Development of Shelf Stable Formulation for Adenovirus Vectored Vaccines and Therapeutics

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Iwashkiw, Aaisha Ameen, Natallia Kazhdan, Sam Afkhami, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7767949/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 15 You are reading this latest preprint version Abstract A major limitation of therapeutic delivery is the cold chain storage requirement. Adenoviruses (AdV) have been demonstrated to be an effective delivery vector for several indications including COVID-19 vaccines but are limited by storage and transportation conditions. Previous work has demonstrated formulation and drying of vectored vaccines with pullulan and trehalose-based (PT) films significantly improves thermostabilization. To increase the accessibility of AdV based therapeutics, we developed a vacuum based drying methodology with optimized PT excipients resulting in a shelf stable product. We demonstrate the thermostability of formulated and dried AdV at 55 o C for 7 weeks with less than 0.5 total log IU loss, and over 44 weeks at 37 o C with less than 0.25 total log IU loss. Additionally, murine vaccination with the ChAd-TriCoV/Mac vaccine showed no difference in response between fresh and aged at 37 o C for 44 weeks. These data demonstrate our formulation methodology’s performance, resulting in a shelf stable formulation for AdV based therapeutics. Biological sciences/Biotechnology Biological sciences/Drug discovery Health sciences/Health care Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Significance Statement In this work, we developed a vacuum drying based protocol to optimize the stability of AdV and demonstrate thermal stability at 37 o C for 450 days, and 55 o C for up to 49 days. These results are the best described thermostability of AdV to date, approximately 2.5x longer at 55 o C than required for the highest VVM 250 category. Finally, we demonstrate equal efficacy of a previously described AdV vectored COVID 19 vaccine in a murine model comparing fresh -80 o C stocks, dried vaccine, and dried vaccine aged for 310 days at 37 o C. Introduction Proactive measures such as vaccination, as demonstrated by programs targeting diseases such as polio, smallpox, and measles, has established the value of prevention of diseases, reducing the global health care burden [ 1 ]. Recently it has been demonstrated global vaccination efforts have saved over 154 million lives since 1974, with 101 million being infants [ 2 ]. Most vaccines produced require stringent cold chain controls to prevent degradation and subsequent loss of efficacy. Vaccines including lipid nanoparticle encapsulated mRNA vaccines, and viral vectored vaccines require a -70 o C cold chain pathway, which is technically, logistically, and economically challenging to disseminate critical vaccines to hard-to-reach places and populations. Up to 80% of vaccination programs funds are directed to funding these cold chain requirements [ 3 ]. In addition is the requirement for public health organizations to create both static and rotating stockpiles of vaccines for different types of outbreaks, which is also heavily dependent on the stability of vaccine products [ 4 ]. To improve vaccine stability, many groups supplement the product with stabilizing excipients and remove water, resulting in decreased mobility of molecules and inhibiting liquid degradation mechanisms [ 5 ]. Solid biopharmaceuticals have greater thermostability and are less sensitive to degradation by both chemical and physical mechanisms [ 5 – 7 ]. Two main theories have been postulated to explain this phenomenon: vitrification and water replacement theory [ 7 ]. The most common drying process employed is lyophilization, where liquid samples are frozen and liquid is removed by sublimation [ 8 , 9 ]. Several biopharmaceuticals are sensitive to ice crystals and cold denaturation causing substantial loss of functionality [ 10 ]. While not commercially employed at scale, vacuum drying techniques have been developed for drying biopharmaceuticals [ 11 ]. Vacuum drying has several advantages over lyophilization including being less energy intensive, less physical stress on the therapeutic during dehydration, and can impart greater thermostability to biologically active organisms such as probiotic bacteria. [ 11 ] Adenoviruses (AdV) are non-enveloped DNA viruses which can infect a wide range of hosts and are often associated with mild-to-moderate respiratory symptoms. However, AdV have been harnessed as a viral vector platform for gene therapy, and as vaccines for cancer and other infectious diseases such as SARS-CoV-2 [ 12 – 14 ]. One drawback of AdV vectored therapeutics is a cold chain requirement for storage and shipment [ 15 ]. Although studies thermostabilizing AdV vectored vaccine have demonstrated 0.17 log IU loss after 2 years at 5 o C with a predicted stability for 5 years at 5 o C, the requirement for cold chain treatment of AdV vaccines is still a limitation for the technology, and delivery of treatments with limitations in cold chain infrastructure [ 15 ]. In this work we describe the development of a stable shelf formulation (PT120-D) utilizing vacuum drying techniques for AdV vectors. Building off our previous work with vesicular stomatitis virus (VSV) vector stabilization [ 16 ], we optimized a drying schedule and excipient formulation for enhanced vacuum drying of human adenovirus serotype 5 vectors, and an inhaled chimpanzee adenovirus serotype 68-based COVID-19 vaccine (ChAd-TriCoV/Mac) that is in phase II clinical trials in Canada. The final formulation demonstrated a total titer loss of less than 0.25 log IU over 44 weeks at 37 o C, as well as less than 0.3 log loss after up to 7 weeks at 55 o C which is > 2.5× longer than the VVM250 requirements [ 17 ]. To our knowledge, no reports have thus far shown comparable thermostability and durability of stabilization. Importantly, we further demonstrated that the formulated and dried vaccine retained its in vivo immunogenicity after 44 weeks of incubation at 37°C—comparable to that of the liquid control—highlighting the potential of this approach to enable long-term storage without cold chain. Together, these data demonstrate the stabilization effectiveness of the PT120-D formulation, resulting in a shelf stable delivery system for AdV based therapeutics. Results PT120-D formulation of HuAd5-GFP has significantly better thermostability when vacuum dried compared to freeze dried without nitrogen gas backfilling We utilized a human adenovirus serotype 5 vector expressing GFP (HuAd5-GFP) for initial comparison of PT120-D stabilization to the lead formulation identified in the literature which utilized a formulation consisting of inulin and mannitol [ 18 ]. We performed two different dry schedules (Schedule 1 and 2; Supplemental Table 1) with the two formulations and assayed thermostability at 37 o C (Fig. 1 ). For Formulation 1, different film morphologies were observed, whereas formulation 2 formed a cake regardless of drying schedule (Fig. 1 A). Formulation 1 vacuum dried had a glassy film compared to the freeze dried being a lyophilized cake. Formulation 1 vacuum dried was the best combination for lowest process and thermostability loss, whereas the same formulation had the greatest loss vacuum dried (Fig. 1 B/C). No significant difference in process loss or thermostability was observed for Formulation 2 between drying schedules. Over a 450-day incubation at 37 o C, no additional loss was observed for the Formulation 1 vacuum dried (total Log IU loss of 0.2), compared to accumulated losses for the other 3 conditions (total Log IU loss > 4). Additionally, both pullulan and trehalose are required for stabilizing adenovirus (Figure S1 ). To the best of our knowledge, this reflects the highest level of stability reported for an adenoviral vector to date. Formulation F1 demonstrates thermostability stability at 45/55 o C, but weaker stability at 37 o C observed with nitrogen backfilling With the robust stability observed at 37 o C, we next sought to determine if a more accelerated thermal challenge was viable for Formulation 1. According to the WHO VVM stability criteria, 55 o C is the hottest thermal challenge utilized for calculated accelerated thermostability of a vaccine product [ 17 ]. For the highest thermostability categorization of VVM250, it would be the equivalent of 250 days at 37 o C, 73 days at 45 o C, or 17 days at 55 o C. The additional step of backfilling the headspace of the vial was employed as it is commonplace to backfill samples with an inert gas such as nitrogen to improve stability by reducing chemical instabilities [ 6 ]. Utilizing HuAd5 with Formulation 1, dry schedule 1, and nitrogen gas backfilling, we observed a similar film morphology as in Fig. 1 (Fig. 2 A). A similar process loss of ~ 0.2 Log IU was consistent with the previous data. However, at 37 o C we observed a greater Log IU loss, and the plateau observed over 375 days was 0.73 log IU loss (Fig. 2 B/C). At more strenuous thermal challenges, we observed a greater IU loss with a calculated plateau at 1.12 Log IU loss for 45 o C and a loss of 1.65 log IU after 21 days at 55 o C. While not outstanding results, utilizing elevated thermal challenges provided a more rapid turnaround time for iterative testing to improve formulation performance. XRD analyses of dried PT120-D formulations demonstrates film crystallinity with F1, and removal of salt ions eliminates crystallinity For commercial dried products, it is commonplace to backfill samples with an inert gas like nitrogen to improve stability by reducing chemical instabilities [ 6 ]. Based off the results from our recent work [ 16 ], we hypothesized that crystallinity of the dried PT120-D film was causing the additional titer loss when vials were backfilled with dried nitrogen gas. Comparing the HuAd5-GFP thermostability results of Figs. 1 and 2 , we had significantly greater loss of AdV titer with nitrogen backfilled vials compared to atmospheric conditions. To understand if the nitrogen backfilling was affecting the structure of the dried film, XRD analysis was performed on a thermal treated time course at 55 o C (Fig. 3 ). No crystallinity was detected at D0 for all conditions assayed, and no crystallinity was detected over the 28-day incubation period for either the atmospheric filled Formulation F1 or nitrogen backfilled F6. Conversely, Formulation 1 had a low percentage crystallinity detected on Day 1 and peaked at 3.25% on day 7. A slow decline in crystallinity was observed until Day 28 when the experiment was completed. A correlation between observed film crystallinity and AdV IU titer loss was shown to be dependent on the combination of salts and nitrogen gas backfilling of vial headspace. HuAd5-GFP stability requires BSA, and removal of salt ions improves thermostability at 55 o C To confirm removal of crystallinity improves thermostability, we formulated and dried HuAd5-GFP with formulation F1, F4, and F5 with dry schedule 3 (Fig. 4 ). Formulation F4 was tested to determine whether BSA was important for HuAd5-GFP stability. No significant difference was observed for process loss between the three conditions, with F4 having slightly greater Log IU loss. Samples were incubated at 55 o C, and no significant difference in total IU loss was observed on Day 7 with all 3 having between 0.9-1.0 log IU loss. By day 