Nef in HIV fitness.

We describe in this paper that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β-Dribofuranosyl)- quinoline-3-carboxylic acid (compound A) inhibits the HIV-1 replication in human primary cells. We initially observed that compound A inhibited HIV-1 infection in peripheral blood mononuclear cells (PBMCs) in a dosedependent manner, resulting in an EC50 of 1.5 ± 0.5 μM and in a selective index of 1134. Likewise, compound A blocked HIV-1BA-L replication in macrophages in a dose-dependent manner, with an EC50 equal to 4.98 ± 0.9 μM. The replication of HIV-1 isolates from subtypes C and F was also inhibited by compound A with the same efficiency. Compound A inhibited an early event of the HIV-1 replicative cycle, since it prevented viral DNA synthesis in PBMCs exposed to HIV-1. Kinetic assays demonstrated that compound A inhibits the HIV-1 enzyme reverse transcriptase (RT) in dose-dependent manner, with a KI equal to 0.5 ± 0.04 μM. Using a panel of HIV-1 isolates harboring NNRTI resistance mutations, we found a low degree of cross-resistance between compound A and clinical available NNRTIs. In addition, compound A exhibited additive effects with the RT inhibitors AZT and nevirapine, and synergized with the protease inhibitor atazanavir. Our results encourage continuous studies about the kinetic impact of compound A towards different catalytic forms of RT enzyme, and suggest that our nucleoside represents a promising molecule for future antiretroviral drug design.

CTB-MPR649-684, a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649-684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB-MPR649-684 was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB-MPR649-684 were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR649-684 raised only a modest level of anti-MPR649-684 Abs. However, a prime-boost immunization with CTB-MPR649-684 and a second MPR649-684-based immunogen elicited a more productive anti-MPR649-684 antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR649-684-based immunogens may provide broad protective value independent of infection neutralization.

Administration of oligodeoxynucleotides (ODNs) containing CpG motifs generates a rapid and potent response of CC-chemokines, known as ligands of the HIV-1 co-receptor CCR5, in the murine female genital tract. The present study explored the potential HIV inhibitory activities of different human CpG prototypes either alone or conjugated to the non-toxic subunit of cholera toxin (CTB). Results showed that in vitro replication of both HIV-1 and HIV-2 can be suppressed by different human CpG prototypes. Importantly, the conjugation of CpG ODN to CTB (CTB-CpG) enhanced the antiviral activity of CpG against primary HIV-1 isolates of both R5 and X4 phenotypes in peripheral blood mononuclear cells (PBMC) as well as U87.CD4 co-receptor indicator cells. CTB-CpGs triggered higher amounts of MIP-1α, and MIP- 1β in PBMC than the corresponding CpG ODNs, which may explain the superior antiviral effect of CTB-CpG against R5 virus in PBMC. Incubation of PBMC with CpG ODN and CTB-CpG did not alter surface expression of HIV-1 receptors indicating that the observed anti-HIV-1 effect is not mediated through down regulation of HIV-1 receptors on target cells. Further, the enhanced antiviral effect of CTB-CpG was dependent on the presence of phosphorothioate backbone in the ODN, whereas the presence of CpG motif in ODNs was dispensable. These results have implications for the development of novel intervention strategies to prevent HIV infection.

