Pavan Muttil

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2

An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2 to 25 μg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37 °C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations.

Preparation and Characterization of Novel Magnetic Nano-in-Microparticles for Site-Specific Pulmonary Drug Delivery

We propose the use of novel inhalable nano-in-microparticles (NIMs) for site-specific pulmonary drug delivery. Conventional lung cancer therapy has failed to achieve therapeutic drug concentrations at tumor sites without causing adverse effects in healthy tissue. To increase targeted drug delivery near lung tumors, we have prepared and characterized a magnetically responsive dry powder vehicle containing doxorubicin. A suspension of lactose, doxorubicin and Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) were spray dried. NIMs were characterized for their size and morphological properties by various techniques: dynamic light scattering (DLS) and laser diffraction (LS) to determine hydrodynamic size of the SPIONs and the NIMs, respectively; next generation cascade impactor (NGI) to determine the aerodynamic diameter and fine particle fraction (FPF); scanning (SEM) and transmission (TEM) electron microscopy to analyze particle surface morphology; electron dispersive X-ray spectroscopy (EDS) to determine iron loading in NIMs; inductively coupled plasma atomic emission spectroscopy (ICP-AES) and superconducting quantum interference device (SQUID) to determine Fe3O4 content in the microparticles; and high performance liquid chromatography (HPLC) to determine doxorubicin loading in the vehicle. NIMs deposition and retention near a magnetic field was performed using a proof-of-concept cylindrical tube to mimic the conducting airway deposition. The hydrodynamic size and zeta potential of SPIONs were 56 nm and -49 mV, respectively. The hydrodynamic and aerodynamic NIM diameters were 1.6 μm and 3.27±1.69 μm, respectively. SEM micrographs reveal spherical particles with rough surface morphology. TEM and focused ion beam-SEM micrographs corroborate the porous nature of NIMs, and surface localization of SPIONs. An in vitro tracheal mimic study demonstrates more than twice the spatial deposition and retention of NIMs, compared to a liquid suspension, in regions under the influence of a strong magnetic gradient. We report the novel formulation of an inhaled and magnetically responsive NIM drug delivery vehicle. This vehicle is capable of being loaded with one or more chemotherapeutic agents, with future translational ability to be targeted to lung tumors using an external magnetic field.

Optimized Formulation of a Thermostable Spray-Dried Virus-Like Particle Vaccine against Human Papillomavirus

Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP)-based vaccine for HPV infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multicomponent excipient system and by optimizing the spray-drying parameters using a half-factorial design approach. Dry-powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry-powder VLPs that were stored at 37 °C for more than a year elicited high anti-L2 IgG antibody titers. Spray-dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ∼84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage.

Oral Tolerance to Environmental Mycobacteria Interferes with Intradermal, but Not Pulmonary, Immunization against Tuberculosis

Bacille Calmette–Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB) and is administered in over 150 countries worldwide. Despite its widespread use, the vaccine has a variable protective efficacy of 0–80%, with the lowest efficacy rates in tropical regions where TB is most prevalent. This variability is partially due to ubiquitous environmental mycobacteria (EM) found in soil and water sources, with high EM prevalence coinciding with areas of poor vaccine efficacy. In an effort to elucidate the mechanisms underlying EM interference with BCG vaccine efficacy, we exposed mice chronically to Mycobacterium avium (M. avium), a specific EM, by two different routes, the oral and intradermal route, to mimic human exposure. After intradermal BCG immunization in mice exposed to oral M. avium, we saw a significant decrease in the pro-inflammatory cytokine IFN-γ, and an increase in T regulatory cells and the immunosuppressive cytokine IL-10 compared to naïve BCG-vaccinated animals. To circumvent the immunosuppressive effect of oral M. avium exposure, we vaccinated mice by the pulmonary route with BCG. Inhaled BCG immunization rescued IFN-γ levels and increased CD4 and CD8 T cell recruitment into airways in M. avium-presensitized mice. In contrast, intradermal BCG vaccination was ineffective at T cell recruitment into the airway. Pulmonary BCG vaccination proved protective against Mtb infection regardless of previous oral M. avium exposure, compared to intradermal BCG immunization. In conclusion, our data indicate that vaccination against TB by the pulmonary route increases BCG vaccine efficacy by avoiding the immunosuppressive interference generated by chronic oral exposure to EM. This has implications in TB-burdened countries where drug resistance is on the rise and health care options are limited due to economic considerations. A successful vaccine against TB is necessary in these areas as it is both effective and economical.

Spray-Dried Multiscale Nano-biocomposites Containing Living Cells

Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines; it also allows the study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes. Here, we report a spray-drying process enabling the large-scale production of functional nano-biocomposites (NBCs) containing living cells within ordered 3D lipid-silica nanostructures. The spray-drying process is demonstrated to work with multiple cell types and results in dry powders exhibiting a unique combination of properties including highly ordered 3D nanostructure, extended lipid fluidity, tunable macromorphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed a Youngs modulus and hardness of 13 and 1.4 GPa, respectively. We hypothesized this high strength would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in a NBC-induced VBNC, less than 1 in 10,000 cells underwent resuscitation. The NBC platform production of large quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBCs may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines.