21, Formulation F4 lacking BSA had significantly great loss, (1.7 Log IU) compared to 1.23 for Formulation F1 and 1.03 for Formulation F4. Overall, BSA was critical for thermostability in PT120-D films, and removal of salt ions modestly improved HuAd5-GFP thermostability. Addition of PMAL significantly increased dialysis loss, but increased trehalose significantly increased thermostability at 55C To further improve the performance of PT120-D at 55 o C, we decided to either increase the amount of trehalose (Formulation F7) or add 1% PMAL (Formulation F6), and surfactant that had shown promise in the literature at stabilizing biologics in thin films [ 19 ]. Unexpectedly, a significant loss of titer (1.3–1.4 Log IU loss) for two differently vectored HuAd5 (GFP and Luciferase transgenes) during buffer exchange with Zeba spin columns (Fig. 5 ). Process loss observed for all three formulations was between 0.05–0.25 Log IU, with formulation F7 having the least observed loss. After 49 days at 55 o C, the formulation with PMAL performed the worst with a total loss of ~ 2 log IU, with the majority of the loss derived from the dialysis step. Formulation F5 had a total loss of 1.1 log IU for both vectors, but the best performing formulation was F7 with a loss of 0.5 log IU. This thermostability result is by far the most stable we have observed compared to the literature to date. Thermostability of a chimpanzee adenovirus-vectored vaccine at 55C comparable to HuAd5 We have thus far developed a novel formulation technology using a representative human AdV. However, there is growing evidence highlighting the utility of non-human AdV, such as those of simian origin, as improved vectors for vaccine development [ 20 ]. Given sequence differences between AdV isolated from different species which can alter capsid sequences, it is important to evaluate such formulations using AdV from different species. To this end, we chose to utilize a chimpanzee AdV serotype 68-based COVID-19 (ChAd-TriCoV/Mac) inhaled aerosol vaccine that is under evaluation in a phase II trial in Canada [ 13 ]. To assay if Formulation F7 provided comparable stabilization between human and chimpanzee AdV, we formulated, dried, and thermally aged the samples at 55 o C (Fig. 6 ). Both sets of samples had minimal process loss observed and impressive thermostability. After 42-day incubation at 55 o C, both AdV’s had less than 0.5 log IU loss. These data support the widespread applicability of the PT120-D Formulation F7 to thermostabilize different species-derived AdV vectored vaccines and therapeutics. Long term thermostability at 37C and immunogenicity of ChAd-triCoV/Mac Vaccine in mice While we have demonstrated the long-term thermostability of our formulated AdV vectors, it is equally important to confirm that they retain their functional activity in vivo , as this is critical for their practical applicability [ 13 ]. To this end, samples were dried and thermally aged at 37 o C. Samples were tested by ICW for viable titer, and over 44 weeks of incubation we observed less than 0.25 total Log IU lost (Fig. 7 ). In congruence with our previous data (Fig. 6 ), no significant difference was observed between the HuAd5-GFP control and the ChAd-TriCoV/Mac vectored vaccine for titer loss over the course of the experiment. We next sought to assess the vaccine formulation in vivo using previously published study designs [ 13 ]. To this end, animals were intranasally (i.n.) vaccinated either with a fresh liquid control (ChAd-TriCoV/Mac (Frozen control)), a freshly dried and reconstituted sample (ChAd-TriCoV/Mac (Dried/Fresh)), or a dried sample that was incubated at 37 o C for 310 days and reconstituted (ChAd-TriCoV/Mac (Dried/Aged)). Prior to vaccination, the dried formulations were reconstituted in PBS and mixed by pipet for 5–10 minutes. Quantification of the − 80 o C control and reconstituted material was performed by ICW, with the freshly dried ChAd-TriCoV/Mac experimental group receiving a slightly higher dose than the other two (Fig. 8 A). Animals were sacrificed 28 days post-vaccination and serum was collected for antibody responses and bronchoalveolar lavage (BAL) fluid was collected for airway antibody responses and T cell responses [ 21 ]. In naïve animals, the airways are largely devoid of T cells and are induced following local respiratory mucosal exposure. As such, we first assessed the induction of airway CD4 and CD8 T cell responses elicited by each formulation following i.n. vaccination with ChAd-TriCoV/Mac. In comparison to animals vaccinated with the liquid control, either dried formulation elicited comparable CD4 and CD8 T cell responses in the airway, suggesting that the dried formulations retain their broad immunogenicity (Fig. 8 B). As this vaccine encodes the S1 domain of the spike antigen of SARS-CoV-2, we next assessed antigen-specific T cell responses following ex vivo stimulation with overlapping S1 peptide pools. Similar to the induction of T cell responses, animals vaccinated with either dried formulation induced levels of antigen-specific CD4 and CD8 T responses that were comparable to control-vaccinated mice (Fig. 8 C/D). We next assessed both serum and airway binding IgG antibody responses by ELISA elicited by each formulation following i.n. vaccination. Brochoalveolar lavage fluid was concentrated prior to assessment to improve detection. Animals vaccinated with either dried formulation induced similar levels of anti-spike IgG responses in the serum (Fig. 8 E) and BAL (Fig. 8 F) that were comparable to control-vaccinated mice. These results indicate the dried vaccine formulation retained its ability to induce robust antigen-specific T cell and antibody responses even after long-term storage at 37 o C. Discussion The goal of this work was to build off our previous work demonstrating the combination of two carbohydrates, pullulan, and trehalose, to thermally stabilize an inherently unstable but therapeutically important viral vector, adenovirus (AdV). Examples in the literature have shown moderate stabilization of AdV[ 15 , 18 ], so our goal was to develop the highest classification of stability for AdV vectored therapeutics. Based off the WHO’s VVM stability temperatures and times, the most stable vaccine status is VVM250 which is attained at either > 250 days at 37 o C, 71 days at 45 o C, or 17 days at 55 o C [ 17 ]. Our initial comparison between the strategy developed to stabilize the VSV viral vector and the best published work at the time demonstrated a significant stabilization improvement at 37 o C [ 16 , 18 ]. With a stability of less than 0.25 log IU loss over 450 days at 37 o C, this would be easily classified as a VVM250 stable product (Fig. 1 ). However, most dried therapeutic products are backfilled with inert gas such as nitrogen. When the HuAd5 with F1 formulation was dried and backfilled with dried nitrogen gas, we observed ~ 0.5 Log IU worse stabilization compared to atmospheric gas backfilling (Fig. 2 ). Interestingly, we observed a similar result of negative impact of dried film crystallinity to viral vector stabilization for AdV as we previously reported with the VSV vector. This result was exasperated at more elevated temperatures (55 o C vs 37 o C) with over 3% dried film being crystalline (Fig. 3 ). By eliminating salt ions to stabilize the AdV, we observed a significant increase in thermostability without additional process loss. To improve our speed of development, we pushed the accelerated aging experiments to temperatures we have not observed successful results reported. While our initial attempts were markedly more stable than we could observe in the published literature (similar losses of 1.25 Log IU Loss from published at ~ 30 days to 355 days for our work at 45 o C; Fig. 2 ), these would not be satisfactory for a successful product. Over several development iterations, we developed both a drying schedule and formulation composition able to maintain thermostability for HuAd5 vector of less than 0.5 log loss IU over 49 days at 55 o C, a stability datapoint unprecedented in the literature (Fig. 5 ). While our development work was performed with a non-targeted HuAd5 vector, we next sought to demonstrate the same stability with a proven AdV viral vectored vaccine. Additionally, several key AdV therapeutic vectors are derived from chimpanzee origins, so demonstration of our formulation strategy with different AdV’s was critical to prove widespread applicability. We pursued our work with a trivalent chimpanzee adenovirus vectored COVID 19 vaccine (ChAd-TriCoV/Mac) which has demonstrated efficacy in mice and currently is being tested in a phase II human trial by inhaled aerosol [ 13 , 22 ]. Currently, this vaccine requires − 80 o C storage to ensure stability. When ChAd-TriCoV/Mac was formulated, dried, and aged at 55 o C, we observe equal or slightly greater stability after 42 days as compared to the HuAd5 vector, with a total loss of 0.29 ± 0.05 Log IU (Fig. 6 ). This result confirms the PT120-D formulation methodology can be used on AdV vectors derived from different mammalian hosts, as well as with proven vector vaccines. To ensure efficacy in a mouse model with a more applicable accelerated aging temperature, we formulated, dried, and aged ChAd-TriCoV/Mac at 37 o C for 44 weeks (Fig. 7 ). Prior to vaccination, we assayed the same sample which revealed only 0.23 ± 0.04 Log IU loss from the initial stock material. Three different test groups were designed, the − 80 o C stock vaccine, a freshly prepared formulated and dried group, and the 44-week aged at 37 o C group (Fig. 8 ). All three groups demonstrated similar antigen-specific T cell and antibody responses. Overall, a demonstration equivalent immunological responses between the − 80 o C stock ChAd-TriCoV/Mac and the 44-week aged at 37 o C group is an outstanding result. These data firmly put the PT120-D formulated ChAd-TriCoV/Mac into the VVM250 stability classification, a first for a COVID-19 targeted vaccine. We project, based off our data showing a plateau of titer loss after the first 1–2 weeks of incubation and Arrhenius accelerated decay rates, the formulated vaccine should have a shelf stability (25 o C) of between 7–10 years. Dried ChAd-TriCoV/Mac vials continue to be incubated at 37 o C, and subsequent follow-up studies will determine if this projection remains accurate. In summary, we demonstrated the drying and formulation strategy involving a pullulan and trehalose-based film to create a shelf stable format for AdV based therapeutics. To the best of our knowledge, no other research has achieved thermostability at 55 o C for 7 weeks and represents a significant advancement in opportunity for storage and delivery of AdV-vectored therapeutics. Demonstration of equivalent immunogenicity with stock material further supports these claims. Future work with clinical trials will elucidate the potential of PT120-D based formulation technology. Materials and Methods Materials Pullulan and trehalose SG dried powders were kindly provided by Nagase Viita Co., Ltd. Purified human adenovirus serotype 5 (HuAd5) expressing either green fluorescent protein (GFP), Luciferase, and chimpanzee adenovirus serotype 68-based COVID-19 vaccine (ChAd-TriCoV/Mac) were manufactured and provided by Dr. Brian Lichty’s research group (McMaster University)[ 13 ]. The stock buffers Tris (1 M pH 7.2; Teknova Cat No: T1072), 1 M CaCl 2 (Sigma Cat No: 21115-100 mL), 1 M MgSO 4 (Sigma; Cat