{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T13:11:55Z","timestamp":1791983515504,"version":"4.4.0"},"reference-count":50,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,6,11]],"date-time":"2024-06-11T00:00:00Z","timestamp":1718064000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Natural Sciences and Engineering Research Council of Canada (NSERC)","award":["RGPIN-2021-02691"],"award-info":[{"award-number":["RGPIN-2021-02691"]}]},{"name":"Natural Sciences and Engineering Research Council of Canada (NSERC)","award":["CRC-240394"],"award-info":[{"award-number":["CRC-240394"]}]},{"name":"Canada Research Chair","award":["RGPIN-2021-02691"],"award-info":[{"award-number":["RGPIN-2021-02691"]}]},{"name":"Canada Research Chair","award":["CRC-240394"],"award-info":[{"award-number":["CRC-240394"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3\u20136% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 \u00b0C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose\/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2\u20138 \u00b0C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.<\/jats:p>","DOI":"10.3390\/v16060942","type":"journal-article","created":{"date-parts":[[2024,6,12]],"date-time":"2024-06-12T06:47:30Z","timestamp":1718174850000},"page":"942","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7152-0468","authenticated-orcid":false,"given":"MD Faizul Hussain","family":"Khan","sequence":"first","affiliation":[{"name":"Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H2X 1Y4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7334-3296","authenticated-orcid":false,"given":"Maryam","family":"Youssef","sequence":"additional","affiliation":[{"name":"Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H2X 1Y4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean","family":"Nesdoly","sequence":"additional","affiliation":[{"name":"Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H2X 1Y4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9110-8815","authenticated-orcid":false,"given":"Amine A.","family":"Kamen","sequence":"additional","affiliation":[{"name":"Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC H2X 1Y4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1038\/s41392-022-00996-y","article-title":"COVID-19 vaccine development: Milestones, lessons and prospects","volume":"7","author":"Li","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kis, Z. (2022). Stability modelling of mRNA vaccine quality based on temperature monitoring throughout the distribution chain. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14020430"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Emmer, K.L., and Ertl, H.C.J. (2020). Recombinant adenovirus vectors as mucosal vaccines. Mucosal Vaccine, Elsevier.","DOI":"10.1016\/B978-0-12-811924-2.00024-9"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Bogale, H.A., Amhare, A.F., and Bogale, A.A. (2019). Assessment of factors affecting vaccine cold chain management practice in public health institutions in east Gojam zone of Amhara region. BMC Public Health, 19.","DOI":"10.1186\/s12889-019-7786-x"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2217","DOI":"10.1016\/j.vaccine.2016.08.045","article-title":"Improving cold chain systems: Challenges and solutions","volume":"35","author":"Ashok","year":"2017","journal-title":"Vaccine"},{"key":"ref_6","unstructured":"World Economic Forum (2024, May 07). Over Half of Vaccines Are Wasted Globally for These Simple Reasons. Available online: https:\/\/www.weforum.org\/agenda\/2018\/07\/the-biggest-hurdle-to-universal-vaccination-might-just-be-a-fridge\/."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2269","DOI":"10.1080\/21645515.2019.1649532","article-title":"Recombinant vesicular stomatitis virus vector vaccines for WHO blueprint priority pathogens","volume":"15","author":"Fathi","year":"2019","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_8","first-page":"97","article-title":"Factors affecting the stability of viral vaccines","volume":"87","author":"Peetermans","year":"1996","journal-title":"Dev. Biol. Stand."},{"key":"ref_9","first-page":"11","article-title":"Rahman Forced degradation studies: An essential tool for the formulation development of vaccines","volume":"3","author":"Hasija","year":"2013","journal-title":"Vaccine Dev. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1615\/CritRevTherDrugCarrierSyst.v16.i1.10","article-title":"Formulation, stability, and delivery of live attenuated vaccines for human use","volume":"16","author":"Volkin","year":"1999","journal-title":"Crit. Rev. Ther. Drug Carr. Syst."},{"key":"ref_11","unstructured":"Warne, N.W., and Mahler, H.-C. (2023, January 24). Challenges in Protein Product Development. Available online: http:\/\/www.springer.com\/series\/8825."