Aswani Vunnava

Intradermal Vaccination with Influenza Virus-Like Particles by Using Microneedles Induces Protection Superior to That with Intramuscular Immunization

ABSTRACT Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (≤40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.

Dysregulated B Cell Expression of RANKL and OPG Correlates with Loss of Bone Mineral Density in HIV Infection

HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.

Antiretroviral therapy induces a rapid increase in bone resorption that is positively associated with the magnitude of immune reconstitution in HIV infection

Objective:Antiretroviral therapy (ART) paradoxically intensifies bone loss in the setting of HIV infection. Although the extent of bone loss varies, it occurs with virtually all ART types, suggesting a common pathway that may be aligned with HIV disease reversal. Using an animal model of immunodeficiency we recently demonstrated that immune activation associated with CD4+ T-cell reconstitution induces increased production of the osteoclastogenic cytokines RANKL and TNF&agr; by immune cells, driving enhanced bone resorption and loss in bone mineral density. Design:To confirm these findings in humans, we investigated the early kinetics of CD4+ T-cell recovery in relation to biomarkers of bone turnover and osteoclastogenic regulators in a prospective 24-week cohort study. Methods:Clinical data and blood sampling for HIV-RNA PCR, CD4+ T-cell counts, bone turnover biomarkers, and osteoclastogenic regulators were obtained from ART-naïve HIV-infected study participants initiating standard doses of lopinavir/ritonavir plus tenofovir disoproxil fumarate/emtricitabine at baseline and at weeks 2, 8, 12, and 24 post ART. Results:C-terminal telopeptide of collagen (CTx) a sensitive biomarker of bone resorption rose by 200% above baseline at week 12, remaining elevated through week 24 (&agr;<0.01), and was associated with significant increases in plasma levels of osteoclastogenic regulators [receptor activator of NF-kB ligand (RANKL), tumor necrosis factor alpha, (TNF&agr;)]. Importantly, the magnitude of CD4+ T-cell recovery correlated significantly with CTx (rs = 0.387, &agr;=0.01). Conclusion:Our data suggest that ART-induced bone loss occurs early, is aligned with early events of immune reconstitution, and these immune changes provide a unifying mechanism to explain in part the skeletal decline common to all ART.

A Single-dose Zoledronic Acid Infusion Prevents Antiretroviral Therapy–induced Bone Loss in Treatment-naive HIV-infected Patients: A Phase IIb Trial

BACKGROUND Human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) are associated with bone loss leading to increased fracture rate among HIV-infected individuals. ART-induced bone loss is most intense within the first 48 weeks of therapy, providing a window for prophylaxis with long-acting antiresorptives. METHODS In a phase 2, double-blind, placebo-controlled trial, we randomized 63 nonosteoporotic, ART-naive adults with HIV initiating ART with atazanavir/ritonavir + tenofovir/emtricitabine to a single zoledronic acid (ZOL) infusion (5 mg) vs placebo to determine the efficacy of ZOL in mitigating ART-induced bone loss. Plasma bone turnover markers and bone mineral density (BMD) were performed at weeks 0, 12, 24, and 48 weeks. Primary outcome was change in C-terminal telopeptide of collagen at 24 weeks. Repeated-measures analyses using mixed linear models were used to estimate and compare study endpoints. RESULTS The ZOL arm had a 65% reduction in bone resorption relative to the placebo arm at 24 weeks (0.117 ng/mL vs 0.338 ng/mL; P < .001). This effect of ZOL occurred as early as 12 weeks (73% reduction; P < .001) and persisted through week 48 (57% reduction; P < .001). The ZOL arm had an 8% higher lumbar spine BMD at 12 weeks relative to the placebo arm (P = .003), and remained 11% higher at 24 and 48 weeks. Similar trends were observed in the hip and femoral neck. CONCLUSIONS A single dose of ZOL administered at ART initiation prevented ART-induced bone loss through the first 48 weeks of ART, the period when ART-induced bone loss is most pronounced. Validation of these results in larger multicenter randomized clinical trials is warranted. CLINICAL TRIALS REGISTRATION NCT01228318.