No: M3409-100 mL), 5 M NaCl (VWR; E529-500 mL), and dry powders of bovine serum albumin (Sigma; A9418-10G) and gelatin (Criterion, C7921) were diluted and resuspended as described in Table 1 . Table 1 Summary of formulation components utilized in this study. All percentages are representative of w/v. Formulation Components Tris (pH 7.2) CaCl 2 MgSO 4 Gelatin BSA NaCl Pullulan Trehalose Other F1 10mM 10mM 10mM 0.05% 0.5% 50mM 2.5% 5% F2 10mM (pH 8.2) 1mM 100mM 5% Inulin, 5% Mannitol F3 10mM 5% F4 10mM 2.5% 5% F5 10mM 0.5% 2.5% 5% F6 10mM 0.5% 2.5% 5% 1% PMAL F7 10mM 0.5% 2.5% 15% AdV Dialysis and Formulation Manufactured 500 µL AdV stock aliquots were stored at -80 o C in 10 mM Tris-Cl, pH 8.0 with 10% glycerol. After gentle thawing on ice, viral particles were buffer exchanged into the formulation buffer using Zeba Spin Desalting columns 7K MWCO (Thermo Scientific). Viral particles were mixed at a 1:9 ratio with the formulation buffer containing Pullulan and/or Trehalose and 100 µL was aliquoted into 2mL vials. Samples were subsequently dried either using a vacuum pump and desiccator or the SP VirTis AdVantage Pro Freeze dryer system. If required, after completion of the dry schedule on the SP VirTis AdVantage Pro, vials were backfilled with dried nitrogen gas and stoppered in system. In-Cell Western (ICW) for Adenovirus infectious unit quantification Human embryonic kidney 293 with deletions in early gene regions 1 and 3 (HEK 293 ΔE1/E3; provided by Dr. Lichty’s Research group) are grown to 70–90% confluency and harvested using 3 mL of 1× citric saline incubated for 4–8 minutes at 37 o C. Resuspended cells are counted using an automated cell counter (Corning) and diluted in completed MEM Earles media for plating at 1.8–2.5 x 10 5 cells/well (3.6–5.0 x 10 5 cells/mL) in tissue-culture treated 24-well plates (Corning Costarplates). Plated cells are incubated overnight at 37 o C/5% CO 2 . The next day, dried formulated AdV samples are reconstituted in 1 mL of warmed Milli-Q water and allowed to fully dissolve for 8–10 minutes. Samples are vortexed at moderate speed for 15s prior to serial dilution in completed media. 250 µL of diluted AdV are gently pipetted onto confluent HEK 293 cells plated in 24-well plates from the day prior and incubated at 37C/5% CO 2 for the 36–48 hour infection course. ICW Staining Procedure The inoculum is swiftly discarded after the 2-day infection course. Infected plates are fixed on dry ice by carefully pipetting 300 µL of chilled methanol and incubated for 14 minutes at -20 o C. The fixative is swiftly discarded and fixed plates undergo 3 rounds of washing in 500 µL of blocking buffer (1% BSA in PBS), with the final wash kept for blocking 30 minutes at RT. Blocking is swiftly discarded prior to a 1-hour incubation in 200 µL of diluted primary antibody (1:3000 Adenovirus Antibody 8C4 in 1% BSA/PBS) at RT. The primary incubation is swiftly discarded followed by 3 washes in blocking buffer prior to incubating in 200 µL of diluted secondary antibody (1:400 IRDye680RD Goat anti-Mouse IgG Secondary Antibody in 1% BSA/PBS) at RT for 1 hour. After discarding the secondary, plates are washed thrice in 500 µL of PBS prior to scanning in the final wash with fluorescent bio-imager (LICOR Odyssey DLx). Outputted signal is measured as raw counts and quantified as IU/mL. XRD Analysis of Dried Formulations for Crystallinity Dried formulations were prepared with a 25°C dry cycle in 2 mL vials. For XRD analysis, the film was crushed into a powder and extracted from the vials. The powder samples were mounted on top of a zero-background Si wafer for the XRD measurements. The data were collected using a Bruker D8 DISCOVER with DAVINCI. A DESIGN diffractometer equipped with a Co sealed-tube source and an Eiger2R 500K area detector was used. A 2D continuous scan from 12–80° 2Th was collected with 0.02 degree steps at 1.2 seconds per step. Mice Age-matched 6–8-week-old wild-type female were purchased from either Charles River Laboratories (Saint Constant, QC, Canada). Animals were housed in a specific pathogen-free level B Facility at McMaster University, Hamilton, ON, Canada. All experimental protocols were approved, and experiments were performed in accordance with institutional guidelines from the Animal Research and Ethics Board. All methods are reported in accordance with ARRIVE guidelines. Vaccination Animals were anesthetized with isoflurane and vaccinated intranasally with 1.1-2.9x10 6 IU of a recombinant a recombinant chimpanzee adenovirus serotype 68 SARS-CoV-2 vaccine (ChAd-TriCoV/Mac). Intranasal vaccinations were performed with a final volume of 40µL diluted in PBS. Where noted, dried formulations were resuspended in PBS prior to vaccination. Bronchoalveolar lavage mononuclear cell isolation Mice were euthanized by exsanguination as previously established [ 13 , 24 ]. Briefly, mice are placed into an anesthetic chamber, and isoflurane is administered as 3–5% in O 2 prior to cervical dislocation as per institutional guidelines. Cells from bronchoalveolar lavage (BAL) as previously described [ 23 ]. Briefly, BAL was performed by instillation with 250 µL, followed by 200 µL of PBS. This fraction was utilized for downstream antibody analysis. Further instillation of 3x 300 µL of PBS was performed for BAL cell retrieval. Isolated cells were resuspended in complete RPMI 1640 (10% FBS, 1% L-glutamine, 100 U/mL penicillin/streptomycin, 1% HEPES pH 7.3, 1% MEM non-essential amino acids (Gibco, Gaithersburg, MD, United States), 1% sodium-pyruvate (Gibco, Gaithersburg, MD, United States). Cell numbers were quantified in Turk’s Blood Dilution Fluid (RICCA Chemical, Arlington, TX, United States) and counted under a microscope. Peptide library construction and stimulation Peptide libraries consisting of 10 amino acid, 15mer synthetic overlapping peptides for vaccine encoded antigen S1 were synthesized by Pepscan (Lelystad, The Netherlands). Peptides were reconstituted in DMSO according to manufacturer’s instructions to a final concentration of 40 µg/µL. Antigen peptide pools were generated with each pool containing 0.2 µg/µL of each peptide. Peptide stimulations were carried utilizing 2 µg of each peptide/mL of culture media. Flow cytometry Cell immunostaining and flow cytometry were performed as previously described [ 23 – 25 ] Briefly, isolated mononuclear cells were plated in U-bottom, 96-well plates at a maximum concentration of 2x10 7 cells/mL. Following staining with The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific Waltham, MA, United States) at room temperature for 30 min, cells were washed and blocked with anti-CD16/CD32 (clone 2.4G2) in 0.5% BSA-PBS for 15 min on ice and then stained with fluorochrome-labeled mAbs for 30 min on ice. Fluorochrome-labeled mAbs used for staining cells were anti-CD3-V450 (clone 17A2), anti-CD4 APC-Cy7 (clone GK1.5), anti-CD8 PE-Cy7 (clone 53 − 6.7), and anti-IFNγ APC (clone XMG1.2). Stained cells were fixed and permeabilized with BD Cytofix/Cytoperm before incubation in BD Perm/Wash buffer (BD Biosciences, San Jose, CA, United States). All mAbs and reagents were purchased from BD Biosciences. Stained cells were processed according to BD Biosciences instructions for flow cytometry and run on a BD LSR II flow cytometer. Data were analyzed using FlowJo software (version 10.1; Tree Star, Ashland, OR, United States). Recombinant antigen production Plasmids encoding mammalian cell codon optimized sequences for full-length spike of SARS-CoV-2 was generously gifted from the lab of Dr. Florian Krammer [ 26 ] (Icahn School of Medicine, NY, United States). Proteins were produced in Expi293F cells (ThermoFisher Scientific Waltham, MA, United States) according to the manufacturers’ instructions and purified as previously described [ 27 ]. Briefly, when culture viability reached 40%, supernatants were collected and spun at 500 x g for 5 minutes. The supernatant was then incubated by shaking overnight at 4°C with 1 mL of Ni-NTA agarose (Qiagen, Germantown MD, United States) per 25 mL of transfected cell supernatant. The following day 10 mL polypropylene gravity flow columns (Qiagen, Germantown, MD, United States) were used to elute the protein. Recombinant RBD was concentrated in a 10 kDa Amicon centrifugal units (Millipore Sigma, Etobicoke, ON, Canada), and recombinant Spike was concentrated in a 50kDa Amicon centrifugal unit (Millipore Sigma, Etobicoke, ON, Canada) prior to being resuspended in phosphate buffered saline (PBS). Enzyme Linked Immunosorbent Assay (ELISAs) for antibody measurement 96-well NUNC- MaxiSorp™ plates (Thermo Scientific, Waltham, MA, United States) were coated overnight at 4°C with SARS-CoV-2 full-length spike, diluted to 2 µg/mL in bicarbonate-carbonate coating buffer (pH 9.4). Plates were blocked by shaking for 1 hour at 37°C with reagent diluent (0.5% bovine serum albumin (BSA), 0.02% sodium azide, in 1X Tris-Tween buffer). Samples were serially diluted from 1:5 (serum), or 1:8 (BAL) starting dilution. BAL samples were first concentrated through Pierce™ Protein Concentrators with a 50 kDa molecular weight cut-off (MWCO) (ThermoFisher Scientific Waltham, MA, United States) according to the manufacturer’s instructions, with volumes normalized prior to concentration. Samples were arranged such that one row contained only antigen and secondary antibodies and served as the plate blank. Following a 1 hour incubation with shaking at 37°C, plates were washed three times with 1X Tris-Tween wash buffer. After washing, goat anti-mouse-biotin antibodies (Southern Biotech, Birmingham, AL, United States), IgG (1:5000) were diluted in reagent diluent and added to all wells. Plates were again incubated for 1 hour, with shaking, at 37°C, followed by three washes with 1X Tris-Tween buffer. A streptavidin-alkaline phosphatase secondary antibody (1:2000, Southern Biotech, Birmingham, AL, United States) was added to all wells for 1 hour with shaking at 37°C. Plates were subsequently washed three times prior to addition of pNPP one component microwell substrate solution (Southern Biotech, Birmingham, AL, United States) to each well. Plates were developed for 10 minutes and the reaction was quenched with an equal volume 3N sodium hydroxide. The optical density (O.D.) at 405 nm was read on a SpectramaxI3 (Molecular Devices, San Jose, CA, United States). Endpoint titers were defined by the lowest dilution at which the O.D. was three standard deviations above the mean of the blank wells. Statistical Analysis and Data Availability Statistical analysis and graphing of data were performed with PRISM software (Graphpad Prism v10.1.2). Comparison of two groups was performed with two-tailed paired t-tests. For analysis of more than two unique groups, ANOVA was utilized with Tukey multiple analyses parameter. All data generated or analysed during this study are included in this published article [and its supplementary information files]. Declarations Acknowledgments We would like to acknowledge Vicky Jarvis for her assistance with the XRD experiments and analysis and Maria Fe C Medina for manufacturing the Adenovirus stocks. For their contribution with organizational management, direction, and manuscript editing, we would like to thank Dr. Robert DeWitte and Brent Lefebvre. For their efforts on experimental assistance, we would like to thank Abdulhamid Mohamud, Arthur Liou, Jason Choi, Divhleen Ruprai, and Ria Baijal. Finally, we thank Dr. Lichty’s research group for discussions and brainstorming. Elarex would like to acknowledge the National Research Council of Canada Industrial Research Assistance Program (NRC IRAP) for providing advisory services and research and development funding for our stabilization work with AdV. Funding: National Research Council Canada IRAP Contribution # 1009921 Ontario Centre for Innovation #36006 Author Contributions: Conceptualization: JAI, AA, SA, JEB, CDMF, BDL Methodology: JAI, AA, SA, JEB, BDL, MSM Investigation: JAI, AA, NK, SA, MD Visualization: JAI, SA Supervision: JEB, BDL, MSM Writing—original draft: JAI, SA Writing—review & editing: JAI, AA, JEB, MRD, SA, CDMF Competing Interest Statement: All other authors declare they have no competing interests. References Orenstein, W.A. and R. Ahmed, Simply put: Vaccination saves lives. Proc Natl Acad Sci U S A, 2017. 