Pharmacokinetics of Ethionamide Delivered in Spray-Dried Microparticles to the Lungs of Guinea Pigs

The use of ethionamide (ETH) in treating multidrug-resistant tuberculosis is limited by severe side effects. ETH disposition after pulmonary administration in spray-dried particles might minimize systemic exposure and side effects. To explore this hypothesis, spray-dried ETH particles were optimized for performance in a dry powder aerosol generator and exposure chamber. ETH particles were administered by the intravenous (IV), oral, or pulmonary routes to guinea pigs. ETH appearance in plasma, bronchoalveolar lavage, and lung tissues was measured and subjected to noncompartmental pharmacokinetic analysis. Dry powder aerosol generator dispersion of 20% ETH particles gave the highest dose at the exposure chamber ports and fine particle fraction of 72.3%. Pulmonary ETH was absorbed more rapidly and to a greater extent than orally administered drug. At Tmax, ETH concentrations were significantly higher in plasma than lungs from IV dosing, whereas insufflation lung concentrations were 5-fold higher than in plasma. AUC(0-t) (area under the curve) and apparent total body clearance (CL) were similar after IV administration and insufflation. AUC(0-t) after oral administration was 6- to 7-fold smaller and CL was 6-fold faster. Notably, ETH bioavailability after pulmonary administration was significantly higher (85%) than after oral administration (17%). These results suggest that pulmonary ETH delivery would potentially enhance efficacy for tuberculosis treatment given the high lung concentrations and bioavailability.

Characterization of a spray-dried candidate HPV L2-VLP vaccine stored for multiple years at room temperature

HPV infections are associated with human cancers. Although three prophylactic vaccines have been approved to protect against HPV infections, the vaccines require cold-chain storage and may not be suitable for third world countries with less developed refrigeration facilities. We previously developed a bacteriophage L2 virus-like particle (VLP)-based candidate vaccine, which elicited broadly protective antibodies against diverse HPV types. Spray-drying of MS2-16L2 VLPs into a dry powder enhanced the stability of these VLPs. Building on these studies, we assessed the long-term stability and immunogenicity of the spray-dried VLPs. Mice immunized with a single dose of spray-dried MS2-16L2 VLPs, which had been stored for 14 months at room temperature (RT), were partially protected from challenge with a high dose of HPV16, one year after immunization. However, immunization with two doses of MS2-16L2 VLPs stored at RT for 34 months elicited high titer anti-HPV antibodies. More importantly, this group of mice showed significant protection from HPV16, 4 months after immunization. These results suggest that spray-dried MS2-16L2 VLPs retain their effectiveness after long-term storage at RT, and may be suitable in third world countries with less developed refrigeration facilities.

A stable live bacterial vaccine

Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most.

In Vivo Pulmonary Delivery and Magnetic-Targeting of Dry Powder Nano-in-Microparticles

This brief communication evaluates the cytotoxicity and targeting capability of a dry powder chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose matrix. In vitro cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH assays to assess viability and toxicity after 48 h of NIMs exposure. In vivo magnetic-field-dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were endotracheally administered fluorescently labeled NIMs either as a dry powder or a liquid aerosol in the presence of an external magnet placed over the left lung. Quantification of fluorescence and iron showed a significant increase in both fluorescence intensity and iron content to the left magnetized lung. In comparison, we observed decreased targeting of fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better targeted to specific regions of the lung in the presence of a magnetic field, compared to a liquid suspension.

Respirable Uranyl-Vanadate-Containing Particulate Matter Derived From a Legacy Uranium Mine Site Exhibits Potentiated Cardiopulmonary Toxicity

Exposure to windblown particulate matter (PM) arising from legacy uranium (U) mine sites in the Navajo Nation may pose a human health hazard due to their potentially high metal content, including U and vanadium (V). To assess the toxic impact of PM derived from Claim 28 (a priority U mine) compared with background PM, and consider the putative role of metal species U and V. Two representative sediment samples from Navajo Nation sites (Background PM and Claim 28 PM) were obtained, characterized in terms of chemistry and morphology, and fractioned to the respirable (≤ 10 μm) fraction. Mice were dosed with either PM sample, uranyl acetate, or vanadyl sulfate via aspiration (100 µg), with assessments of pulmonary and vascular toxicity 24 h later. Particulate matter samples were also examined for in vitro effects on cytotoxicity, oxidative stress, phagocytosis, and inflammasome induction. Claim 28 PM10 was highly enriched with U and V and exhibited a unique nanoparticle ultrastructure compared with background PM10. Claim 28 PM10 exhibited enhanced pulmonary and vascular toxicity relative to background PM10. Both U and V exhibited complementary pulmonary inflammatory potential, with U driving a classical inflammatory cytokine profile (elevated interleukin [IL]-1β, tumor necrosis factor-α, and keratinocyte chemoattractant/human growth-regulated oncogene) while V preferentially induced a different cytokine pattern (elevated IL-5, IL-6, and IL-10). Claim 28 PM10 was more potent than background PM10 in terms of in vitro cytotoxicity, impairment of phagocytosis, and oxidative stress responses. Resuspended PM10 derived from U mine waste exhibit greater cardiopulmonary toxicity than background dusts. Rigorous exposure assessment is needed to gauge the regional health risks imparted by these unremediated sites.