Multi-Antigen Viral-Vectored Vaccine Protects Against SARS-CoV-2 and Variants in a Lethal hACE2 Transgenic Mouse Model
Abstract
1. Introduction
2. Materials and Methods
2.1. Vaccine Construction and Characterization
2.2. In Vivo Mouse Vaccination and Infection Experiments
2.3. Plaque Reduction Neutralization Test (PRNT)
2.4. Binding Antibody
2.5. T Cell Analysis
2.6. Viral Load Analysis
2.7. Immunohistochemistry
2.8. Luminex
2.9. Statistical Analysis
3. Results
3.1. Design and Characterization of Multi-Antigen MVA-VLP Vaccine Candidate
3.2. GEO-CM02 Vaccination Elicits Neutralizing Antibodies and Functional T Cell Responses in hACE2 Mice
3.3. The GEO-CM02 Vaccine Provides Protection Against Lethal SARS-CoV-2 Variants in K18-hACE2 Mice
3.4. Dramatic Reduction in Lung Pathology and Inflammatory Markers in GEO-CM02-Vaccinated Mice
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Singh, D.; Yi, S.V. On the origin and evolution of SARS-CoV-2. Exp. Mol. Med. 2021, 53, 537–547. [Google Scholar] [CrossRef] [PubMed]
- FDA. COVID-19 Vaccines for 2023–2024. Available online: https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/covid-19-vaccines-2023-2024 (accessed on 1 January 2025).
- Baden, L.R.; El Sahly, H.M.; Essink, B.; Kotloff, K.; Frey, S.; Novak, R.; Diemert, D.; Spector, S.A.; Rouphael, N.; Creech, C.B. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021, 384, 403–416. [Google Scholar] [CrossRef]
- Corbett, K.S.; Edwards, D.K.; Leist, S.R.; Abiona, O.M.; Boyoglu-Barnum, S.; Gillespie, R.A.; Himansu, S.; Schäfer, A.; Ziwawo, C.T.; DiPiazza, A.T. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature 2020, 586, 567–571. [Google Scholar] [CrossRef] [PubMed]
- Polack, F.P.; Thomas, S.J.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Perez, J.L.; Pérez Marc, G.; Moreira, E.D.; Zerbini, C. Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N. Engl. J. Med. 2020, 383, 2603–2615. [Google Scholar] [CrossRef]
- McMahan, K.; Yu, J.; Mercado, N.B.; Loos, C.; Tostanoski, L.H.; Chandrashekar, A.; Liu, J.; Peter, L.; Atyeo, C.; Zhu, A. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature 2021, 590, 630–634. [Google Scholar] [CrossRef]
- Shen, X.; Tang, H.; Pajon, R.; Smith, G.; Glenn, G.M.; Shi, W.; Korber, B.; Montefiori, D.C. Neutralization of SARS-CoV-2 variants B. 1.429 and B. 1.351. N. Engl. J. Med. 2021, 384, 2352–2354. [Google Scholar] [CrossRef]
- Cui, Z.; Liu, P.; Wang, N.; Wang, L.; Fan, K.; Zhu, Q.; Wang, K.; Chen, R.; Feng, R.; Jia, Z. Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron. Cell 2022, 185, 860–871.e13. [Google Scholar] [CrossRef]
- Hachmann, N.P.; Miller, J.; Collier, A.Y.; Ventura, J.D.; Yu, J.; Rowe, M.; Bondzie, E.A.; Powers, O.; Surve, N.; Hall, K. Neutralization escape by SARS-CoV-2 Omicron subvariants BA. 2.12. 1, BA. 4, and BA. 5. N. Engl. J. Med. 2022, 387, 86–88. [Google Scholar] [CrossRef] [PubMed]
- Cao, Y.; Yisimayi, A.; Jian, F.; Song, W.; Xiao, T.; Wang, L.; Du, S.; Wang, J.; Li, Q.; Chen, X. BA. 2.12. 1, BA. 4 and BA. 5 escape antibodies elicited by Omicron infection. Nature 2022, 608, 593–602. [Google Scholar] [CrossRef]
- Yang, J.; Han, M.; Wang, L.; Wang, L.; Xu, T.; Wu, L.; Ma, J.; Wong, G.; Liu, W.; Gao, G.F. Relatively rapid evolution rates of SARS-CoV-2 spike gene at the primary stage of massive vaccination. Biosaf. Health 2022, 4, 228–233. [Google Scholar] [CrossRef]