114 (16): p. 4031-4033. Shattock, A.J., et al., Contribution of vaccination to improved survival and health: modelling 50 years of the Expanded Programme on Immunization. Lancet, 2024. 403 (10441): p. 2307-2316. Pelliccia, M., et al., Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months. Nat Commun, 2016. 7 : p. 13520. Jarrett, S., et al., The importance of vaccine stockpiling to respond to epidemics and remediate global supply shortages affecting immunization: strategic challenges and risks identified by manufacturers. Vaccine X, 2021. 9 : p. 100119. Ghaemmaghamian, Z., et al., Stabilizing vaccines via drying: Quality by design considerations. Adv Drug Deliv Rev, 2022. 187 : p. 114313. Lai, M.C. and E.M. Topp, Solid-state chemical stability of proteins and peptides. J Pharm Sci, 1999. 88 (5): p. 489-500. Mensink, M.A., et al., How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm, 2017. 114 : p. 288-295. Qi, Y. and C.B. Fox, Development of thermostable vaccine adjuvants. Expert Rev Vaccines, 2021. 20 (5): p. 497-517. Emami, F., et al., Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals. Pharmaceutics, 2018. 10 (3). Jangle, R.D. and S.S. Pisal, Vacuum foam drying: an alternative to lyophilization for biomolecule preservation. Indian J Pharm Sci, 2012. 74 (2): p. 91-100. Walters, R.H., et al., Next generation drying technologies for pharmaceutical applications. J Pharm Sci, 2014. 103 (9): p. 2673-2695. Salauddin, M., et al., Clinical Application of Adenovirus (AdV): A Comprehensive Review. Viruses, 2024. 16 (7). Afkhami, S., et al., Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2. Cell, 2022. 185 (5): p. 896-915 e19. Hackett, N.R. and R.G. Crystal, Four Decades of Adenovirus Gene Transfer Vectors: History and Current Use. Mol Ther, 2025. Mathot, F., et al., A lyophilised formulation of chimpanzee adenovirus vector for long-term stability outside the deep-freeze cold chain. Commun Med (Lond), 2025. 5 (1): p. 23. Iwashkiw, J.A., et al., Improved thermal stabilization of VSV-vector with enhanced vacuum drying in pullulan and trehalose-based films. Sci Rep, 2024. 14 (1): p. 18522. PQS performance specification WHO, Editor. 2018. Berg, A., et al., Stability of Chimpanzee Adenovirus Vectored Vaccines (ChAdOx1 and ChAdOx2) in Liquid and Lyophilised Formulations. Vaccines (Basel), 2021. 9 (11). Bajrovic, I., et al., Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature. Sci Adv, 2020. 6 (10): p. eaau4819. Farina, S.F., et al., Replication-defective vector based on a chimpanzee adenovirus. J Virol, 2001. 75 (23): p. 11603-13. D'Agostino, M.R., et al., Protocol for isolation and characterization of lung tissue resident memory T cells and airway trained innate immunity after intranasal vaccination in mice. STAR Protoc, 2022. 3 (3): p. 101652. A Trial of a Next Generation COVID-19 Vaccine Delivered by Inhaled Aerosol (AeroVax) . 2024; Available from: https://clinicaltrials.gov/study/NCT06381739?term=aerovax&rank=1. D'Agostino, M.R., et al., Airway Macrophages Mediate Mucosal Vaccine-Induced Trained Innate Immunity against Mycobacterium tuberculosis in Early Stages of Infection. J Immunol, 2020. 205 (10): p. 2750-2762. Jeyanathan, M., et al., Novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent TB protection. Mucosal Immunol, 2015. 8 (6): p. 1373-87. Yao, Y., et al., Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity. Cell, 2018. 175 (6): p. 1634-1650 e17. Amanat, F., et al., A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med, 2020. 26 (7): p. 1033-1036. Stadlbauer, D., et al., Repeated cross-sectional sero-monitoring of SARS-CoV-2 in New York City. Nature, 2021. 590 (7844): p. 146-150. Additional Declarations No competing interests reported. Supplementary Files 20250915NPJAdVStabilizationSI.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 Mar, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviewers agreed at journal 18 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 25 Nov, 2025 Reviewers agreed at journal 25 Nov, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers invited by journal 24 Nov, 2025 Editor assigned by journal 01 Nov, 2025 Editor invited by journal 29 Oct, 2025 Submission checks completed at journal 28 Oct, 2025 First submitted to journal 28 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7767949","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":537243061,"identity":"636513bf-e3f8-4b33-9d3f-6b0c699bc127","order_by":0,"name":"Jeremy A. 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A) Dried film morphology for both formulations F1 and F2 either vacuum dried or freeze dried. B) Process and thermostability loss of titer incubated at 37\u003csup\u003eo\u003c/sup\u003eC over 450 days as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/dd6f698353d8c35bdb145fab.png"},{"id":94837842,"identity":"ee4e23c0-fc81-4e89-ac7d-736d8cf6f3c6","added_by":"auto","created_at":"2025-10-31 08:51:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":316211,"visible":true,"origin":"","legend":"\u003cp\u003eStability time course at 3 different incubation temperatures of HuAd5-GFP dried on a 4/25°C dry schedule and backfilled with dry N2 gas. A) Dried film morphology for formulation F1 vacuum dried and backfilled with dried nitrogen gas. B) Process and thermostability loss of titer incubated at 37,45, and 55\u003csup\u003eo\u003c/sup\u003eC over up to 355 days as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Using a one-stage decay model (Graphpad PRISM), a calculated plateau was determined for each of the three thermal treatments.\u0026nbsp; Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/468605120cbd05f97a15c70c.png"},{"id":94985006,"identity":"efe11b49-18a7-4ae4-aeab-7d6ac9d04089","added_by":"auto","created_at":"2025-11-03 06:57:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157225,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;XRD crystallinity time course of PT120-D dried films incubated at 55\u003csup\u003eo\u003c/sup\u003eC for 28 days. Samples were dried in 2 mL vials using the 4/25\u003csup\u003eo\u003c/sup\u003eC 24 hour dry schedule and backfilled with dried N2 gas.\u0026nbsp; The samples were crimped and incubated at 55\u003csup\u003eo\u003c/sup\u003eC.\u0026nbsp; Samples were extracted from vials and analyzed by XRD. Formulation F1 sample that had significant crystallinity present, whereas F6 did not have any crystallinity observed.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/b4250d9e77d8ec3fe3ae2b89.png"},{"id":94837845,"identity":"5032a02a-d2b2-4918-93b1-e2367af2b087","added_by":"auto","created_at":"2025-10-31 08:51:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":350033,"visible":true,"origin":"","legend":"\u003cp\u003eThermostability at 55\u003csup\u003eo\u003c/sup\u003eC with 25\u003csup\u003eo\u003c/sup\u003eC drying temperature and the role of BSA.\u0026nbsp; A) Dried film morphology for formulation F1, F4, and F5 vacuum dried and backfilled with dried nitrogen gas. B) Process and thermostability loss of titer incubated at 55\u003csup\u003eo\u003c/sup\u003eC over 21 days as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/4f9f1dbf0995d7a4e88ccce5.png"},{"id":94837848,"identity":"1914c2fb-9f19-46eb-8888-3f15c90a7d10","added_by":"auto","created_at":"2025-10-31 08:51:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":476098,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of Pullulan and Trehalose concentrations to improve 2 different HuAd5 vectors at 55\u003csup\u003eo\u003c/sup\u003eC A) Dried film morphology for formulation F1, F6, and F7 vacuum dried and backfilled with dried nitrogen gas. B) Process and thermostability loss of titer incubated at 55\u003csup\u003eo\u003c/sup\u003eC over 49 days as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/db9aad6fca5df10485d92289.png"},{"id":94837863,"identity":"645b992f-7125-4898-88c4-c3400c6477ca","added_by":"auto","created_at":"2025-10-31 08:51:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":225503,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the thermostability of HuAd5-GFP and ChAd-TriCoV/Mac thermostability at 55\u003csup\u003eo\u003c/sup\u003eC. A) Dried film morphology for HuAd5-GFP and ChAd-TriCoV/Mac with formulation F7 vacuum dried and backfilled with dried nitrogen gas. B) Process and thermostability loss of titer incubated at 55\u003csup\u003eo\u003c/sup\u003eC over 42 days as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/b4ba0c90f2f5ca4a73cafc20.png"},{"id":94985173,"identity":"37151824-c3bc-420b-900f-54e14d1a6df6","added_by":"auto","created_at":"2025-11-03 06:57:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":344636,"visible":true,"origin":"","legend":"\u003cp\u003eThermostability of ChAd-TriCoV/Mac challenged at 37\u003csup\u003eo\u003c/sup\u003eC for VVM250 stability. A) Dried film morphology for HuAd5-GFP and ChAd-TriCoV/Mac with formulation F7 vacuum dried and backfilled with dried nitrogen gas. B) Process and thermostability loss of titer incubated at 37\u003csup\u003eo\u003c/sup\u003eC over 44 weeks as determined by ICW. C) Calculated means and standard deviation (SD) for each data point of accumulated Log PFU loss for each condition. Each data point is collected from duplicate serial dilution plating of biological duplicate vials.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/bd5b2dc850f8574d52982701.png"},{"id":94837855,"identity":"ab0bf2a9-a20a-43db-b37f-d29aefd689c5","added_by":"auto","created_at":"2025-10-31 08:51:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":381312,"visible":true,"origin":"","legend":"\u003cp\u003eFormulated ChAd-TriCoV/Mac aged for 310 days at 37\u003csup\u003eo\u003c/sup\u003eC retains its immunogenicity \u003cem\u003ein vivo\u003c/em\u003e. A) Dose of ChAd-TriCoV/Mac given per mouse as calculated by ICW. B) Total T cell responses in the airways four weeks post-intranasal (i.n.) vaccination with either formulated vaccines or frozen control. C) Absolute number of S1-specific CD8 and CD4 T cells in the BAL following \u003cem\u003eex vivo\u003c/em\u003e stimulation with S1 peptide pools. D) Frequency of IFNγ+ T cells following \u003cem\u003eex vivo \u003c/em\u003estimulation with S1 peptide pools. E) Serum anti-spike IgG endpoint titers. F) BAL fluid anti-spike IgG endpoint titers. Data presented in (B, C, E, F) represent mean ± SEM. Statistical analysis were one-way ANOVA with Tukey’s multiple comparisons test. Data is representative of 1 experiment, n=5 animals per group. ns = not significant.