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) responses provide an important defense in controlling HIV-1 replication, but they fail to control the progression of AIDS in advanced HIV-1 infection. To uncover the situation of these responses in patients with advanced HIV-1 infection, we assessed HIV-1- specific CTL responses in 20 individuals with advanced HIV-1 infection using 407 overlapping peptides spanning all expressed HIV-1 proteins using a gamma interferon-enzyme-linked immunospot (ELISpot) assay. In comparison to 20 individuals with moderately advanced HIV-1 infection, HIV-1-specific CTL responses were significantly decreased (P=0.044) and less peptides could be recognized (P=0.05) in advanced HIV-1 infection. Weakening of Env-gp120 and Gag-specific CTL responses contributed importantly to the decrease of CTL magnitude (P=0.042 and 0.078, respectively), while Env-gp41-specific CTL responses were relatively stronger during the end-stage of HIV-1 infection (P<0.001). Nef and Env-gp41 represented the most frequently targeted HIV-1 proteins in advanced HIV-1 infection. The entropy scores of peptides targeted in two groups were not significantly different. Only the breadth and magnitude of Envgp41- specific CTL responses were positively correlated with viral loads in advanced HIV-1 infection (P=0.005 and 0.001, respectively). These findings suggest that progressive HIV-1 infection is associated with a weakening of Env-gp120- and Gag-specific CTL responses, and a simultaneous expansion of Env-gp41-specific CTL which is likely driven by high level viral replication.

The cyclotriazadisulfonamide (CADA) compounds are a new class of specific CD4-targeted HIV entry inhibitors. The in vitro anti-HIV activity of CADA was shown to correlate with its ability to specifically downmodulate cell surface expression of the CD4 receptor in human cells. Here, we evaluated its potential as an anti-HIV microbicide. CADA exerted a clear CD4 receptor downregulating effect in dendritic cells (DC) and subsequently inhibited HIV-1BaL replication in DC/T cell co-cultures. The compound proved to be active against a variety of clinical isolates belonging to the HIV-1 subtypes A, B, C, D, F, G, H, AE and O. Furthermore, it prevented human T cells from being infected with the laboratory-adapted strains X4 HIV-1NL4.3 and R5 SIVmac251. Flow cytometric analysis demonstrated a significant and dosedependent downregulation of CD4 on macaque PBMCs. In addition, the compound exerted a marked anti-SIVmac251 activity in these cells from simian origin. The combination of CADA with cellulose acetate phthalate (CAP) resulted in a synergistic inhibition of HIV-1 and SIV infection. Finally, gel formulated CADA proved to preserve the CD4 downmodulating and antiviral activity of this compound when formulated as a microbicide gel. Thus, our data suggest that CADA may have potential as a broad-spectrum anti-HIV microbicide drug candidate. The preservation of the activity of gel formulated CADA will make it now feasible for testing this unique entry inhibitor in non-human primates, not only as a single drug but also in a synergistic conjunction with other anti-HIV compounds.

HAART has substantially decreased mortality and morbidity among HIV-infected patients. We retrospectively analyzed morbidity and mortality in a cohort of HIV-infected adult patients with prolonged and frequent follow up (1987- 2006). The study was divided in pre-HAART and HAART period for comparative reasons. In total, 615 HIV-infected patients (54 females) were included in our study. 144 died during the pre-HAART period (51.4 deaths per 100 patients). During the HAART period only 38 patients died from a total of 335 patients receiving HAART (11.3 deaths per 100 patients); the follow up in this part of the cohort was 2139 persons-years and the death incidence 1.77 deaths/per 100 personyears. The subanalysis excluding patients who died within 3 months from admission showed that death incidence among patients that have been receiving HAART from the time of diagnosis (1.2 deaths per 100 person-years) was slightly lower, compared to the death incidence of patients treated for some time with non-HAART as well (1.58 deaths per 100 personsyears). After the availability of HAART in this unit, the proportion of non-AIDS related deaths increased significantly from 8% to 40% (p<0.001); infections remained the leading cause of death in both groups of patients. The most common non-AIDS related causes of deaths were cancer and coronary disease. Our data from the studied cohort adds to the relevant literature regarding the dramatic reduction of morbidity and mortality that occurred after the availability of HAART.