- Choi, S.J.; Kim, D.-U.; Noh, J.Y.; Kim, S.; Park, S.-H.; Jeong, H.W.; Shin, E.-C. T cell epitopes in SARS-CoV-2 proteins are substantially conserved in the Omicron variant. Cell. Mol. Immunol. 2022, 19, 447–448. [Google Scholar] [CrossRef] [PubMed]
- Primorac, D.; Brlek, P.; Matišić, V.; Molnar, V.; Vrdoljak, K.; Zadro, R.; Parčina, M. Cellular immunity—The key to long-term protection in individuals recovered from SARS-CoV-2 and after vaccination. Vaccines 2022, 10, 442. [Google Scholar] [CrossRef] [PubMed]
- Bertoletti, A.; Le Bert, N.; Qui, M.; Tan, A.T. SARS-CoV-2-specific T cells in infection and vaccination. Cell. Mol. Immunol. 2021, 18, 2307–2312. [Google Scholar] [CrossRef]
- Liao, M.; Liu, Y.; Yuan, J.; Wen, Y.; Xu, G.; Zhao, J.; Cheng, L.; Li, J.; Wang, X.; Wang, F. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nat. Med. 2020, 26, 842–844. [Google Scholar] [CrossRef] [PubMed]
- Oja, A.E.; Saris, A.; Ghandour, C.A.; Kragten, N.A.; Hogema, B.M.; Nossent, E.J.; Heunks, L.M.; Cuvalay, S.; Slot, E.; Linty, F. Divergent SARS-CoV-2-specific T-and B-cell responses in severe but not mild COVID-19 patients. Eur. J. Immunol. 2020, 50, 1998–2012. [Google Scholar] [CrossRef]
- Moderbacher, C.R.; Ramirez, S.I.; Dan, J.M.; Grifoni, A.; Hastie, K.M.; Weiskopf, D.; Belanger, S.; Abbott, R.K.; Kim, C.; Choi, J. Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity. Cell 2020, 183, 996–1012.E19. [Google Scholar] [CrossRef]
- Sekine, T.; Perez-Potti, A.; Rivera-Ballesteros, O.; Strålin, K.; Gorin, J.-B.; Olsson, A.; Llewellyn-Lacey, S.; Kamal, H.; Bogdanovic, G.; Muschiol, S. Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell 2020, 183, 158–168.E14. [Google Scholar] [CrossRef]
- Tan, Y.; Liu, F.; Xu, X.; Ling, Y.; Huang, W.; Zhu, Z.; Guo, M.; Lin, Y.; Fu, Z.; Liang, D. Durability of neutralizing antibodies and T-cell response post SARS-CoV-2 infection. Front. Med. 2020, 14, 746–751. [Google Scholar] [CrossRef]
- Ni, L.; Ye, F.; Cheng, M.-L.; Feng, Y.; Deng, Y.-Q.; Zhao, H.; Wei, P.; Ge, J.; Gou, M.; Li, X. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity 2020, 52, 971–977.E3. [Google Scholar] [CrossRef]
- Altmann, D.M.; Boyton, R.J.; Beale, R. Immunity to SARS-CoV-2 variants of concern. Science 2021, 371, 1103–1104. [Google Scholar] [CrossRef]
- Chiuppesi, F.; Nguyen, V.H.; Park, Y.; Contreras, H.; Karpinski, V.; Faircloth, K.; Nguyen, J.; Kha, M.; Johnson, D.; Martinez, J. Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in Syrian hamsters and non-human primates. npj Vaccines 2022, 7, 7. [Google Scholar] [CrossRef] [PubMed]
- Tan, A.T.; Linster, M.; Tan, C.W.; Le Bert, N.; Chia, W.N.; Kunasegaran, K.; Zhuang, Y.; Tham, C.Y.; Chia, A.; Smith, G.J. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Rep. 2021, 34, 108728. [Google Scholar] [CrossRef] [PubMed]
- Grifoni, A.; Weiskopf, D.; Ramirez, S.I.; Mateus, J.; Dan, J.M.; Moderbacher, C.R.; Rawlings, S.A.; Sutherland, A.; Premkumar, L.; Jadi, R.S. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell 2020, 181, 1489–1501.E15. [Google Scholar] [CrossRef] [PubMed]
- Xu, R.; Shi, M.; Li, J.; Song, P.; Li, N. Construction of SARS-CoV-2 virus-like particles by mammalian expression system. Front. Bioeng. Biotechnol. 2020, 8, 862. [Google Scholar]
- Roldão, A.; Mellado, M.C.M.; Castilho, L.R.; Carrondo, M.J.; Alves, P.M. Virus-like particles in vaccine development. Expert Rev. Vaccines 2010, 9, 1149–1176. [Google Scholar] [CrossRef]