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/356e836d83b4ae7b4b37b5bb.png"},{"id":95000480,"identity":"c1cccde8-8767-43c2-948f-6cbba77bc370","added_by":"auto","created_at":"2025-11-03 08:58:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3743422,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/124ff40b-219a-41fa-ab99-bd67f8390efe.pdf"},{"id":94985039,"identity":"6c1f8bee-854b-43f6-a7bd-31a36a009e34","added_by":"auto","created_at":"2025-11-03 06:57:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":280149,"visible":true,"origin":"","legend":"","description":"","filename":"20250915NPJAdVStabilizationSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7767949/v1/cc14d9fbd7d8161750bde2d9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Shelf Stable Formulation for Adenovirus Vectored Vaccines and Therapeutics","fulltext":[{"header":"Significance Statement","content":"\u003cp\u003eIn this work, we developed a vacuum drying based protocol to optimize the stability of AdV and demonstrate thermal stability at 37\u003csup\u003eo\u003c/sup\u003eC for 450 days, and 55\u003csup\u003eo\u003c/sup\u003eC for up to 49 days. These results are the best described thermostability of AdV to date, approximately 2.5x longer at 55\u003csup\u003eo\u003c/sup\u003eC than required for the highest VVM 250 category. \u0026nbsp; Finally, we demonstrate equal efficacy of a previously described AdV vectored COVID 19 vaccine in a murine model comparing fresh \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -80\u003csup\u003eo\u003c/sup\u003eC stocks, dried vaccine, and dried vaccine aged for 310 days at 37\u003csup\u003eo\u003c/sup\u003eC.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eProactive measures such as vaccination, as demonstrated by programs targeting diseases such as polio, smallpox, and measles, has established the value of prevention of diseases, reducing the global health care burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recently it has been demonstrated global vaccination efforts have saved over 154\u0026nbsp;million lives since 1974, with 101\u0026nbsp;million being infants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most vaccines produced require stringent cold chain controls to prevent degradation and subsequent loss of efficacy. Vaccines including lipid nanoparticle encapsulated mRNA vaccines, and viral vectored vaccines require a -70\u003csup\u003eo\u003c/sup\u003eC cold chain pathway, which is technically, logistically, and economically challenging to disseminate critical vaccines to hard-to-reach places and populations. Up to 80% of vaccination programs funds are directed to funding these cold chain requirements [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition is the requirement for public health organizations to create both static and rotating stockpiles of vaccines for different types of outbreaks, which is also heavily dependent on the stability of vaccine products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo improve vaccine stability, many groups supplement the product with stabilizing excipients and remove water, resulting in decreased mobility of molecules and inhibiting liquid degradation mechanisms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Solid biopharmaceuticals have greater thermostability and are less sensitive to degradation by both chemical and physical mechanisms [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Two main theories have been postulated to explain this phenomenon: vitrification and water replacement theory [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The most common drying process employed is lyophilization, where liquid samples are frozen and liquid is removed by sublimation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several biopharmaceuticals are sensitive to ice crystals and cold denaturation causing substantial loss of functionality [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While not commercially employed at scale, vacuum drying techniques have been developed for drying biopharmaceuticals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Vacuum drying has several advantages over lyophilization including being less energy intensive, less physical stress on the therapeutic during dehydration, and can impart greater thermostability to biologically active organisms such as probiotic bacteria. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eAdenoviruses (AdV) are non-enveloped DNA viruses which can infect a wide range of hosts and are often associated with mild-to-moderate respiratory symptoms. However, AdV have been harnessed as a viral vector platform for gene therapy, and as vaccines for cancer and other infectious diseases such as SARS-CoV-2 [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. One drawback of AdV vectored therapeutics is a cold chain requirement for storage and shipment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although studies thermostabilizing AdV vectored vaccine have demonstrated 0.17 log IU loss after 2 years at 5\u003csup\u003eo\u003c/sup\u003eC with a predicted stability for 5 years at 5\u003csup\u003eo\u003c/sup\u003eC, the requirement for cold chain treatment of AdV vaccines is still a limitation for the technology, and delivery of treatments with limitations in cold chain infrastructure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this work we describe the development of a stable shelf formulation (PT120-D) utilizing vacuum drying techniques for AdV vectors. Building off our previous work with vesicular stomatitis virus (VSV) vector stabilization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we optimized a drying schedule and excipient formulation for enhanced vacuum drying of human adenovirus serotype 5 vectors, and an inhaled chimpanzee adenovirus serotype 68-based COVID-19 vaccine (ChAd-TriCoV/Mac) that is in phase II clinical trials in Canada. The final formulation demonstrated a total titer loss of less than 0.25 log IU over 44 weeks at 37\u003csup\u003eo\u003c/sup\u003eC, as well as less than 0.3 log loss after up to 7 weeks at 55\u003csup\u003eo\u003c/sup\u003eC which is \u0026gt;\u0026thinsp;2.5\u0026times; longer than the VVM250 requirements [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To our knowledge, no reports have thus far shown comparable thermostability and durability of stabilization. Importantly, we further demonstrated that the formulated and dried vaccine retained its \u003cem\u003ein vivo\u003c/em\u003e immunogenicity after 44 weeks of incubation at 37\u0026deg;C\u0026mdash;comparable to that of the liquid control\u0026mdash;highlighting the potential of this approach to enable long-term storage without cold chain. Together, these data demonstrate the stabilization effectiveness of the PT120-D formulation, resulting in a shelf stable delivery system for AdV based therapeutics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePT120-D formulation of HuAd5-GFP has significantly better thermostability when vacuum dried compared to freeze dried without nitrogen gas backfilling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe utilized a human adenovirus serotype 5 vector expressing GFP (HuAd5-GFP) for initial comparison of PT120-D stabilization to the lead formulation identified in the literature which utilized a formulation consisting of inulin and mannitol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We performed two different dry schedules (Schedule 1 and 2; Supplemental Table\u0026nbsp;1) with the two formulations and assayed thermostability at 37\u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For Formulation 1, different film morphologies were observed, whereas formulation 2 formed a cake regardless of drying schedule (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Formulation 1 vacuum dried had a glassy film compared to the freeze dried being a lyophilized cake. Formulation 1 vacuum dried was the best combination for lowest process and thermostability loss, whereas the same formulation had the greatest loss vacuum dried (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB/C). No significant difference in process loss or thermostability was observed for Formulation 2 between drying schedules. Over a 450-day incubation at 37\u003csup\u003eo\u003c/sup\u003eC, no additional loss was observed for the Formulation 1 vacuum dried (total Log IU loss of 0.2), compared to accumulated losses for the other 3 conditions (total Log IU loss\u0026thinsp;\u0026gt;\u0026thinsp;4). Additionally, both pullulan and trehalose are required for stabilizing adenovirus (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To the best of our knowledge, this reflects the highest level of stability reported for an adenoviral vector to date.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFormulation F1 demonstrates thermostability stability at 45/55\u003c/b\u003e\u003csup\u003e\u003cb\u003eo\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC, but weaker stability at 37\u003c/b\u003e\u003csup\u003e\u003cb\u003eo\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC observed with nitrogen backfilling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWith the robust stability observed at 37\u003csup\u003eo\u003c/sup\u003eC, we next sought to determine if a more accelerated thermal challenge was viable for Formulation 1. According to the WHO VVM stability criteria, 55\u003csup\u003eo\u003c/sup\u003eC is the hottest thermal challenge utilized for calculated accelerated thermostability of a vaccine product [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For the highest thermostability categorization of VVM250, it would be the equivalent of 250 days at 37\u003csup\u003eo\u003c/sup\u003eC, 73 days at 45\u003csup\u003eo\u003c/sup\u003eC, or 17 days at 55\u003csup\u003eo\u003c/sup\u003eC. The additional step of backfilling the headspace of the vial was employed as it is commonplace to backfill samples with an inert gas such as nitrogen to improve stability by reducing chemical instabilities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUtilizing HuAd5 with Formulation 1, dry schedule 1, and nitrogen gas backfilling, we observed a similar film morphology as in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A similar process loss of ~\u0026thinsp;0.2 Log IU was consistent with the previous data. However, at 37\u003csup\u003eo\u003c/sup\u003eC we observed a greater Log IU loss, and the plateau observed over 375 days was 0.73 log IU loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB/C). At more strenuous thermal challenges, we observed a greater IU loss with a calculated plateau at 1.12 Log IU loss for 45\u003csup\u003eo\u003c/sup\u003eC and a loss of 1.65 log IU after 21 days at 55\u003csup\u003eo\u003c/sup\u003eC. While not outstanding results, utilizing elevated thermal challenges provided a more rapid turnaround time for iterative testing to improve formulation performance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eXRD analyses of dried PT120-D formulations demonstrates film crystallinity with F1, and removal of salt ions eliminates crystallinity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor commercial dried products, it is commonplace to backfill samples with an inert gas like nitrogen to improve stability by reducing chemical instabilities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Based off the results from our recent work [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we hypothesized that crystallinity of the dried PT120-D film was causing the additional titer loss when vials were backfilled with dried nitrogen gas. Comparing the HuAd5-GFP thermostability results of Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we had significantly greater loss of AdV titer with nitrogen backfilled vials compared to atmospheric conditions. To understand if the nitrogen backfilling was affecting the structure of the dried film, XRD analysis was performed on a thermal treated time course at 55\u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No crystallinity was detected at D0 for all conditions assayed, and no crystallinity was detected over the 28-day incubation period for either the atmospheric filled Formulation F1 or nitrogen backfilled F6. Conversely, Formulation 1 had a low percentage crystallinity detected on Day 1 and peaked at 3.25% on day 7. A slow decline in crystallinity was observed until Day 28 when the experiment was completed. A correlation between observed film crystallinity and AdV IU titer loss was shown to be dependent on the combination of salts and nitrogen gas backfilling of vial headspace.