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5507","DOI":"10.1016\/j.vaccine.2015.08.085","article-title":"Freeze-drying of live virus vaccines: A review","volume":"33","author":"Hansen","year":"2015","journal-title":"Vaccine"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/0264-410X(88)90006-0","article-title":"Stability of oral polio vaccine at different temperatures","volume":"6","author":"Sokhey","year":"1988","journal-title":"Vaccine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1128\/jcm.18.3.658-662.1983","article-title":"Effect of sucrose phosphate and sorbitol on infectivity of enveloped viruses during storage","volume":"18","author":"Howell","year":"1983","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","unstructured":"Rey, L., and May, J.C. (2010). Freeze Drying\/Lyophilization of Pharmaceutical and Biological Products, CRC Press. [3rd ed.]."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/j.mimet.2006.02.017","article-title":"Preservation of micro-organisms by drying; A review","volume":"66","author":"Morgan","year":"2006","journal-title":"J. Microbiol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Adams, G.D.J. (2003). Lyophilization of vaccines. Vaccine Protocols, Humana Press.","DOI":"10.1385\/1-59259-399-2:223"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1002\/jps.20364","article-title":"Mechanism of protein stabilization by sugars during freeze-drying and storage: Native structure preservation, specific interaction, and\/or immobilization in a glassy matrix?","volume":"94","author":"Chang","year":"2005","journal-title":"J. Pharm. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Pikal, M.J. (1994). Freeze-Drying of Proteins, ACS Publications.","DOI":"10.1021\/bk-1994-0567.ch008"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1615","DOI":"10.1021\/js9800174","article-title":"Sugar\u2013polymer hydrogen bond interactions in lyophilized amorphous mixtures","volume":"87","author":"Taylor","year":"1998","journal-title":"J. Pharm. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.ijpharm.2019.02.035","article-title":"Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders","volume":"561","author":"Toniolo","year":"2019","journal-title":"Int. J. Pharm."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.3382\/ps.2009-00620","article-title":"Development of a stabilizer for lyophilization of an attenuated duck viral hepatitis vaccine","volume":"89","author":"Kang","year":"2010","journal-title":"Poult. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1038\/s41541-022-00508-7","article-title":"Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2","volume":"7","author":"Taddeo","year":"2022","journal-title":"NPJ Vaccines"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6696","DOI":"10.1016\/j.vaccine.2019.09.027","article-title":"Stabilization and formulation of a recombinant Human Cytomegalovirus vector for use as a candidate HIV-1 vaccine","volume":"37","author":"Kumru","year":"2019","journal-title":"Vaccine"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"13189","DOI":"10.1128\/JVI.01704-14","article-title":"A Histidine Residue of the Influenza Virus Hemagglutinin Controls the pH Dependence of the Conformational Change Mediating Membrane Fusion","volume":"88","author":"Mair","year":"2014","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1007\/s10517-018-4097-7","article-title":"Composition of the stabilizer and conditions of lyophilization for preserving infectious activity of influenza virus","volume":"165","author":"Dubrovina","year":"2018","journal-title":"Bull. Exp. Biol. Med."},{"key":"ref_27","first-page":"299","article-title":"Sorbitol-gelatin and glutamic acid-lactose solutions for stabilization of reference preparations of measles virus","volume":"23","author":"Mendes","year":"1989","journal-title":"Bull. Pan Am. Health Organ."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2070","DOI":"10.1016\/j.xphs.2018.04.019","article-title":"Urea improves stability of inactivated polio vaccine serotype 3 during lyophilization and storage in dried formulations","volume":"107","author":"Qi","year":"2018","journal-title":"J. Pharm. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/jcm.4.1.1-5.1976","article-title":"Stability of pseudorabies virus during freeze-drying and storage: Effect of suspending media","volume":"4","author":"Scott","year":"1976","journal-title":"J. Clin. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1021\/bp034362x","article-title":"Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization","volume":"20","author":"Zhai","year":"2004","journal-title":"Biotechnol. Prog."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1128\/am.20.5.723-726.1970","article-title":"Lyophilization of cell-free marek\u2019s disease herpesvirus and a herpesvirus from turkeys","volume":"20","author":"Calnek","year":"1970","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_32","first-page":"257","article-title":"Stabilizer for lyophilization of rubella virus","volume":"29","author":"Hekker","year":"1970","journal-title":"Arch. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1099\/0022-1317-17-3-281","article-title":"The effects of various protecting agents on the inactivation of foot-and-mouth disease virus in aerosols and during freeze-drying","volume":"17","author":"Barlow","year":"1972","journal-title":"J. Gen. