Off the beaten path: Novel mRNA-nanoformulations for therapeutic vaccination against HIV.

Over the last few years, immunotherapy for HIV in general and therapeutic vaccination in particular, has received a tremendous boost, both in preclinical research and in clinical applications. This interest is based on the evidence that the immune system plays a crucial role in controlling HIV infection, as shown for long-term non-progressors and elite controllers, and that immune responses can be manipulated towards targeting conserved epitopes. So far, the most successful approach has been vaccination with autologous dendritic cells (DCs) loaded ex vivo with antigens and activation signals. Although this approach offers much promise, it also comes with significant drawbacks such as the requirement of a specialized infrastructure and expertise, as well as major challenges for logistics and storage, making it extremely time consuming and costly. Therefore, methods are being developed to avoid the use of ex vivo generated, autologous DCs. One of these methods is based on mRNA for therapeutic vaccination. mRNA has proven to be a very promising vaccine platform, as the coding information for any desired protein, including antigens and activation signals, can be generated in a very short period of time, showing promise both as an off-the-shelf therapy and as a personalized approach. However, an important drawback of this approach is the short half-life of native mRNA, due to the presence of ambient RNases. In addition, proper immunization requires that the antigens are expressed, processed and presented at the right immunological site (e.g. the lymphoid tissues). An ambivalent aspect of mRNA as a vaccine is its capacity to induce type I interferons, which can have beneficial adjuvant effects, but also deleterious effects on mRNA stability and translation. Thus, proper formulation of the mRNA is crucially important. Many approaches for RNA formulation have already been tested, with mixed success. In this review we discuss the state-of-the-art and future trends for mRNA-nanoparticle formulations for HIV vaccination, both in the prophylactic and in the therapeutic setting.

Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection.

Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics.

A Classifier to Predict Viral Control After Antiretroviral Treatment Interruption in Chronic HIV-1-Infected Patients.

OBJECTIVES: To construct a classifier that predicts the probability of viral control after analytical treatment interruptions (ATI) in HIV research trials. METHODS: Participants of a dendritic cell-based therapeutic vaccine trial (DCV2) constituted the derivation cohort. One of the primary endpoints of DCV2 was the drop of viral load (VL) set point after 12 weeks of ATI (delta VL12). We classified cases as "controllers" (delta VL12 > 1 log10 copies/mL, n = 12) or "noncontrollers" (delta VL12 < 0.5 log10 copies/mL, n = 10) and compared 190 variables (clinical data, lymphocyte subsets, inflammatory markers, viral reservoir, ELISPOT, and lymphoproliferative responses) between the 2 groups. Naive Bayes classifiers were built from combinations of significant variables. The best model was subsequently validated on an independent cohort. RESULTS: Controllers had significantly higher pre-antiretroviral treatment VL [110,250 (IQR 71,968-275,750) vs. 28,600 (IQR 18737-39365) copies/mL, P = 0.003] and significantly lower proportion of some T-lymphocyte subsets than noncontrollers: prevaccination CD4CD45RA+RO+ (1.72% vs. 7.47%, P = 0.036), CD8CD45RA+RO+ (7.92% vs. 15.69%, P = 0.017), CD4+CCR5+ (4.25% vs. 7.40%, P = 0.011), and CD8+CCR5+ (14.53% vs. 27.30%, P = 0.043), and postvaccination CD4+CXCR4+ (12.44% vs. 22.80%, P = 0.021). The classifier based on pre-antiretroviral treatment VL and prevaccine CD8CD45RA+RO+ T cells was the best predictive model (overall accuracy: 91%). In an independent validation cohort of 107 ATI episodes, the model correctly identified nonresponders (negative predictive value = 94%), while it failed to identify responders (positive predictive value = 20%). CONCLUSIONS: Our simple classifier could correctly classify those patients with low probability of control of VL after ATI. These data could be helpful for HIV research trial design.

Immunomodulatory Activity of Tyrosine Kinase Inhibitors to Elicit Cytotoxicity Against Cancer and Viral Infection.