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuAd5-GFP stability requires BSA, and removal of salt ions improves thermostability at 55\u003csup\u003eo\u003c/sup\u003eC\u003c/h2\u003e\u003cp\u003eTo confirm removal of crystallinity improves thermostability, we formulated and dried HuAd5-GFP with formulation F1, F4, and F5 with dry schedule 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Formulation F4 was tested to determine whether BSA was important for HuAd5-GFP stability. No significant difference was observed for process loss between the three conditions, with F4 having slightly greater Log IU loss. Samples were incubated at 55\u003csup\u003eo\u003c/sup\u003eC, and no significant difference in total IU loss was observed on Day 7 with all 3 having between 0.9-1.0 log IU loss. By day 21, Formulation F4 lacking BSA had significantly great loss, (1.7 Log IU) compared to 1.23 for Formulation F1 and 1.03 for Formulation F4. Overall, BSA was critical for thermostability in PT120-D films, and removal of salt ions modestly improved HuAd5-GFP thermostability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAddition of PMAL significantly increased dialysis loss, but increased trehalose significantly increased thermostability at 55C\u003c/h3\u003e\n\u003cp\u003eTo further improve the performance of PT120-D at 55\u003csup\u003eo\u003c/sup\u003eC, we decided to either increase the amount of trehalose (Formulation F7) or add 1% PMAL (Formulation F6), and surfactant that had shown promise in the literature at stabilizing biologics in thin films [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Unexpectedly, a significant loss of titer (1.3\u0026ndash;1.4 Log IU loss) for two differently vectored HuAd5 (GFP and Luciferase transgenes) during buffer exchange with Zeba spin columns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Process loss observed for all three formulations was between 0.05\u0026ndash;0.25 Log IU, with formulation F7 having the least observed loss. After 49 days at 55\u003csup\u003eo\u003c/sup\u003eC, the formulation with PMAL performed the worst with a total loss of ~\u0026thinsp;2 log IU, with the majority of the loss derived from the dialysis step. Formulation F5 had a total loss of 1.1 log IU for both vectors, but the best performing formulation was F7 with a loss of 0.5 log IU. This thermostability result is by far the most stable we have observed compared to the literature to date.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eThermostability of a chimpanzee adenovirus-vectored vaccine at 55C comparable to HuAd5\u003c/h3\u003e\n\u003cp\u003eWe have thus far developed a novel formulation technology using a representative human AdV. However, there is growing evidence highlighting the utility of non-human AdV, such as those of simian origin, as improved vectors for vaccine development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given sequence differences between AdV isolated from different species which can alter capsid sequences, it is important to evaluate such formulations using AdV from different species. To this end, we chose to utilize a chimpanzee AdV serotype 68-based COVID-19 (ChAd-TriCoV/Mac) inhaled aerosol vaccine that is under evaluation in a phase II trial in Canada [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo assay if Formulation F7 provided comparable stabilization between human and chimpanzee AdV, we formulated, dried, and thermally aged the samples at 55\u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Both sets of samples had minimal process loss observed and impressive thermostability. After 42-day incubation at 55\u003csup\u003eo\u003c/sup\u003eC, both AdV\u0026rsquo;s had less than 0.5 log IU loss. These data support the widespread applicability of the PT120-D Formulation F7 to thermostabilize different species-derived AdV vectored vaccines and therapeutics.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eLong term thermostability at 37C and immunogenicity of ChAd-triCoV/Mac Vaccine in mice\u003c/h3\u003e\n\u003cp\u003eWhile we have demonstrated the long-term thermostability of our formulated AdV vectors, it is equally important to confirm that they retain their functional activity \u003cem\u003ein vivo\u003c/em\u003e, as this is critical for their practical applicability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To this end, samples were dried and thermally aged at 37\u003csup\u003eo\u003c/sup\u003eC. Samples were tested by ICW for viable titer, and over 44 weeks of incubation we observed less than 0.25 total Log IU lost (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In congruence with our previous data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), no significant difference was observed between the HuAd5-GFP control and the ChAd-TriCoV/Mac vectored vaccine for titer loss over the course of the experiment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next sought to assess the vaccine formulation \u003cem\u003ein vivo\u003c/em\u003e using previously published study designs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To this end, animals were intranasally (i.n.) vaccinated either with a fresh liquid control (ChAd-TriCoV/Mac (Frozen control)), a freshly dried and reconstituted sample (ChAd-TriCoV/Mac (Dried/Fresh)), or a dried sample that was incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 310 days and reconstituted (ChAd-TriCoV/Mac (Dried/Aged)). Prior to vaccination, the dried formulations were reconstituted in PBS and mixed by pipet for 5\u0026ndash;10 minutes. Quantification of the \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003eC control and reconstituted material was performed by ICW, with the freshly dried ChAd-TriCoV/Mac experimental group receiving a slightly higher dose than the other two (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Animals were sacrificed 28 days post-vaccination and serum was collected for antibody responses and bronchoalveolar lavage (BAL) fluid was collected for airway antibody responses and T cell responses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn na\u0026iuml;ve animals, the airways are largely devoid of T cells and are induced following local respiratory mucosal exposure. As such, we first assessed the induction of airway CD4 and CD8 T cell responses elicited by each formulation following i.n. vaccination with ChAd-TriCoV/Mac. In comparison to animals vaccinated with the liquid control, either dried formulation elicited comparable CD4 and CD8 T cell responses in the airway, suggesting that the dried formulations retain their broad immunogenicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). As this vaccine encodes the S1 domain of the spike antigen of SARS-CoV-2, we next assessed antigen-specific T cell responses following \u003cem\u003eex vivo\u003c/em\u003e stimulation with overlapping S1 peptide pools. Similar to the induction of T cell responses, animals vaccinated with either dried formulation induced levels of antigen-specific CD4 and CD8 T responses that were comparable to control-vaccinated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC/D).\u003c/p\u003e\u003cp\u003eWe next assessed both serum and airway binding IgG antibody responses by ELISA elicited by each formulation following i.n. vaccination. Brochoalveolar lavage fluid was concentrated prior to assessment to improve detection. Animals vaccinated with either dried formulation induced similar levels of anti-spike IgG responses in the serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) and BAL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF) that were comparable to control-vaccinated mice. These results indicate the dried vaccine formulation retained its ability to induce robust antigen-specific T cell and antibody responses even after long-term storage at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe goal of this work was to build off our previous work demonstrating the combination of two carbohydrates, pullulan, and trehalose, to thermally stabilize an inherently unstable but therapeutically important viral vector, adenovirus (AdV). Examples in the literature have shown moderate stabilization of AdV[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], so our goal was to develop the highest classification of stability for AdV vectored therapeutics. Based off the WHO\u0026rsquo;s VVM stability temperatures and times, the most stable vaccine status is VVM250 which is attained at either \u0026gt;\u0026thinsp;250 days at 37\u003csup\u003eo\u003c/sup\u003eC, 71 days at 45\u003csup\u003eo\u003c/sup\u003eC, or 17 days at 55\u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our initial comparison between the strategy developed to stabilize the VSV viral vector and the best published work at the time demonstrated a significant stabilization improvement at 37\u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. With a stability of less than 0.25 log IU loss over 450 days at 37\u003csup\u003eo\u003c/sup\u003eC, this would be easily classified as a VVM250 stable product (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, most dried therapeutic products are backfilled with inert gas such as nitrogen. When the HuAd5 with F1 formulation was dried and backfilled with dried nitrogen gas, we observed\u0026thinsp;~\u0026thinsp;0.5 Log IU worse stabilization compared to atmospheric gas backfilling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, we observed a similar result of negative impact of dried film crystallinity to viral vector stabilization for AdV as we previously reported with the VSV vector. This result was exasperated at more elevated temperatures (55\u003csup\u003eo\u003c/sup\u003eC vs 37\u003csup\u003eo\u003c/sup\u003eC) with over 3% dried film being crystalline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By eliminating salt ions to stabilize the AdV, we observed a significant increase in thermostability without additional process loss. To improve our speed of development, we pushed the accelerated aging experiments to temperatures we have not observed successful results reported. While our initial attempts were markedly more stable than we could observe in the published literature (similar losses of 1.25 Log IU Loss from published at ~\u0026thinsp;30 days to 355 days for our work at 45\u003csup\u003eo\u003c/sup\u003eC; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), these would not be satisfactory for a successful product. Over several development iterations, we developed both a drying schedule and formulation composition able to maintain thermostability for HuAd5 vector of less than 0.5 log loss IU over 49 days at 55\u003csup\u003eo\u003c/sup\u003eC, a stability datapoint unprecedented in the literature (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile our development work was performed with a non-targeted HuAd5 vector, we next sought to demonstrate the same stability with a proven AdV viral vectored vaccine. Additionally, several key AdV therapeutic vectors are derived from chimpanzee origins, so demonstration of our formulation strategy with different AdV\u0026rsquo;s was critical to prove widespread applicability. We pursued our work with a trivalent chimpanzee adenovirus vectored COVID 19 vaccine (ChAd-TriCoV/Mac) which has demonstrated efficacy in mice and currently is being tested in a phase II human trial by inhaled aerosol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Currently, this vaccine requires \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003eC storage to ensure stability. When ChAd-TriCoV/Mac was formulated, dried, and aged at 55\u003csup\u003eo\u003c/sup\u003eC, we observe equal or slightly greater stability after 42 days as compared to the HuAd5 vector, with a total loss of 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 Log IU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This result confirms the PT120-D formulation methodology can be used on AdV vectors derived from different mammalian hosts, as well as with proven vector vaccines.