Measles (Me) and rubella (Ru) viral diseases are targeted for elimination by ensuring a high level of vaccination coverage worldwide. Less costly, more convenient MeRu vaccine delivery systems should improve global vaccine coverage, especially in low – and middle – income countries (LMICs). In this work, we examine formulating a live, attenuated Me and Ru combination viral vaccine with Nanopatch™, a solid polymer micro-projection array for intradermal delivery. First, high throughput, qPCR-based viral infectivity and genome assays were established to enable formulation development to stabilize Me and Ru in a scaled-down, custom-built evaporative drying system to mimic the Nanopatch™ vaccine coating process. Second, excipient screening and optimization studies identified virus stabilizers for use during the drying process and upon storage in the dried state. Finally, a series of real-time and accelerated stability studies identified eight candidate formulations that met a target thermal stability criterion for live vaccines (<1 log₁₀ loss after 1 week storage at 37°C). Compared to −80°C control samples, the top candidate formulations resulted in minimal viral infectivity titer losses after storage at 2–8°C for 6 months (i.e., <0.1 log₁₀ for Me, and ~0.4 log₁₀ for Ru). After storage at 25°C over 6 months, ~0.3–0.5 and ~1.0–1.4 log₁₀ titer losses were observed for Me and Ru, respectively, enabling the rank-ordering of the stability of candidate formulations. These results are discussed in the context of future formulation challenges for developing microneedle-based dosage forms containing stabilized live, attenuated viral vaccines for use in LMICs.

Rotavirus infections remain a leading cause of morbidity and mortality among infants residing in low- and middle-income countries. To address the large need for protection from this vaccine-preventable disease we are developing a trivalent subunit rotavirus vaccine which is currently being evaluated in a multinational Phase 3 clinical trial for prevention of serious rotavirus gastroenteritis. Currently, there are no universally accepted in vivo or in vitro models that allow for correlation of field efficacy to an immune response against serious rotavirus gastroenteritis. As a new generation of non-replicating rotavirus vaccines are developed the lack of an established model for evaluating vaccine efficacy becomes a critical issue related to how vaccine potency and stability can be assessed. Our previous publication described the development of an in vitro ELISA to quantify individual vaccine antigens adsorbed to an aluminum hydroxide adjuvant to address the gap in vaccine potency methods for this non-replicating rotavirus vaccine candidate. In the present study, we report on concordance between ELISA readouts and in vivo immunogenicity in a guinea pig model as it relates to vaccine dosing levels and sensitivity to thermal stress. We found correlation between in vitro ELISA values and neutralizing antibody responses engendered after animal immunization. Furthermore, this in vitro assay could be used to demonstrate the effect of thermal stress on vaccine potency, and such results could be correlated with physicochemical analysis of the recombinant protein antigens. This work demonstrates the suitability of the in vitro ELISA to measure vaccine potency and the correlation of these measurements to an immunologic outcome.

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2-8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery.

Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2–8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50–60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitro gastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2–8°C and −20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities.

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector’s physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.