Effect of protein binding on unbound atazanavir and darunavir cerebrospinal fluid concentrations

HIV‐1 protease inhibitors (PIs) exhibit different protein binding affinities and achieve variable plasma and tissue concentrations. Degree of plasma protein binding may impact central nervous system penetration. This cross‐sectional study assessed cerebrospinal fluid (CSF) unbound PI concentrations, HIV‐1 RNA, and neopterin levels in subjects receiving either ritonavir‐boosted darunavir (DRV), 95% plasma protein bound, or atazanavir (ATV), 86% bound. Unbound PI trough concentrations were measured using rapid equilibrium dialysis and liquid chromatography/tandem mass spectrometry. Plasma and CSF HIV‐1 RNA and neopterin were measured by Ampliprep/COBAS® Taqman® 2.0 assay (Roche) and enzyme‐linked immunosorbent assay (ALPCO), respectively. CSF/plasma unbound drug concentration ratio was higher for ATV, 0.09 [95% confidence interval (CI) 0.06–0.12] than DRV, 0.04 (95%CI 0.03–0.06). Unbound CSF concentrations were lower than protein adjusted wild‐type inhibitory concentration‐50 (IC50) in all ATV and 1 DRV‐treated subjects (P < 0.001). CSF HIV‐1 RNA was detected in 2/15 ATV and 4/15 DRV subjects (P = 0.65). CSF neopterin levels were low and similar between arms. ATV relative to DRV had higher CSF/plasma unbound drug ratio. Low CSF HIV‐1 RNA and neopterin suggest that both regimens resulted in CSF virologic suppression and controlled inflammation.

Impact of protease inhibitors on intracellular concentration of tenofovir-diphosphate among HIV-1 infected patients

Intracellular nucleoside reverse transcriptase inhibitor (NRTI) concentrations are associated with plasma HIV-1 response. Coadministration of protease inhibitors with NRTIs can affect intracellular concentrations due to protease inhibitor inhibition of efflux transporters. Tenofovir-diphosphate (TFV-DP) concentrations within peripheral blood mononuclear cells were compared among individuals receiving either atazanavir or darunavir-based regimens. There was a trend towards higher TFV-DP concentrations among women and among participants receiving atazanavir. TFV-DP intracellular concentrations were positively associated with undetectable plasma HIV-1 RNA.

Cerebrospinal fluid concentrations of tenofovir and emtricitabine in the setting of HIV‐1 protease inhibitor‐based regimens

In the combination antiretroviral therapy (cART) era, prevalence of HIV-associated neurocognitive disorders (HAND) remains high: 36.2-44.8% 1, contributing to morbidity 1 and mortality 2. Suboptimal control of HIV in the central nervous system (CNS) likely plays a role in this phenomenon, as supported by elevated neopterin and detectable HIV-1 RNA in cerebrospinal fluid (CSF) of patients on long term cART 3. Studies have shown that limited CNS penetration by antiretroviral (ARV) drugs is associated with HAND 4,5. Multiple factors impact CNS drug penetration, including molecular size, lipophilicity, plasma protein binding, and affinity for efflux pump transporters 6. For example, the ATP-binding cassette (ABC) superfamily of efflux transporters, including p-glycoprotein and multidrug-resistance associated proteins (MRPs), are expressed at blood-brain and blood-CSF barriers and can limit drug disposition7. Because many ARVs are substrates and/or inhibitors of ABC transporters, complex drug-drug interactions can occur. Specifically, protease inhibitors (PIs) atazanavir (ATV) and darunavir (DRV) are common in cART regimens when given with ritonavir (RTV) plus a backbone of tenofovir disoproxil fumarate (TDF), the tenofovir (TFV) pro-drug, and emtricitabine (FTC) 8. Both ATV and DRV inhibit p-glycoprotein and MRP-1 9-11 ; these transporters are found at blood-brain and blood-CSF barriers and are important for TFV 12 and FTC transport 13. We compared CSF TFV and FTC concentrations between patients receiving two different RTV-boosted PI regimens: ATV vs DRV. We also examined associations between CSF drug concentrations and CSF HIV-1 RNA detection.