Tyrosine kinase inhibitors (TKIs) of aberrant tyrosine kinase (TK) activity have been widely used to treat chronic myeloid leukemia (CML) for decades in clinic. An area of growing interest is the reported ability of TKIs to induce immunomodulatory effects with anti-tumor and anti-viral activity, which appears to be mediated by directly or indirectly acting on immune cells. In selected cases of patients with CML, TKI treatment may be interrupted and a non-drug remission may be observed. In these patients, an immune mechanism of increased anti-tumor cytotoxic activity induced by chronic administration of TKIs has been suggested. TKIs increase some populations of natural killer (NK), NK-LGL, and T-LGLs cells especially in dasatinib treated CML patients infected with cytomegalovirus (CMV). In addition, dasatinib increases responses against CMV and is able to inhibit HIV replication in vitro. Recent studies suggest that subclinical reactivation of CMV could drive expansion of specific subsets of NK- and T-cells with both anti-tumoral and anti-viral function. Therefore, the underlying mechanisms implicated in the expansion of this increased anti-tumor and anti-viral cytotoxic activity induced by TKIs could be a new therapeutic approach to take into account against cancer and viral infections such as HIV-1 infection. The present review will briefly summarize the immunomodulatory effects of TKIs on T cells, NKs, and B cells. Therapeutic implications for modulating immunity against cancer and viral infections and critical open questions are also discussed.

Evaluation of resistance to HIV-1 infection ex vivo of PBMCs isolated from patients with chronic myeloid leukemia treated with different tyrosine kinase inhibitors.

Current antiretroviral treatment (ART) may control HIV-1 replication but it cannot cure the infection due to the formation of a reservoir of latently infected cells. CD4+ T cell activation during HIV-1 infection eliminates the antiviral function of the restriction factor SAMHD1, allowing proviral integration and the reservoir establishment. The role of tyrosine kinases during T-cell activation is essential for these processes. Therefore, the inhibition of tyrosine kinases could control HIV-1 infection and restrict the formation of the reservoir. A family of tyrosine kinase inhibitors (TKIs) is successfully used in clinic for treating chronic myeloid leukemia (CML). The safety and efficacy against HIV-1 infection of five TKIs was assayed in PBMCs isolated from CML patients on prolonged treatment with these drugs that were infected ex vivo with HIV-1. We determined that the most potent and safe TKI against HIV-1 infection was dasatinib, which preserved SAMHD1 antiviral function and avoid T-cell activation through TCR engagement and homeostatic cytokines. Imatinib and nilotinib showed lower potency and bosutinib was quite toxic in vitro. Ponatinib presented similar profile to dasatinib but as it has been associated with higher incidence of arterial ischemic events, dasatinib would be the better choice of TKI to be used as adjuvant of ART in order to avoid the establishment and replenishment of HIV-1 reservoir and move forward towards an HIV cure.

Increased expression with differential subcellular location of cytidine deaminase APOBEC3G in human CD4(+) T-cell activation and dendritic cell maturation.

APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells.

Dasatinib inhibits HIV-1 replication through the interference of SAMHD1 phosphorylation in CD4+ T cells.

Massive activation of infected CD4+ T cells during acute HIV-1 infection leads to reservoir seeding and T-cell destruction. During T-cell activation, the antiviral effect of the innate factor SAMHD1 is neutralized through phosphorylation at T592, allowing HIV-1 infection. Dasatinib, a tyrosine kinase inhibitor currently used for treating chronic myeloid leukemia, has been described to control HIV-1 replication through its negative effect on T-cell proliferation and viral entry. We demonstrate that Dasatinib can actually interfere with SAMHD1 phosphorylation in human peripheral blood lymphocytes, preserving its antiviral activity against HIV-1. Dasatinib prevented SAMHD1 phosphorylation in vitro and ex vivo, impairing HIV-1 reverse transcription and proviral integration. This was the major mechanism of action because the presence of Vpx, which degrades SAMHD1, in HIV-1 virions impeded the inhibitory effect of Dasatinib on HIV-1 replication. In fact, infection with VSV-pseudotyped HIV-1 virions and fusion of BlaM-Vpr-containing HIV-1 viruses with activated PBMCs in the presence of Dasatinib suggested that Dasatinib was not acting at fusion level. Finally, PBMCs from patients on chronic treatment with Dasatinib showed a lower level of SAMHD1 phosphorylation in response to activating stimuli and low susceptibility to HIV-1 infection ex vivo. Consequently, Dasatinib is a compound currently used in clinic that preserves the antiviral function of SAMHD1. Using Dasatinib as adjuvant of antiretroviral therapy during early primary HIV-1 infection would contribute to reduce viral replication and spread, prevent reservoir seeding, and preserve CD4 counts and CTL responses. These events would create a more favorable virologic and immunologic environment for future interventional studies aiming at HIV-1 eradication.

Structured Treatment Interruptions and Low Doses of IL-2 in Patients with Primary HIV Infection. Inflammatory, Virological and Immunological Outcomes.