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6996","DOI":"10.1016\/j.vaccine.2019.07.003","article-title":"Development of suspension adapted Vero cell culture process technology for production of viral vaccines","volume":"37","author":"Shen","year":"2019","journal-title":"Vaccine"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Kim, G.N., Choi, J.-A., Wu, K., Saeedian, N., Yang, E., Park, H., Woo, S.-J., Lim, G., Kim, S.-G., and Eo, S.-K. (2021). A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2. PLOS Pathog., 17.","DOI":"10.1371\/journal.ppat.1011000"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2649","DOI":"10.1002\/bit.27785","article-title":"Bioreactor production of rVSV-based vectors in Vero cell suspension cultures","volume":"118","author":"Kiesslich","year":"2021","journal-title":"Biotechnol. Bioeng."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Yang, Z., Paes, B.C.M.F., Fulber, J.P.C., Tran, M.Y., Farn\u00f3s, O., and Kamen, A.A. (2023). Development of an Integrated Continuous Manufacturing Process for the rVSV-Vectored SARS-CoV-2 Candidate Vaccine. Vaccines, 11.","DOI":"10.3390\/vaccines11040841"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Farn\u00f3s, O., Paes, B.C.M.F., Getachew, B., Rourou, S., Chaabene, A., Gelaye, E., Tefera, T.A., and Kamen, A.A. (2023). Intranasally Delivered Adenoviral Vector Protects Chickens against Newcastle Disease Virus: Vaccine Manufacturing and Stability Assessments for Liquid and Lyophilized Formulations. Vaccines, 12.","DOI":"10.20944\/preprints202311.0739.v1"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Bullen, C.K., Davis, S.L., and Looney, M.M. (2022). Quantification of Infectious SARS-CoV-2 by the 50% Tissue Culture Infectious Dose Endpoint Dilution Assay. SARS-CoV-2: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-0716-2111-0_9"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Klepzig, L.S., Juckers, A., Knerr, P., Harms, F., and Strube, J. (2020). Digital twin for lyophilization by process modeling in manufacturing of biologics. Processes, 8.","DOI":"10.3390\/pr8101325"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1006\/biol.1998.0157","article-title":"Stability of 17D yellow fever virus vaccine using different stabilizers","volume":"26","author":"Adebayo","year":"1998","journal-title":"Biologicals"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1016\/j.xphs.2020.11.015","article-title":"The science is there: Key considerations for stabilizing viral vector-based COVID-19 vaccines","volume":"110","author":"Crommelin","year":"2020","journal-title":"J. Pharm. Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1128\/am.20.6.935-938.1970","article-title":"Stabilities of suspensions of influenza virus dried by sublimation of ice in vacuo to different contents of residual moisture and sealed under different gases","volume":"20","author":"Greiff","year":"1970","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1038\/sj.gt.3301527","article-title":"Development of formulations that enhance physical stability of viral vectors for gene therapy","volume":"8","author":"Croyle","year":"2001","journal-title":"Gene Ther."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1006\/abbi.1994.1224","article-title":"Role of cellular energy status in tocopheryl hemisuccinate cytoprotection against ethyl methanesulfonate-induced toxicity","volume":"311","author":"Ray","year":"1994","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/0021-9673(94)01257-F","article-title":"Moisture content in proteins: Its effects and measurement","volume":"705","author":"Towns","year":"1995","journal-title":"J. Chromatogr. A"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Adams, G. (2007). The principles of freeze-Drying. Cryopreservation and Freeze-Drying Protocols, Springer.","DOI":"10.1007\/978-1-59745-362-2_2"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Wasserman, A., Sarpal, R., and Phillips, B.R. (2014). Lyophilization in Vaccine Processes. Vaccine Development and Manufacturing, Wiley.","DOI":"10.1002\/9781118870914.ch9"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/j.jfoodeng.2012.03.001","article-title":"Time-scale modeling and optimal control of freeze\u2013drying","volume":"111","author":"Antelo","year":"2012","journal-title":"J. Food Eng."},{"key":"ref_50","unstructured":"Centers for Disease Control and Prevention (2024, February 24). Administration of ERVEBO\u00ae, Available online: https:\/\/www.cdc.gov\/vhf\/ebola\/clinicians\/vaccine\/vaccine-administration.html."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/6\/942\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:57:09Z","timestamp":1760108229000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/6\/942"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,11]]},"references-count":50,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["v16060942"],"URL":"https:\/\/doi.org\/10.3390\/v16060942","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,11]]}}}