Converging and evolving immuno-genomic routes toward immune escape in breast cancer.

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals.

Mass vaccination campaigns reduced COVID-19 incidence and severity. Here, we evaluated the immune responses developed in SARS-CoV-2-uninfected patients with predominantly antibody-deficiencies (PAD) after three mRNA-1273 vaccine doses. PAD patients were classified based on their immunodeficiency: unclassified primary antibody-deficiency (unPAD, n = 9), common variable immunodeficiency (CVID, n = 12), combined immunodeficiency (CID, n = 1), and thymoma with immunodeficiency (TID, n = 1). unPAD patients and healthy controls (HCs, n = 10) developed similar vaccine-induced humoral responses after two doses. However, CVID patients showed reduced binding and neutralizing titers compared to HCs. Of interest, these PAD groups showed lower levels of Spike-specific IFN-γ-producing cells. CVID individuals also presented diminished CD8+T cells. CID and TID patients developed cellular but not humoral responses. Although the third vaccine dose boosted humoral responses in most PAD patients, it had limited effect on expanding cellular immunity. Vaccine-induced immune responses in PAD individuals are heterogeneous, and should be immunomonitored to define a personalized therapeutic strategies.

Safety, immunogenicity and effect on viral rebound of HTI vaccines in early treated HIV-1 infection: a randomized, placebo-controlled phase 1 trial.

HIVACAT T-cell immunogen (HTI) is a novel human immunodeficiency virus (HIV) vaccine immunogen designed to elicit cellular immune responses to HIV targets associated with viral control in humans. The AELIX-002 trial was a randomized, placebo-controlled trial to evaluate as a primary objective the safety of a combination of DNA.HTI (D), MVA.HTI (M) and ChAdOx1.HTI (C) vaccines in 45 early-antiretroviral (ART)-treated individuals (44 men, 1 woman; NCT03204617). Secondary objectives included T-cell immunogenicity, the effect on viral rebound and the safety of an antiretroviral treatment interruption (ATI). Adverse events were mostly mild and transient. No related serious adverse events were observed. We show here that HTI vaccines were able to induce strong, polyfunctional and broad CD4 and CD8 T-cell responses. All participants experienced detectable viral rebound during ATI, and resumed ART when plasma HIV-1 viral load reached either >100,000 copies ml-1, >10,000 copies ml-1 for eight consecutive weeks, or after 24 weeks of ATI. In post-hoc analyses, HTI vaccines were associated with a prolonged time off ART in vaccinees without beneficial HLA (human leukocyte antigen) class I alleles. Plasma viral load at the end of ATI and time off ART positively correlated with vaccine-induced HTI-specific T-cell responses at ART cessation. Despite limited efficacy of the vaccines in preventing viral rebound, their ability to elicit robust T-cell responses towards HTI may be beneficial in combination cure strategies, which are currently being tested in clinical trials.

Limited Humoral and Specific T-Cell Responses After SARS-CoV-2 Vaccination in PWH With Poor Immune Reconstitution.

BACKGROUND: We analyzed humoral and cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in people with human immunodeficiency virus (HIV; PWH) who had CD4+ T-cell counts <200/µL (HIV<200 group). METHODS: This prospective cohort study included 58 PWH in the HIV<200 group, 36 with CD4+ T-cell counts >500/µL (HIV>500 group), and 33 HIV-1-negative controls (control group). Antibodies against the SARS-CoV-2 spike protein (anti-S immunoglobulin [Ig] G) and the receptor-binding domain (anti-RBD IgG) were quantified before and 4 weeks after the first and the second doses of BNT162b2 or mRNA-1273 (at week 8). Viral neutralization activity and T-cell responses were also determined. RESULTS: At week 8, anti-S/anti-RBD IgG responses increased in all groups (P < .001). Median (interquartile range) anti-S and anti-RBD IgG levels at week 8 were 153.6 (26.4-654.9) and 171.9 (61.8-425.8) binding antibody units (BAU)/mL, respectively, in the HIV<200 group, compared with 245.6 (145-824) and 555.8 (166.4-1751) BAU/mL in the HIV>500 group and 274.7 (193.7-680.4) and 281.6 (181-831.8) BAU/mL in controls (P < .05). Neutralizing capacity and specific T-cell immune responses were absent or reduced in 33% of those in the HIV<200 group, compared with 3.7% in the HIV>500 group (P < .01). CONCLUSIONS: One-third of PWH with CD4+ T-cell counts <200/µL show low anti-S/anti-RBD IgG levels, reduced in vitro neutralization activity against SARS-CoV-2, and no vaccine-induced T cells after receiving coronavirus disease 2019 mRNA vaccines.