BACKGROUND: Interventions during primary HIV infection (PHI) can modify the clinical course during the chronic phase. The long-term effect of structured treatment interruptions (STI) followed by low doses of interleukin-2 (IL-2) in treated PHI patients is unknown. METHODS: Twelve PHI patients with viral load (VL) <20 copies/mL, CD4 cells >500 cells/mm3, and CD4/CD8 ratio >1, on antiretroviral therapy (ART) initiated within the first 90 days of infection and continued for at least 12 months were included. They underwent four STI and were then allocated (week 0 of the study) to ART alone or ART plus low doses of IL-2. ART was stopped once VL <20 copies/mL ('final stop'). Primary endpoints were VL<3000 copies/mL and CD4 cells >500 cells/mm3 at 48 weeks; secondary endpoints were immune activation, inflammatory markers until 48 weeks and the time before resuming ART (CD4 <350 cells/mm3 or AIDS) after 'final stop', compared between groups. RESULTS: Ten out of 12 patients were males, median age was 35 years and the main risk was men-who-have-sex-with-men. Only one out of 12 patients (in the STI group) maintained VL<3000 copies/mL and CD4 cells >500 cells/mm3 without ART at 48 weeks. All other virological and immunological parameters were comparable between groups at week 0, 'final stop' and week 48. However, the proportion of CD8-CD38+ cells, tumor necrosis factor and srIL-2 were higher in the IL-2 group at 'final stop' and week 24. All these differences vanished during follow-up. At 5 years after the final stop 3 out of 6 patients in the IL-2 group and 6 out of 6 patients in the STI group have resumed ART (P = 0.19). CONCLUSIONS: STI and IL-2 failed to achieve virological control after ART interruption. STI were not deleterious in long-term follow-up, an important issue for eradication and functional cure trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02300623.

Opportunistic infections and immune reconstitution inflammatory syndrome in HIV-1-infected adults in the combined antiretroviral therapy era: a comprehensive review.

Despite the availability of effective combined antiretroviral treatment, many patients still present with advanced HIV infection, often accompanied by an AIDS-defining disease. A subgroup of patients starting antiretroviral treatment under these clinical conditions may experience paradoxical worsening of their disease as a result of an exaggerated immune response towards an active (but also subclinical) infectious agent, despite an appropriate virological and immunological response to the treatment. This clinical condition, known as immune reconstitution inflammatory syndrome, may cause significant morbidity and even mortality if it is not promptly recognized and treated. This review updates current knowledge about the incidence, diagnostic criteria, risk factors, clinical manifestations, and management of opportunistic infections and immune reconstitution inflammatory syndrome in the combined antiretroviral treatment era.

Safety and immunogenicity of a modified vaccinia Ankara-based HIV-1 vaccine (MVA-B) in HIV-1-infected patients alone or in combination with a drug to reactivate latent HIV-1.

OBJECTIVES: The safety, immunogenicity, impact on the latent reservoir and rebound of viral load after therapeutic HIV-1 vaccination with recombinant modified vaccinia Ankara-based (MVA-B) HIV-1 vaccine expressing monomeric gp120 and the fused Gag-Pol-Nef polyprotein of clade B with or without a drug to reactivate latent HIV-1 (disulfiram) were assessed. METHODS: HIV-1-infected patients were randomized to receive three injections of MVA-B (n = 20) or placebo (n = 10). Twelve patients (eight who received vaccine and four who were given placebo) received a fourth dose of MVA-B followed by 3 months of disulfiram. Combined ART (cART) was discontinued 8 weeks after the last dose of MVA-B. Clinical Trials.gov identifier: NCT01571466. RESULTS: MVA-B was safe and well tolerated. A minor, but significant, increase in the T cell responses targeting vaccine inserts of Gag was observed [a median of 290, 403 and 435 spot-forming-cells/10(6) PBMCs at baseline, after two vaccinations and after three vaccinations, respectively; P = 0.02 and P = 0.04]. After interruption of cART, a modest delay in the rebound of the plasma viral load in participants receiving vaccine but not disulfiram was observed compared with placebo recipients (P = 0.01). The dynamics of the viral load rebound did not change in patients receiving MVA-B/disulfiram. No changes in the proviral reservoir were observed after disulfiram treatment. CONCLUSIONS: MVA-B vaccination was a safe strategy to increase Gag-specific T cell responses in chronically HIV-1-infected individuals, but it did not have a major impact on the latent reservoir or the rebound of plasma viral load after interruption of cART when given alone or in combination with disulfiram.

Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.

UNLABELLED: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. IMPORTANCE: HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

Rate and predictors of non-AIDS events in a cohort of HIV-infected patients with a CD4 T cell count above 500 cells/mm³.

