Identification of film-based formulations that move mRNA lipid nanoparticles out of the freezer.

COVID-19 vaccines consisting of mRNA lipid nanoparticles (LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antigen protected millions of people from severe disease; however, they must be stored frozen prior to use. The objective of this study was to evaluate the compatibility and stability of mRNA LNPs within a polymer-based film matrix. An optimized formulation of polymer base, glycerol, surfactants, and PEGylated lipid that prevents damage to the LNP due to physical changes during the film-forming process (osmotic stress, surface tension, spatial stress, and water loss) was identified.

Physical characteristics and stability profile of recombinant plasmid DNA within a film matrix.

Plasmids are essential source material for production of biological drugs, vaccines and vectors for gene therapy. They are commonly formulated as frozen solutions. Considering the cost associated with maintenance of cold chain conditions during storage and transport, there is a significant need for alternative methods for stabilization of plasmids at ambient temperature.

Thermostability and in vivo performance of AAV9 in a film matrix.

Background: Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection.

Methods: AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions.

COVID-19 Vaccines and the Virus: Impact on Drug Metabolism and Pharmacokinetics.

This article reports on an American Society of Pharmacology and Therapeutics, Division of Drug Metabolism and Disposition symposium held at Experimental Biology on April 2, 2022, in Philadelphia. As of July 2022, over 500 million people have been infected with SARS-CoV-2 (the virus causing COVID-19) and over 12 billion vaccine doses have been administered. Clinically significant interactions between viral infections and hepatic drug metabolism were first recognized over 40 years ago during a cluster of pediatric theophylline toxicity cases attributed to reduced hepatic drug metabolism amid an influenza B outbreak.

Evaluation of intermolecular interactions required for thermostability of a recombinant adenovirus within a film matrix.

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal.

Immunization and Drug Metabolizing Enzymes: Focus on Hepatic Cytochrome P450 3A.

Objective: Infectious disease emergencies like the 2013-2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism.

Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature.

A novel, thin-film platform that preserves live viruses, bacteria, antibodies, and enzymes without refrigeration for extended periods of time is described. Studies with recombinant adenovirus in an optimized formulation that supports recovery of live virus through 16 freeze-thaw cycles revealed that production of an amorphous solid with a glass transition above room temperature and nitrogen-hydrogen bonding between virus and film components are critical determinants of stability. Administration of live influenza virus in the optimized film by the sublingual and buccal routes induced antibody-mediated immune responses as good as or better than those achieved by intramuscular injection.

Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A.

Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection.

A long-lasting, single-dose nasal vaccine for Ebola: a practical armament for an outbreak with significant global impact.

In response to the severity and scale of the 2014 Ebola outbreak, several experimental vaccines were granted fast-track status for clinical testing. Although they may provide long-lasting protection from Ebola, they are, in their current states, far from optimal for populations that need them the most. In this context, nasal immunization addresses the: immune response required at the mucosa where Ebola initiates infection; needs of a population in terms of cost and compliance; and potency of each platform as they contain viruses that naturally infect the respiratory tract.

Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization.

A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.

Evaluation of the HC-04 cell line as an in vitro model for mechanistic assessment of changes in hepatic cytochrome P450 3A during adenovirus infection.

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection.

Modeling pre-existing immunity to adenovirus in rodents: immunological requirements for successful development of a recombinant adenovirus serotype 5-based ebola vaccine.

Pre-existing immunity (PEI) to human adenovirus serotype 5 (Ad5) worldwide is the primary limitation to routine clinical use of Ad5-based vectors in immunization platforms. Using systemic and mucosal PEI induction models in rodents (mice and guinea pigs), we assessed the influence of PEI on the type of adaptive immune response elicited by an Ad5-based vaccine for Ebola with respect to immunization route. Splenocytes isolated from vaccinated animals revealed that immunization by the same route in which PEI was induced significantly compromised Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ CD8+ T cells and ZGP-specific multifunctional CD8+ T cell populations.

Emerging targets and novel approaches to Ebola virus prophylaxis and treatment.

Ebola is a highly virulent pathogen causing severe hemorrhagic fever with a high case fatality rate in humans and non-human primates (NHPs). Although safe and effective vaccines or other medicinal agents to block Ebola infection are currently unavailable, a significant effort has been put forth to identify several promising candidates for the treatment and prevention of Ebola hemorrhagic fever. Among these, recombinant adenovirus-based vectors have been identified as potent vaccine candidates, with some affording both pre- and post-exposure protection from the virus.

Induction of broad cytotoxic T cells by protective DNA vaccination against Marburg and Ebola.

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported.

A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs.

Sublingual (SL) delivery, a noninvasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa.

Optimized adenovirus-antibody complexes stimulate strong cellular and humoral immune responses against an encoded antigen in naive mice and those with preexisting immunity.

The immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity. In vitro and in vivo assays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 10(11) adenovirus particles mixed with different concentrations of neutralizing antibodies.

Species differences in the pharmacology and toxicology of PEGylated helper-dependent adenovirus.

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h.

Recent advances in Ebolavirus vaccine development.

Ebolavirus is a highly infectious pathogen with a case fatality rate as high as 90%. Currently there is a lack of licensed Ebolavirus vaccines as well as pre- and post-exposure treatments. Recent increases in the frequency of natural human Ebolavirus infections and its potential use as a bioterrorism agent makes vaccine development a priority for many nations.

PEGylated Adenoviruses: From Mice to Monkeys.

Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

Development of a nasal adenovirus-based vaccine: Effect of concentration and formulation on adenovirus stability and infectious titer during actuation from two delivery devices.

A nasal adenovirus-based vaccine is under development. To determine if aggregation occurs during vaccination, infectious titer (limiting dilution) and capsid integrity (dynamic light scattering) were assessed after extrusion of a model vector from two intranasal delivery devices. Preparations of 2.

Long-term virus-induced alterations of CYP3A-mediated drug metabolism: a look at the virology, immunology and molecular biology of a multi-faceted problem.

Virus infections are on the rise. Although the first description of CYP expression during virus infection was recorded 50 years ago, mechanistic studies of this phenomenon only began to appear in the last decade due to breakthroughs in molecular biology, genomic and transgenic technology. This review describes the relationship(s) among CYP-mediated drug metabolism, virus infection and the immune response and evaluates in vitro and in vivo models for mechanistic studies.

Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

Background: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection.

Nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice.

Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.