Epigenetic landscape in the kick-and-kill therapeutic vaccine BCN02 clinical trial is associated with antiretroviral treatment interruption (ATI) outcome.

BACKGROUND: The BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late (< or > 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP. METHODS: PBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n = 5) were included as a control group for RMD administration alone. FINDINGS: DNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion. INTERPRETATION: This study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy. FUNDING: European Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health-National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568.

Incoming HIV virion-derived Gag Spacer Peptide 2 (p1) is a target of effective CD8+ T cell antiviral responses.

Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial" HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A∗02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8+ T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Immune Profiles Identification by Vaccinomics After MVA Immunization in Randomized Clinical Study.

Background: Our previous work has demonstrated the benefits of transcutaneous immunization in targeting Langerhans cells and preferentially inducing CD8 T-cell responses. Methods: In this randomized phase Ib clinical trial including 20 HIV uninfected volunteers, we compared the safety and immunogenicity of the MVA recombinant vaccine expressing HIV-B antigen (MVA-B) by transcutaneous and intramuscular routes. We hypothesized that the quality of innate and adaptive immunity differs according to the route of immunization and explored the quality of the vector vaccine-induced immune responses. We also investigated the early blood transcriptome and serum cytokine levels to identify innate events correlated with the strength and quality of adaptive immunity. Results: We demonstrate that MVA-B vaccine is safe by both routes, but that the quality and intensity of both innate and adaptive immunity differ significantly. Transcutaneous vaccination promoted CD8 responses in the absence of antibodies and slightly affected gene expression, involving mainly genes associated with metabolic pathways. Intramuscular vaccination, on the other hand, drove robust changes in the expression of genes involved in IL-6 and interferon signalling pathways, mainly those associated with humoral responses, and also some levels of CD8 response. Conclusion: Thus, vaccine delivery route perturbs early innate responses that shape the quality of adaptive immunity. Clinical Trial Registration: http://ClinicalTrials.gov, identifier PER-073-13.

HIVconsv Vaccines and Romidepsin in Early-Treated HIV-1-Infected Individuals: Safety, Immunogenicity and Effect on the Viral Reservoir (Study BCN02).

Kick&kill strategies combining drugs aiming to reactivate the viral reservoir with therapeutic vaccines to induce effective cytotoxic immune responses hold potential to achieve a functional cure for HIV-1 infection. Here, we report on an open-label, single-arm, phase I clinical trial, enrolling 15 early-treated HIV-1-infected individuals, testing the combination of the histone deacetylase inhibitor romidepsin as a latency-reversing agent and the MVA.HIVconsv vaccine. Romidepsin treatment resulted in increased histone acetylation, cell-associated HIV-1 RNA, and T-cell activation, which were associated with a marginally significant reduction of the viral reservoir. Vaccinations boosted robust and broad HIVconsv-specific T cells, which were strongly refocused toward conserved regions of the HIV-1 proteome. During a monitored ART interruption phase using plasma viral load over 2,000 copies/ml as a criterium for ART resumption, 23% of individuals showed sustained suppression of viremia up to 32 weeks without evidence for reseeding the viral reservoir. Results from this pilot study show that the combined kick&kill intervention was safe and suggest a role for this strategy in achieving an immune-driven durable viremic control.

In vivo Effects of Romidepsin on T-Cell Activation, Apoptosis and Function in the BCN02 HIV-1 Kick&Kill Clinical Trial.

Romidepsin (RMD) is a well-characterized histone deacetylase inhibitor approved for the treatment of cutaneous T-cell lymphoma. in vitro and in vivo studies have demonstrated that it is able to induce HIV-1 gene expression in latently infected CD4+ T cells from HIV-1+ individuals on suppressive antiretroviral therapy. However, in vitro experiments suggested that RMD could also impair T-cell functionality, particularly of activated T cells. Thus, the usefulness of RMD in HIV-1 kick&kill strategies, that aim to enhance the immune system elimination of infected cells after inducing HIV-1 viral reactivation, may be limited. In order to address whether the in vitro observations are replicated in vivo, we determined the effects of RMD on the total and HIV-1-specific T-cell populations in longitudinal samples from the BCN02 kick&kill clinical trial (NCT02616874). BCN02 was a proof-of-concept study in 15 early treated HIV-1+ individuals that combined MVA.HIVconsv vaccination with three weekly infusions of RMD given as a latency reversing agent. Our results show that RMD induced a transient increase in the frequency of apoptotic T cells and an enhanced activation of vaccine-induced T cells. Although RMD reduced the number of vaccine-elicited T cells secreting multiple cytokines, viral suppressive capacity of CD8+ T cells was preserved over the RMD treatment. These observations have important implications for the design of effective kick&kill strategies for the HIV-1 cure.