\u003c/p\u003e\u003cp\u003eTo ensure efficacy in a mouse model with a more applicable accelerated aging temperature, we formulated, dried, and aged ChAd-TriCoV/Mac at 37\u003csup\u003eo\u003c/sup\u003eC for 44 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Prior to vaccination, we assayed the same sample which revealed only 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 Log IU loss from the initial stock material. Three different test groups were designed, the \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003eC stock vaccine, a freshly prepared formulated and dried group, and the 44-week aged at 37\u003csup\u003eo\u003c/sup\u003eC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). All three groups demonstrated similar antigen-specific T cell and antibody responses. Overall, a demonstration equivalent immunological responses between the \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003eC stock ChAd-TriCoV/Mac and the 44-week aged at 37\u003csup\u003eo\u003c/sup\u003eC group is an outstanding result. These data firmly put the PT120-D formulated ChAd-TriCoV/Mac into the VVM250 stability classification, a first for a COVID-19 targeted vaccine. We project, based off our data showing a plateau of titer loss after the first 1\u0026ndash;2 weeks of incubation and Arrhenius accelerated decay rates, the formulated vaccine should have a shelf stability (25\u003csup\u003eo\u003c/sup\u003eC) of between 7\u0026ndash;10 years. Dried ChAd-TriCoV/Mac vials continue to be incubated at 37\u003csup\u003eo\u003c/sup\u003eC, and subsequent follow-up studies will determine if this projection remains accurate.\u003c/p\u003e\u003cp\u003eIn summary, we demonstrated the drying and formulation strategy involving a pullulan and trehalose-based film to create a shelf stable format for AdV based therapeutics. To the best of our knowledge, no other research has achieved thermostability at 55\u003csup\u003eo\u003c/sup\u003eC for 7 weeks and represents a significant advancement in opportunity for storage and delivery of AdV-vectored therapeutics. Demonstration of equivalent immunogenicity with stock material further supports these claims. Future work with clinical trials will elucidate the potential of PT120-D based formulation technology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003ePullulan and trehalose SG dried powders were kindly provided by Nagase Viita Co., Ltd. Purified human adenovirus serotype 5 (HuAd5) expressing either green fluorescent protein (GFP), Luciferase, and chimpanzee adenovirus serotype 68-based COVID-19 vaccine (ChAd-TriCoV/Mac) were manufactured and provided by Dr. Brian Lichty\u0026rsquo;s research group (McMaster University)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The stock buffers Tris (1 M pH 7.2; Teknova Cat No: T1072), 1 M CaCl\u003csub\u003e2\u003c/sub\u003e (Sigma Cat No: 21115-100 mL), 1 M MgSO\u003csub\u003e4\u003c/sub\u003e (Sigma; Cat No: M3409-100 mL), 5 M NaCl (VWR; E529-500 mL), and dry powders of bovine serum albumin (Sigma; A9418-10G) and gelatin (Criterion, C7921) were diluted and resuspended as described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of formulation components utilized in this study. All percentages are representative of w/v.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFormulation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e\u003cp\u003eComponents\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTris\u003c/p\u003e\u003cp\u003e(pH 7.2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGelatin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBSA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNaCl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePullulan\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eTrehalose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eOther\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.05%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003cp\u003e(pH 8.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5% Inulin,\u003c/p\u003e\u003cp\u003e5% Mannitol\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1% PMAL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10mM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e15%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAdV Dialysis and Formulation\u003c/h3\u003e\n\u003cp\u003eManufactured 500 \u0026micro;L AdV stock aliquots were stored at -80\u003csup\u003eo\u003c/sup\u003eC in 10 mM Tris-Cl, pH 8.0 with 10% glycerol. After gentle thawing on ice, viral particles were buffer exchanged into the formulation buffer using Zeba Spin Desalting columns 7K MWCO (Thermo Scientific). Viral particles were mixed at a 1:9 ratio with the formulation buffer containing Pullulan and/or Trehalose and 100 \u0026micro;L was aliquoted into 2mL vials. Samples were subsequently dried either using a vacuum pump and desiccator or the SP VirTis AdVantage Pro Freeze dryer system. If required, after completion of the dry schedule on the SP VirTis AdVantage Pro, vials were backfilled with dried nitrogen gas and stoppered in system.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIn-Cell Western (ICW) for Adenovirus infectious unit quantification\u003c/h2\u003e\u003cp\u003eHuman embryonic kidney 293 with deletions in early gene regions 1 and 3 (HEK 293 ΔE1/E3; provided by Dr. Lichty\u0026rsquo;s Research group) are grown to 70\u0026ndash;90% confluency and harvested using 3 mL of 1\u0026times; citric saline incubated for 4\u0026ndash;8 minutes at 37\u003csup\u003eo\u003c/sup\u003eC. Resuspended cells are counted using an automated cell counter (Corning) and diluted in completed MEM Earles media for plating at 1.8\u0026ndash;2.5 x 10\u003csup\u003e5\u003c/sup\u003ecells/well (3.6\u0026ndash;5.0 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL) in tissue-culture treated 24-well plates (Corning Costarplates). Plated cells are incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC/5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe next day, dried formulated AdV samples are reconstituted in 1 mL of warmed Milli-Q water and allowed to fully dissolve for 8\u0026ndash;10 minutes. Samples are vortexed at moderate speed for 15s prior to serial dilution in completed media. 250 \u0026micro;L of diluted AdV are gently pipetted onto confluent HEK 293 cells plated in 24-well plates from the day prior and incubated at 37C/5% CO\u003csub\u003e2\u003c/sub\u003e for the 36\u0026ndash;48 hour infection course.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eICW Staining Procedure\u003c/h2\u003e\u003cp\u003eThe inoculum is swiftly discarded after the 2-day infection course. Infected plates are fixed on dry ice by carefully pipetting 300 \u0026micro;L of chilled methanol and incubated for 14 minutes at -20\u003csup\u003eo\u003c/sup\u003eC. The fixative is swiftly discarded and fixed plates undergo 3 rounds of washing in 500 \u0026micro;L of blocking buffer (1% BSA in PBS), with the final wash kept for blocking 30 minutes at RT. Blocking is swiftly discarded prior to a 1-hour incubation in 200 \u0026micro;L of diluted primary antibody (1:3000 Adenovirus Antibody 8C4 in 1% BSA/PBS) at RT. The primary incubation is swiftly discarded followed by 3 washes in blocking buffer prior to incubating in 200 \u0026micro;L of diluted secondary antibody (1:400 IRDye680RD Goat anti-Mouse IgG Secondary Antibody in 1% BSA/PBS) at RT for 1 hour. After discarding the secondary, plates are washed thrice in 500 \u0026micro;L of PBS prior to scanning in the final wash with fluorescent bio-imager (LICOR Odyssey DLx). Outputted signal is measured as raw counts and quantified as IU/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eXRD Analysis of Dried Formulations for Crystallinity\u003c/h2\u003e\u003cp\u003eDried formulations were prepared with a 25\u0026deg;C dry cycle in 2 mL vials. For XRD analysis, the film was crushed into a powder and extracted from the vials. The powder samples were mounted on top of a zero-background Si wafer for the XRD measurements. The data were collected using a Bruker D8 DISCOVER with DAVINCI. A DESIGN diffractometer equipped with a Co sealed-tube source and an Eiger2R 500K area detector was used. A 2D continuous scan from 12\u0026ndash;80\u0026deg; 2Th was collected with 0.02 degree steps at 1.2 seconds per step.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eAge-matched 6\u0026ndash;8-week-old wild-type female were purchased from either Charles River Laboratories (Saint Constant, QC, Canada). Animals were housed in a specific pathogen-free level B Facility at McMaster University, Hamilton, ON, Canada. All experimental protocols were approved, and experiments were performed in accordance with institutional guidelines from the Animal Research and Ethics Board. All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eVaccination\u003c/h2\u003e\u003cp\u003eAnimals were anesthetized with isoflurane and vaccinated intranasally with 1.1-2.9x10\u003csup\u003e6\u003c/sup\u003e IU of a recombinant a recombinant chimpanzee adenovirus serotype 68 SARS-CoV-2 vaccine (ChAd-TriCoV/Mac). Intranasal vaccinations were performed with a final volume of 40\u0026micro;L diluted in PBS. Where noted, dried formulations were resuspended in PBS prior to vaccination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBronchoalveolar lavage mononuclear cell isolation\u003c/h2\u003e\u003cp\u003eMice were euthanized by exsanguination as previously established [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, mice are placed into an anesthetic chamber, and isoflurane is administered as 3\u0026ndash;5% in O\u003csub\u003e2\u003c/sub\u003e prior to cervical dislocation as per institutional guidelines. Cells from bronchoalveolar lavage (BAL) as previously described [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, BAL was performed by instillation with 250 \u0026micro;L, followed by 200 \u0026micro;L of PBS. This fraction was utilized for downstream antibody analysis. Further instillation of 3x 300 \u0026micro;L of PBS was performed for BAL cell retrieval. Isolated cells were resuspended in complete RPMI 1640 (10% FBS, 1% L-glutamine, 100 U/mL penicillin/streptomycin, 1% HEPES pH 7.3, 1% MEM non-essential amino acids (Gibco, Gaithersburg, MD, United States), 1% sodium-pyruvate (Gibco, Gaithersburg, MD, United States). Cell numbers were quantified in Turk\u0026rsquo;s Blood Dilution Fluid (RICCA Chemical, Arlington, TX, United States) and counted under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePeptide library construction and stimulation\u003c/h2\u003e\u003cp\u003ePeptide libraries consisting of 10 amino acid, 15mer synthetic overlapping peptides for vaccine encoded antigen S1 were synthesized by Pepscan (Lelystad, The Netherlands). Peptides were reconstituted in DMSO according to manufacturer\u0026rsquo;s instructions to a final concentration of 40 \u0026micro;g/\u0026micro;L. Antigen peptide pools were generated with each pool containing 0.2 \u0026micro;g/\u0026micro;L of each peptide. Peptide stimulations were carried utilizing 2 \u0026micro;g of each peptide/mL of culture media.