The reduction of risk of non-AIDS events after combined antiretroviral therapy (cART) initiation and the crude incidence rate (CIR) of these events in patients who control the viral load without cART (controllers) in a cohort of 574 antiretroviral-naive patients with a baseline CD4 T cell count above 500 cells/mm³ were assessed. Non-AIDS severe events were defined as a first admission to the hospital due to non-AIDS-defining malignancies, cardiovascular, neuropsychiatric, liver-related, or end-stage renal disease events. Potential determinants of non-AIDS/death events were studied using Cox regression models. Eighty-five non-AIDS/death events occurred during 6,062 persons-years of follow-up (PYFU) with a CIR of 1.4 per 100 PYFU. Factors associated with non-AIDS/death event were age (HR 3.4; 95% CI: 1.6-6.9), nadir CD4 below 350 cells/mm³ (HR 2.5; 95% CI: 1.4-4.6), and a last determination of viral load above the median (HR 1.9; 95% CI: 1.0-3.3). The CIR of non-AIDS/death events was 2.1 and 1.8 per 100 PYFU before and after cART in patients who started cART (n=446). A reduction of CIR of non-AIDS events after cART initiation was observed only in patients with a nadir of CD4 above 350 cells/mm³ (2.5 vs. 0.6 per 100 PYFU, p=0.004, and remained stable after cART in patients with a median nadir of CD4 below 350 cells/mm³. CIR was similar in elite, viremic, and noncontrollers (1.1, 1.0, and 1.5 per 100 PYFU, respectively, p=0.25). Reduction of CIR of non-AIDS events after cART initiation depends on nadir CD4 T cell count. Most of the controllers patients had a CIR similar to noncontrollers. These data support the early initiation of cART in HIV-infected patients.

Influence of episodes of intermittent viremia ("blips") on immune responses and viral load rebound in successfully treated HIV-infected patients.

Presenting episodes of intermittent viremia (EIV) under combination antiretroviral therapy (cART) is frequent, but there exists some controversy about their consequences. They have been described as inducing changes in immune responses potentially associated with a better control of HIV infection. Conversely, it has been suggested that EIV increases the risk of virological failure. A retrospective analysis of a prospective, randomized double-blinded placebo-controlled study was performed. Twenty-six successfully treated HIV-infected adults were randomized to receive an immunization schedule or placebo, and after 1 year of follow-up cART was discontinued. The influence of EIV on T cell subsets, HIV-1-specific T cell immune responses, and viral load rebound, and the risk of developing genotypic mutations were evaluated, taking into account the immunization received. Patients with EIV above 200 copies/ml under cART had a lower proportion of CD4(+) and CD4(+)CD45RA(+)RO(-) T cells, a higher proportion of CD8(+) and CD4(+)CD38(+)HLADR(+) T cells, and higher HIV-specific CD8(+) T cell responses compared to persistently undetectable patients. After cART interruption, patients with EIV presented a significantly higher viral rebound (p=0.007), associated with greater increases in HIV-specific lymphoproliferative responses and T cell populations with activation markers. When patients with EIV between 20 and 200 copies/ml were included, most of the differences disappeared. Patients who present EIV above 200 copies/ml showed a lower CD4(+) T cell count and higher activation markers under cART. After treatment interruption, they showed greater specific immune responses against HIV, which did not prevent a higher virological rebound. EIV between 20 and 200 copies/ml did not have this deleterious effect.

The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens.

Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.

Resumption of HIV replication is associated with monocyte/macrophage derived cytokine and chemokine changes: results from a large international clinical trial.

BACKGROUND: There is increasing interest in the role of immune activation and inflammation in HIV disease, but data on direct effects of HIV replication on immune cell activation are limited. METHODS: High sensitivity multiplex bead array assays (MBAAs) were used to measure changes in plasma cytokines and chemokines [interleukin (IL)-1ß, IL-2, IL-6, IL-7, IL-8, IL-12p70, IL-17, tumor necrosis factor-α (TNFα), interferon-γ, granulocyte macrophage colony-stimulating factor, IL-4, IL-5, IL-10, IL-13, CXCL10] from randomization (month 0) to month 2 in a random sample of 200 patients from both the drug conservation (DC) and viral suppression (VS) arms of the Strategies for Management of Antiretroviral Therapy (SMART) trial. IL-6 was also measured by ELISA. Data were evaluated using nonparametric correlation and censored parametric analysis of covariance and associations were declared as statistically significant when the Bonferroni-adjusted P-value was less than 0.003. RESULTS: Compared with the VS arm, significant increases were seen in the DC arm for TNFα (+0.34 log(e) pg/ml, P = 0.0001), IL-10 (+0.33 log(e) pg/ml, P = 0.00001) and CXCL10 (+0.66 log(e) pg/ml, P = 0.00001). IL-6 ELISA poorly correlated with IL-6 MBAA (Spearman's rho = 0.29, P = 0.0001). CONCLUSION: Resumption of HIV replication after ceasing antiretroviral therapy is associated predominantly with an increase of monocyte/macrophage-derived cytokines. Measurement of IL-6 levels may be affected by assay method and this should be considered in future studies of biomarkers.