Does Antigen Glycosylation Impact the HIV-Specific T Cell Immunity?

It is largely unknown how post-translational protein modifications, including glycosylation, impacts recognition of self and non-self T cell epitopes presented by HLA molecules. Data in the literature indicate that O- and N-linked glycosylation can survive epitope processing and influence antigen presentation and T cell recognition. In this perspective, we hypothesize that glycosylation of viral proteins and processed epitopes contribute to the T cell response to HIV. Although there is some evidence for T cell responses to glycosylated epitopes (glyco-epitopes) during viral infections in the literature, this aspect has been largely neglected for HIV. To explore the role of glyco-epitope specific T cell responses in HIV infection we conducted in silico and ex vivo immune studies in individuals with chronic HIV infection. We found that in silico viral protein segments with potentially glycosylable epitopes were less frequently targeted by T cells. Ex vivo synthetically added glycosylation moieties generally masked T cell recognition of HIV derived peptides. Nonetheless, in some cases, addition of simple glycosylation moieties produced neo-epitopes that were recognized by T cells from HIV infected individuals. Herein, we discuss the potential importance of these observations and compare limitations of the employed technology with new methodologies that may have the potential to provide a more accurate assessment of glyco-epitope specific T cell immunity. Overall, this perspective is aimed to support future research on T cells recognizing glycosylated epitopes in order to expand our understanding on how glycosylation of viral proteins could alter host T cell immunity against viral infections.

A 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 confers protections against HIV-1 infection.

We investigated whether a 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 (T-cell immunoglobulin and mucin domain 1), modulates susceptibility to HIV-1 infection. The polymorphism was genotyped in three case/control cohorts of HIV-1 exposed seronegative individuals (HESN) and HIV-1 infected subjects from Italy, Peru, and Colombia; data from a Thai population were retrieved from the literature. Across all cohorts, homozygosity for the short TIM-1 allele was more common in HESNs than in HIV-1 infected subjects. A meta-analysis of the four association analyses yielded a p value of 0.005. In vitro infection assays of CD4+ T lymphocytes indicated that homozygosity for the short allele is associated with lower rate of HIV-1 replication. These results suggest that the deletion allele protects from HIV-1 infection with a recessive effect.

Population pharmacokinetic modelling of the changes in atazanavir plasma clearance caused by ritonavir plasma concentrations in HIV-1 infected patients.

AIMS: The aim of the present study was to develop a simultaneous population pharmacokinetic model for atazanavir (ATV) incorporating the effect of ritonavir (RTV) on clearance to predict ATV concentrations under different dosing regimens in HIV-1-infected patients. METHODS: A Cross-sectional study was carried out in 83 HIV-1-infected adults taking ATV 400 mg or ATV 300 mg/RTV 100 mg once daily. Demographic and clinical characteristics were registered and blood samples collected to measure drug concentrations. A population pharmacokinetic model was constructed using nonlinear mixed-effects modelling and used to simulate six dosing scenarios. RESULTS: The selected one-compartmental model described the pharmacokinetics of RTV and ATV simultaneously, showing exponential, direct inhibition of ATV clearance according to the RTV plasma concentration, which explained 17.5% of the variability. A mean RTV plasma concentration of 0.63 mg l-1 predicted an 18% decrease in ATV clearance. The percentages of patients with an end-of-dose-interval concentration of ATV below or above the minimum and maximum target concentrations of 0.15 mg l-1 and 0.85 mg l-1 favoured the selection of the simulated ATV/RTV once-daily regimens (ATV 400 mg, ATV 300 mg/RTV 100 mg, ATV 300 mg/RTV 50 mg, ATV 200/RTV 100 mg) over the unboosted twice-daily regimens (ATV 300 mg, ATV 200 mg). CONCLUSIONS: A one-compartment simultaneous model can describe the pharmacokinetics of RTV and ATV, including the effect of RTV plasma concentrations on ATV clearance. This model is promising for predicting individuals' ATV concentrations in clinical scenarios, and supports further clinical trials of once-daily doses of ATV 300 mg/RTV 50 mg or ATV 200 mg/RTV 100 mg to confirm efficacy and safety.

P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load.