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eCell immunostaining and flow cytometry were performed as previously described [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Briefly, isolated mononuclear cells were plated in U-bottom, 96-well plates at a maximum concentration of 2x10\u003csup\u003e7\u003c/sup\u003e cells/mL. Following staining with The LIVE/DEAD\u0026trade; Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific Waltham, MA, United States) at room temperature for 30 min, cells were washed and blocked with anti-CD16/CD32 (clone 2.4G2) in 0.5% BSA-PBS for 15 min on ice and then stained with fluorochrome-labeled mAbs for 30 min on ice. Fluorochrome-labeled mAbs used for staining cells were anti-CD3-V450 (clone 17A2), anti-CD4 APC-Cy7 (clone GK1.5), anti-CD8 PE-Cy7 (clone 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7), and anti-IFNγ APC (clone XMG1.2). Stained cells were fixed and permeabilized with BD Cytofix/Cytoperm before incubation in BD Perm/Wash buffer (BD Biosciences, San Jose, CA, United States). All mAbs and reagents were purchased from BD Biosciences. Stained cells were processed according to BD Biosciences instructions for flow cytometry and run on a BD LSR II flow cytometer. Data were analyzed using FlowJo software (version 10.1; Tree Star, Ashland, OR, United States).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eRecombinant antigen production\u003c/h2\u003e\u003cp\u003ePlasmids encoding mammalian cell codon optimized sequences for full-length spike of SARS-CoV-2 was generously gifted from the lab of Dr. Florian Krammer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] (Icahn School of Medicine, NY, United States). Proteins were produced in Expi293F cells (ThermoFisher Scientific Waltham, MA, United States) according to the manufacturers\u0026rsquo; instructions and purified as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, when culture viability reached 40%, supernatants were collected and spun at 500 x g for 5 minutes. The supernatant was then incubated by shaking overnight at 4\u0026deg;C with 1 mL of Ni-NTA agarose (Qiagen, Germantown MD, United States) per 25 mL of transfected cell supernatant. The following day 10 mL polypropylene gravity flow columns (Qiagen, Germantown, MD, United States) were used to elute the protein. Recombinant RBD was concentrated in a 10 kDa Amicon centrifugal units (Millipore Sigma, Etobicoke, ON, Canada), and recombinant Spike was concentrated in a 50kDa Amicon centrifugal unit (Millipore Sigma, Etobicoke, ON, Canada) prior to being resuspended in phosphate buffered saline (PBS).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme Linked Immunosorbent Assay (ELISAs) for antibody measurement\u003c/h2\u003e\u003cp\u003e96-well NUNC- MaxiSorp\u0026trade; plates (Thermo Scientific, Waltham, MA, United States) were coated overnight at 4\u0026deg;C with SARS-CoV-2 full-length spike, diluted to 2 \u0026micro;g/mL in bicarbonate-carbonate coating buffer (pH 9.4). Plates were blocked by shaking for 1 hour at 37\u0026deg;C with reagent diluent (0.5% bovine serum albumin (BSA), 0.02% sodium azide, in 1X Tris-Tween buffer). Samples were serially diluted from 1:5 (serum), or 1:8 (BAL) starting dilution. BAL samples were first concentrated through Pierce\u0026trade; Protein Concentrators with a 50 kDa molecular weight cut-off (MWCO) (ThermoFisher Scientific Waltham, MA, United States) according to the manufacturer\u0026rsquo;s instructions, with volumes normalized prior to concentration. Samples were arranged such that one row contained only antigen and secondary antibodies and served as the plate blank. Following a 1 hour incubation with shaking at 37\u0026deg;C, plates were washed three times with 1X Tris-Tween wash buffer. After washing, goat anti-mouse-biotin antibodies (Southern Biotech, Birmingham, AL, United States), IgG (1:5000) were diluted in reagent diluent and added to all wells. Plates were again incubated for 1 hour, with shaking, at 37\u0026deg;C, followed by three washes with 1X Tris-Tween buffer. A streptavidin-alkaline phosphatase secondary antibody (1:2000, Southern Biotech, Birmingham, AL, United States) was added to all wells for 1 hour with shaking at 37\u0026deg;C. Plates were subsequently washed three times prior to addition of pNPP one component microwell substrate solution (Southern Biotech, Birmingham, AL, United States) to each well. Plates were developed for 10 minutes and the reaction was quenched with an equal volume 3N sodium hydroxide. The optical density (O.D.) at 405 nm was read on a SpectramaxI3 (Molecular Devices, San Jose, CA, United States). Endpoint titers were defined by the lowest dilution at which the O.D. was three standard deviations above the mean of the blank wells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis and Data Availability\u003c/h2\u003e\u003cp\u003eStatistical analysis and graphing of data were performed with PRISM software (Graphpad Prism v10.1.2). Comparison of two groups was performed with two-tailed paired t-tests. For analysis of more than two unique groups, ANOVA was utilized with Tukey multiple analyses parameter. All data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Vicky Jarvis for her assistance with the XRD experiments and analysis and Maria Fe C Medina for manufacturing the Adenovirus stocks. For their contribution with organizational management, direction, and manuscript editing, we would like to thank Dr. Robert DeWitte and Brent Lefebvre. For their efforts on experimental assistance, we would like to thank Abdulhamid Mohamud, Arthur Liou, Jason Choi, Divhleen Ruprai, and Ria Baijal. Finally, we thank Dr. Lichty\u0026rsquo;s research group for discussions and brainstorming. Elarex would like to acknowledge the National Research Council of Canada Industrial Research Assistance Program (NRC IRAP) for providing advisory services and research and development funding for our stabilization work with AdV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Research Council Canada IRAP Contribution # 1009921\u003c/p\u003e\n\u003cp\u003eOntario Centre for Innovation #36006\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JAI, AA, SA, JEB, CDMF, BDL\u003c/p\u003e\n\u003cp\u003eMethodology: JAI, AA, SA, JEB, BDL, MSM\u003c/p\u003e\n\u003cp\u003eInvestigation: JAI, AA, NK, SA, MD\u003c/p\u003e\n\u003cp\u003eVisualization: JAI, SA\u003c/p\u003e\n\u003cp\u003eSupervision: JEB, BDL, MSM\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: JAI, SA\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review \u0026amp; editing: JAI, AA, JEB, MRD, SA, CDMF \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement: \u003c/strong\u003eAll other authors declare they have no competing interests. \u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOrenstein, W.A. and R. 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Adenoviruses (AdV) have been demonstrated to be an effective delivery vector for several indications including COVID-19 vaccines but are limited by storage and transportation conditions. Previous work has demonstrated formulation and drying of vectored vaccines with pullulan and trehalose-based (PT) films significantly improves thermostabilization. To increase the accessibility of AdV based therapeutics, we developed a vacuum based drying methodology with optimized PT excipients resulting in a shelf stable product. We demonstrate the thermostability of formulated and dried AdV at 55\u003csup\u003eo\u003c/sup\u003eC for 7 weeks with less than 0.5 total log IU loss, and over 44 weeks at 37\u003csup\u003eo\u003c/sup\u003eC with less than 0.25 total log IU loss. Additionally, murine vaccination with the ChAd-TriCoV/Mac vaccine showed no difference in response between fresh and aged at 37\u003csup\u003eo\u003c/sup\u003eC for 44 weeks. These data demonstrate our formulation methodology’s performance, resulting in a shelf stable formulation for AdV based therapeutics.\u003c/p\u003e","manuscriptTitle":"Development of Shelf Stable Formulation for Adenovirus Vectored Vaccines and Therapeutics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 08:51:46","doi":"10.21203/rs.3.rs-7767949/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-04T11:47:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T08:49:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50251804008347862427599221614574815940","date":"2026-01-20T12:53:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T07:55:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182305548453969457413194681631762290275","date":"2026-01-18T20:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282215503875295768430096737291643909766","date":"2026-01-14T08:03:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T23:15:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191435570531730995599023253028270945115","date":"2025-11-25T09:07:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102167321455720748428128159676296368708","date":"2025-11-25T00:17:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250673955434032998010845764053751373868","date":"2025-11-24T22:13:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-24T21:12:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-02T03:40:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-30T02:59:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-28T13:06:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-28T13:02:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3fb96384-f55b-40e4-963a-fca3b18a5c09","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":57132460,"name":"Biological sciences/Biotechnology"},{"id":57132461,"name":"Biological sciences/Drug discovery"},{"id":57132462,"name":"Health sciences/Health care"},{"id":57132463,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-05-08T08:40:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 08:51:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7767949","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7767949","identity":"rs-7767949","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}