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.

Influence of a vaccination schedule on viral load rebound and immune responses in successfully treated HIV-infected patients.

Vaccination is recommended for HIV-infected patients. Transient increases of viral load (VL) and risk of developing resistance to HAART have been described. In addition, VL rebounds could increase HIV-specific immune responses. Twenty-six successfully treated HIV-infected adults were randomized to receive a vaccination schedule or placebo during 12 months. Afterward, HAART was discontinued. Influences of vaccination over VL, genotypic mutations, different T cell subsets, and HIV-1-specific immune responses were evaluated. Patients did not present any secondary effect. No differences in incidence of detectable VL determinations were detected between groups [relative risk 0.54 (95% CI 0.23-1.26)]. No relevant resistance mutations were detected. The vaccinated group showed a significant drop in CD4(+) T cells (p = 0.046) associated with increases in activated T cells. HIV-1-specific lymphoproliferative responses increased more in the vaccinated group during the vaccination period. Viral rebound dynamics after interrupting HAART were similar in both groups. A vaccination schedule in successfully treated HIV patients was safe, was not associated with an increase in detectable VL, and did not increase the risk of developing resistance mutations. However, it induced an increase in T cell activation and a drop in CD4(+) T cells, although these changes did not influence the VL rebound dynamics after HAART interruption.

Immunological dysfunction in HIV-1-infected individuals caused by impairment of adenosine deaminase-induced costimulation of T-cell activation.

The cell surface association between CD26 and adenosine deaminase (ADA) has a costimulatory function during T-cell activation. Several studies have revealed correlations among CD4(+) CD26(+) T-cell depletion, increased serum levels of ADA, and the evolution of human immunodeficiency virus (HIV) infection, implicating CD26 and ADA in HIV disease progression. In this context, we aimed to determine whether ADA costimulation could be altered during HIV infection. ADA costimulation was investigated in cells from HIV-infected patients (n = 36) in terms of proliferation and cytokine secretion. An effect of ADA on T-cell proliferation was found in HIV-1-infected patients and correlated positively with the CD4(+) percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADA-induced increase in cytokine production [interferon (IFN)-gamma, interleukin (IL)-6 and IL-10] was markedly reduced in T cells from HIV-1-infected subjects. To eliminate some of the variables associated with immunological defects in HIV-1-infected patients, anti-CD3 plus ADA assays with T cells from healthy volunteers were performed in the presence of recombinant glycoprotein 120 (gp120). It was found that gp120 was responsible for the impairment of the ADA-CD26 interaction and consequently of the ADA-induced effect on both costimulation and cytokine production. The gp120-mediated disruption of the CD26-ADA interaction is a novel mechanism that might explain, at least in part, the altered immunological features observed in HIV-1-infected patients and may have significant relevance in AIDS pathogenesis.

Immunological profile of heterosexual highly HIV-exposed uninfected individuals: predominant role of CD4 and CD8 T-cell activation.

BACKGROUND: Altered T cell subset distribution patterns in uninfected individual highly exposed to human immunodeficiency virus (HIV) have been explained either as a consequence of viral exposure or as a surrogate marker of low susceptibility to infection. METHODS: Multiple genetic and immunological parameters were studied prospectively in 21 HIV-serodiscordant heterosexual couples. RESULTS: We found changes of both CD4(+) and CD8(+) T cells in highly HIV-exposed, uninfected individuals, with a lower level of naive and CD28(+) T cells and higher levels of HLA-DR(+) T cells and CD4(+) T cells expressing CCR5 and memory CD4(+) T cells than in control subjects. The changes in memory and activated T cells observed in highly HIV-exposed, uninfected partners were directly correlated with plasma viral load (PVL) of the HIV-1-infected partners, whereas changes in naive and CD4(+) CD28(+) T cells observed in highly HIV-exposed uninfected partners were inversely correlated with PVL of the HIV-1-infected partners. We were only able to detect HIV-1-specific T-cell responses in a few highly HIV-exposed uninfected partners. CONCLUSIONS: These data suggest that the peripheral immune cells of highly exposed, uninfected individuals responded according to the level of HIV exposure from the partner, even though evidence of specific HIV stimulation is rarely seen.