OBJECTIVES: To evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. METHODS: The cellular accumulation ratio of [(3)H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gp(high)) and low P-gp activity (P-gp(low)); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. RESULTS: [(3)H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gp(high) cells accumulated less raltegravir (38.4% ±â€Š9.6%) than P-gp(low) cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gp(high) T cells sustained a higher HIV-1 replication than P-gp(low) cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). CONCLUSIONS: Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gp(high) T cells eliminate intracellular raltegravir more readily than P-gp(low) cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gp(high) T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance.

Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells.

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development.

Variants in the CYP7B1 gene region do not affect natural resistance to HIV-1 infection.

BACKGROUND: The genetic bases of natural resistance to HIV-1 infection remain largely unknown. Recently, two genome-wide association studies suggested a role for variants within or in the vicinity of the CYP7B1 gene in modulating HIV susceptibility. CYP7B1 is an appealing candidate for this due to its contribution to antiviral immune responses. We analyzed the frequency of two previously described CYP7B1 variants (rs6996198 and rs10808739) in three independent cohorts of HIV-1 infected subjects and HIV-1 exposed seronegative individuals (HESN). FINDINGS: rs6996198 and rs10808739 were genotyped in three case/control cohorts of sexually-exposed HESN and HIV-1-infected individuals from Italy, Peru and Colombia. Comparison of the allele and genotype frequencies of the two SNPs under different models showed that the only significant difference was seen for rs6996198 in the Peruvian sample (nominal p = 0.048, dominant model). For this variant, a random-effect meta-analysis yielded non-significant results (dominant model, p = 0.78) and revealed substantial heterogeneity among cohorts. No significant effect of the rs10808739 allelic status on HIV-1 infection susceptibility (additive model, p = 0.30) emerged from the meta-analysis. CONCLUSIONS: Although our study had limited power to detect association due to the small sample size, comparisons among the three cohorts revealed very similar allelic and genotypic frequencies in HESN and HIV-1 positive subjects. Overall, these data indicate that the two GWAS-defined variants in the CYP7B1 region do not strongly influence HIV-1 infection susceptibility.

Determinants of virological failure and antiretroviral drug resistance in Mozambique.

OBJECTIVES: The objective of this study was to inform public health actions to limit first-line ART failure and HIV drug resistance in Mozambique. METHODS: This was a cross-sectional study. HIV-1-infected adults on first-line ART for at least 1 year attending routine visits in the Manhiça District Hospital, in a semi-rural area in southern Mozambique with no HIV-1 RNA monitoring available, were evaluated for clinical, socio-demographic, therapeutic, immunological and virological characteristics. Factors associated with HIV-1 RNA ≥1000 copies/mL and HIV drug resistance were determined using multivariate logistic regression. RESULTS: The study included 334 adults on first-line ART for a median of 3 years, of which 65% (214/332) had suppressed viraemia, 11% (37/332) had low-level viraemia (HIV-1 RNA 150-999 copies/mL) and 24% (81/332) had overt virological failure (HIV-1 RNA ≥1000 copies/mL). HIV drug resistance was detected in 89% of subjects with virological failure, but in none with low-level viraemia. Younger age [OR = 0.97 per additional year (95% CI = 0.94-1.00), P = 0.039], ART initiation at WHO stage III/IV [OR = 2.10 (95% CI = 1.23-3.57), P = 0.003] and low ART adherence [OR = 2.69 (95% CI = 1.39-5.19), P = 0.003] were associated with virological failure. Longer time on ART [OR = 1.55 per additional year (95% CI = 1.00-2.43), P = 0.052] and illiteracy [OR = 0.24 (95% CI = 0.07-0.89), P = 0.033] were associated with HIV drug resistance. Compared with HIV-1 RNA, clinician's judgement of ART failure, based on clinical and immunological outcomes, only achieved 29% sensitivity and misdiagnosed 1 out of every 4.5 subjects. CONCLUSIONS: Public health programmes in Mozambique should focus on early HIV diagnosis, early ART initiation and adherence support. Virological monitoring drastically improves the diagnosis of ART failure, enabling a better use of resources.

Immune screening identifies novel T cell targets encoded by antisense reading frames of HIV-1.

Cytotoxic-T lymphocyte (CTL) responses to epitopes in alternative HIV reading frames have been reported. However, the extent of CTL responses to putative proteins encoded in antisense reading frames is unknown. Using sequence alignments and computational approaches, we here predict five potential antisense HIV proteins and characterize common CTL responses against them. Results suggest that antisense-derived sequences are commonly transcribed and translated and could encode functional proteins that contain important targets of anti-HIV cellular immunity.