Low levels of plasma viremia (below 50 copies/ml of HIV-1 RNA) can be detected in the majority of HIV+ subjects successfully treated with HAART. Aim of our study was to evaluate the impact of different antiretroviral regimens on this residual viremia and on proviral HIV-1 DNA in HAART-treated subjects with plasma HIV RNA <400 cp/ml and no history of virological failure. To this purpose, a cross-sectional analysis of 319 HIV-positive patients on HAART with plasma HIV RNA <400 cp/ml was performed. Subjects had been on HAART for a median of 3.6 years: the current regimen included two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor (PI) in 104 (32.6%) cases, of which 73 treated with a boosted PI; two NRTIs plus a non-NRTI (NNRTI) were prescribed in 166 (52.2%) cases, and NRTIs-only in 49 cases (15.4%). Patients treated with PI had the lowest nadir CD4 cell count (237+191 cells/μl) compared to patients treated with NNRTI (384+192 cells/μl) or NRTIs-only (387+222 cells/μl). Cell-associated HIV-1 DNA was measured in 231 subjects. Residual viremia was measured in 238 subjects with plasma HIV-1 RNA levels < 50 copies/ml. Multivariate analysis showed that the use of NNRTI was independently associated to low levels of residual viremia and high levels of HIV-1DNA, whereas the use of PI was independently associated to low levels of HIV-1 DNA. The better virological performance of NNRTI in terms of low residual viremia is consistent with specific literature data, whereas the greater impact of PI on the viral reservoirs is noteworthy and needs further investigations.

Atherosclerosis is a multifactor disease. Lately, infectious factors such as C. pneumoniae have been found to be involved. To determine whether the infection by C. pneumoniae is a risk factor for atherosclerosis in patients with AIDS. Case-control study on 43 patients with AIDS under HAART (16 cases and 27 controls). To document atherosclerosis, a carotid and transcranial Doppler ultrasound was performed. Anti-C pneumoniae antibodies were searched using a microimmunofluorescence test for IgM and IgG levels. To study the associations with risk of atherosclerosis, Odds Ratios were calculated for each IgG anti-C. pneumoniae antibody titre. A titre of 1:64 significantly increased the risk of atherosclerosis. These results suggest that hypertriglyceridemia and C. pneumoniae infection coexistence significantly increases the risk of atherosclerosis. The inverse geometric average of the antibodies titre against C. pneumoniae in individuals with atheromatous plaque fell to 64, two titres above the controls. This difference turned out to be statistically significant. Exposure to C. pneumoniae with antibodies (IgG) should be considered in any HIV diagnosed patient as a risk factor for atherosclerosis, having found that the inverse geometric averages of antibodies titre are significantly different comparing cases and controls, especially in patients with dyslipidemia, hypertriglyceridemia or in patients whose treatments could cause these conditions. In patients with concomitant hypertriglyceridemia, the association increases up to three times. It is advisable that AIDS patients take a serological test to determine exposure to C. pneumoniae, and to assess treatment options.

DG17 is an orally available prodrug of DG35 (a novel HIV protease inhibitor with variable pharmacokinetics). These studies aimed to optimize DG17 pharmacokinetics by gastric acid neutralization and ritonavir pharmacoenhancement. Both studies were conducted using a randomized, cross-over design in which 6 healthy individuals were administered a single dose of 100mg or 200mg DG17, half with the study intervention (sodium bicarbonate solution in the first study, low dose ritonavir in the second). After a one week washout period, each subject was then administered a second dose of DG17, with the study intervention only administered to the other half. Cmax and AUC increases with gastric acid neutralization were greatest in those with the lowest absorption of DG17 alone. All doses were subsequently given with sodium bicarbonate solution in the second study. Low-dose ritonavir co-administration with DG17 increased DG35 Cmax (median 1437 versus 100 ng/ml, p=0.028) and AUC (median 6975 versus 154ng/ml*hr, p=0.028) compared with DG17 without ritonavir. Plasma DG35 exceeded the IC90 for HIV for ≥ 12 hours following a single DG17/ritonavir dose. No significant adverse events occurred. Single dose DG17 is safe and best administered in a manner preventing gastric acid degradation and with low-dose ritonavir.