Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16^INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection

<div><p>Objectives</p><p>As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16^INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16^INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations.</p><p>Methods</p><p>Samples from 73 HIV-infected adults receiving daily tenofovir/emtricitabine (TFV/FTC) with either efavirenz (EFV) or atazanavir/ritonavir (ATV/r) were tested for p16^INK4a expression, and plasma cytokine and intracellular drug concentrations. Associations between p16^INK4a expression and cytokine concentrations were assessed using maximum likelihood methods, and elastic net regression was applied to assess whether cytokines were predictive of intracellular metabolite/endogenous nucleotide exposures.</p><p>Results</p><p>Enrolled participants had a median age of 48 years (range 23–73). There were no significant associations between p16^INK4a expression and cytokines. Results of the elastic net regression showed weak relationships between IL-1Ra and FTC-triphosphate and deoxyadenosine triphosphate exposures, and MIP-1β, age and TFV-diphosphate exposures.</p><p>Conclusions</p><p>In this clinical evaluation, we found no relationships between p16^INK4a expression and cytokines, or cytokines and intracellular nucleotide concentrations. While inflammation is known to play a role in this population, it is not a major contributor to the p16^INK4a association with decreased IM/EN exposures in these HIV-infected participants.</p></div

Correlation between natural-log transformed cytokines and log₂ transformed p16^INK4a expression.

<p>Correlation between natural-log transformed cytokines and log₂ transformed p16^INK4a expression.</p

Boxplots of measured cytokine concentrations.

<p>Boxplots of measured cytokine concentrations.</p

Scatterplots of p16^INK4a expression versus cytokine concentrations.

<p>Observations with quantifiable cytokine concentrations are displayed as blue circles; concentrations below the LLQ are displayed as red + symbols. Cytokine concentrations and p16^INK4a expression were natural-log transformed and log₂ transformed, respectively. A descriptive LOESS curve is plotted handling below LLQ values as observed. Correlation estimates were less than or equal to 0.22 for all associations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168709#pone.0168709.t001" target="_blank">Table 1</a>). LLQ, lower limit of quantification.</p

Elastic net results for TFV-dp AUC, FTC-tp AUC, dATP AUC, and dCTP AUC

<p>Elastic net results for TFV-dp AUC, FTC-tp AUC, dATP AUC, and dCTP AUC</p

Intracellular cytokine analysis shows the presence of antigen-specific cytokine production by CD4+ cells.

<p>Peripheral blood mononuclear cells from vaccinated subjects were stimulated with VMP001 and production of IL-2, IFN-γ and TNF-α was assessed. Pie chart depicts the percentage of CD4⁺ cytokine-producing cells for each cohort 2 weeks post 3^rd vaccination (Day of challenge). G, 2 and T indicate IFN-γIL-2 and TNF-α, respectively. Cytokine producers are represented by a “+” and non-producers by a “-”.</p

Vaccinated subjects who showed a delayed time to parasitemia had higher anti-Type 1 repeat antibodies.

<p>A. Vaccinated individuals from all three cohorts could be grouped into those that showed a delay (green, n = 16) compared to the controls, and those that did not show a delay (red, n = 11). B. Individuals with a delay in patency had higher anti-type 1 repeat antibodies compared to those with no delay (p = 0.02). Each symbol represents an individual. Cohorts 1, 2 and 3 are represented by red, blue, and green, respectively.</p

Adverse events.

<p>Solicited Adverse Events (AEs) were recorded following each vaccination and are reported as the percentage of subjects in each cohort reporting the event. Pain was the most frequently reported AE, being reported by 80–100% of the subjects. A majority of the AEs presented as Grade 1 (G1, mild). Grade 2 (G2, moderate) erythema, fever, headache and Grade 3 (G3, severe) erythema were seen in a small percentage of individuals. AEs are shown post 1^st (V1), 2^nd (V2) and 3^rd (V3) vaccine dose.</p

Fine-specificity analysis demonstrates that all domains of CSP are recognized following immunization with VMP001.

<p>Day of Challenge (DOC, or 2 weeks post-3^rd vaccination) sera from all subjects were reactive to the N-term, type-1 Repeat peptide, as well as C-term region of the protein with the highest reactivity to the C-term. C- term GMTs were significantly higher in all three cohorts compared to the N- term and Type 1 repeat GMTs. Cohort 2 had significantly higher anti-Repeat antibodies compared to Cohorts 1 and 3 (p = 0.003, p = 0.01, respectively).</p

All volunteers immunized with VMP001/AS01_B generated antibodies to VMP001.

<p>Anti-VMP001 antibodies were detected in 80% of vaccinated individuals starting at two weeks post-1^st immunization. Titers were boosted to peak levels post-2^nd immunization, with 100% of subjects developing antibodies. A decrease in antibody titers was observed on the day of third immunization (8, 6 and 4 weeks post 2^nd immunization for cohorts 1, 2 and 3, respectively) followed by a slight increase post 3^rd immunization. Antibody titers, defined as a serum dilution that gives an OD₄₁₄ of 1.0, showed a continual decline post challenge (Po Ch). Box plot represents the 25–75 percentiles and Whiskers indicate the minimum and maximum values. GMTs of anti-VMP001 antibody were significantly higher in group 1 compared to group 2 at 2 time points (2weeks post 2nd, p = 0.01, and on the day of 3rd, vaccination, p = 0.002). GMTs of anti-VMP001 antibody were significantly higher in group 2 compared to group 3 at 2 time points (4weeks post DOC, p = 0.03, and 6 months post-DOC, P = 0.04). GMTs of anti-VMP001 antibody were significantly higher in group 1 compared to group 3 at 5 time points (2 weeks post 1st vaccination, p = 0.02, on the day of 2nd vaccination, p = 0.01, 2 weeks post 2nd vaccination, p = 0.04, on the day of 3rd vaccination p = 0.002, and 6 months post DOC, p = 0.034).</p