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.

Simultaneous pharmacogenetics-based population pharmacokinetic analysis of darunavir and ritonavir in HIV-infected patients.

BACKGROUND: Darunavir is a potent protease inhibitor of HIV. To enhance its pharmacokinetic profile, darunavir must be co-administered with ritonavir. There is wide inter-patient variability in darunavir pharmacokinetics among HIV-infected individuals, however. Darunavir is a known substrate for influx transporters, such as the 1A2 and the 1B1 members of the solute carrier organic anion transporter family (SLCO1A2, SLCO1B1), as well as for efflux transporters such as the multi-drug resistance protein 1 (MRP1). OBJECTIVE: The aim of this study was to develop a semi-mechanistic population pharmacokinetic model for darunavir and ritonavir administered in HIV-infected adults. The desired model would incorporate patient characteristics and pharmacogenetic data contributing to variability in drug concentrations and also take into account the interaction between the two compounds. METHODS: A population pharmacokinetic analysis was performed with 705 plasma samples from 75 Caucasian individuals receiving darunavir/ritonavir (600/100 mg twice daily) for at least 4 weeks. At least one full pharmacokinetic profile was obtained for each participant, and darunavir and ritonavir concentrations in plasma were determined by high performance liquid chromatography. Genotyping for 148 polymorphisms in genes coding for transporters or metabolizing enzymes was conducted by two methods: MALDI-TOF mass spectrometry and real-time polymerase chain reaction-based allelic discrimination. A population pharmacokinetic model was developed for darunavir and for ritonavir. The effect of single nucleotide polymorphisms on the post hoc individual pharmacokinetic parameters was first explored using graphic methods and regression analysis. Those covariates related to changes in darunavir or ritonavir pharmacokinetic parameters were then further evaluated using non-linear mixed effects modeling (NONMEM version VII). RESULTS: Darunavir and ritonavir pharmacokinetics were best described by a two- and one-compartment model, respectively, both with first-order absorption and elimination. The darunavir peripheral volume of distribution decreased as α1-acid glycoprotein concentrations increased. Darunavir clearance was 12 % lower in patients with SLCO3A1 rs8027174 GT/TT genotypes, while homozygosity for the rs4294800 A allele was associated with 2.5-fold higher central volume of distribution. Body weight influenced ritonavir clearance. Ritonavir inhibited darunavir clearance following a maximum-effect model. CONCLUSION: A population pharmacokinetic model to simultaneously describe the pharmacokinetics of darunavir and ritonavir was developed in HIV-infected patients. The model provides better understanding of the interaction between darunavir and ritonavir and suggests an association between SLCO3A1 polymorphisms and darunavir pharmacokinetics. Bayesian estimates of individual darunavir parameters and ritonavir may be useful to predict darunavir exposure.

Herb-drug interaction between Echinacea purpurea and etravirine in HIV-infected patients.

The aim of this open-label, fixed-sequence study was to investigate the potential of the botanical supplement Echinacea purpurea to interact with etravirine, a nonnucleoside reverse transcriptase inhibitor of HIV. Fifteen HIV-infected patients receiving antiretroviral therapy with etravirine (400 mg once daily) for at least 4 weeks were included. E. purpurea root/extract-containing capsules were added to the antiretroviral treatment (500 mg every 8 h) for 14 days. Etravirine concentrations in plasma were determined by high-performance liquid chromatography immediately before and 1, 2, 4, 6, 8, 10, 12, and 24 h after a morning dose of etravirine on day 0 and etravirine plus E. purpurea on day 14. Individual etravirine pharmacokinetic parameters were calculated by noncompartmental analysis and compared between days 0 and 14 by means of the geometric mean ratio (GMR) and its 90% confidence interval (CI). The median age was 46 years (interquartile range, 41 to 50), and the median body weight was 76 kg (interquartile range, 68 to 92). Echinacea was well tolerated, and all participants completed the study. The GMR for etravirine coadministered with E. purpurea relative to etravirine alone was 1.07 (90% CI, 0.81 to 1.42) for the maximum concentration, 1.04 (90% CI, 0.79 to 1.38) for the area under the concentration-time curve from 0 to 24 h, and 1.04 (90% CI, 0.74 to 1.44) for the concentration at the end of the dosing interval. In conclusion, the coadministration of E. purpurea with etravirine was safe and well tolerated in HIV-infected patients; our data suggest that no dose adjustment for etravirine is necessary.