- Zepeda-Cervantes, J.; Ramírez-Jarquín, J.O.; Vaca, L. Interaction Between Virus-Like Particles (VLPs) and Pattern Recognition Receptors (PRRs) From Dendritic Cells (DCs): Toward Better Engineering of VLPs. Front. Immunol. 2020, 11, 1100. [Google Scholar] [CrossRef]
- Liu, L.; Chavan, R.; Feinberg, M.B. Dendritic cells are preferentially targeted among hematolymphocytes by Modified Vaccinia Virus Ankara and play a key role in the induction of virus-specific T cell responses in vivo. BMC Immunol. 2008, 9, 15. [Google Scholar] [CrossRef]
- Kastenmuller, W.; Drexler, I.; Ludwig, H.; Erfle, V.; Peschel, C.; Bernhard, H.; Sutter, G. Infection of human dendritic cells with recombinant vaccinia virus MVA reveals general persistence of viral early transcription but distinct maturation-dependent cytopathogenicity. Virology 2006, 350, 276–288. [Google Scholar] [CrossRef]
- Altenburg, A.F.; van de Sandt, C.E.; Li, B.W.; MacLoughlin, R.J.; Fouchier, R.A.; van Amerongen, G.; Volz, A.; Hendriks, R.W.; de Swart, R.L.; Sutter, G. Modified vaccinia virus Ankara preferentially targets antigen presenting cells in vitro, ex vivo and in vivo. Sci. Rep. 2017, 7, 8580. [Google Scholar] [CrossRef]
- Gasteiger, G.; Kastenmuller, W.; Ljapoci, R.; Sutter, G.; Drexler, I. Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines. J. Virol. 2007, 81, 11925–11936. [Google Scholar] [CrossRef]
- Shen, X.; Wong, S.; Buck, C.B.; Zhang, J.; Siliciano, R.F. Direct priming and cross-priming contribute differentially to the induction of CD8+ CTL following exposure to vaccinia virus via different routes. J. Immunol. 2002, 169, 4222–4229. [Google Scholar] [CrossRef] [PubMed]
- Goepfert, P.A.; Elizaga, M.L.; Sato, A.; Qin, L.; Cardinali, M.; Hay, C.M.; Hural, J.; DeRosa, S.C.; DeFawe, O.D.; Tomaras, G.D. Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J. Infect. Dis. 2011, 203, 610–619. [Google Scholar] [CrossRef]
- Goepfert, P.A.; Elizaga, M.L.; Seaton, K.; Tomaras, G.D.; Montefiori, D.C.; Sato, A.; Hural, J.; DeRosa, S.C.; Kalams, S.A.; McElrath, M.J. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J. Infect. Dis. 2014, 210, 99–110. [Google Scholar] [CrossRef] [PubMed]
- Salvato, M.S.; Domi, A.; Guzmán-Cardozo, C.; Medina-Moreno, S.; Zapata, J.C.; Hsu, H.; McCurley, N.; Basu, R.; Hauser, M.; Hellerstein, M. A single dose of modified vaccinia Ankara expressing Lassa virus-like particles protects mice from lethal intra-cerebral virus challenge. Pathogens 2019, 8, 133. [Google Scholar] [CrossRef] [PubMed]
- Domi, A.; Feldmann, F.; Basu, R.; McCurley, N.; Shifflett, K.; Emanuel, J.; Hellerstein, M.S.; Guirakhoo, F.; Orlandi, C.; Flinko, R. A single dose of modified vaccinia Ankara expressing Ebola virus like particles protects nonhuman primates from lethal Ebola virus challenge. Sci. Rep. 2018, 8, 864. [Google Scholar] [CrossRef]
- Ramírez, J.C.; Gherardi, M.M.; Esteban, M. Biology of attenuated modified vaccinia virus Ankara recombinant vector in mice: Virus fate and activation of B-and T-cell immune responses in comparison with the Western Reserve strain and advantages as a vaccine. J. Virol. 2000, 74, 923–933. [Google Scholar] [CrossRef]
- Ramírez, J.C.; Gherardi, M.M.; Rodríguez, D.; Esteban, M. Attenuated modified vaccinia virus Ankara can be used as an immunizing agent under conditions of preexisting immunity to the vector. J. Virol. 2000, 74, 7651–7655. [Google Scholar] [CrossRef]
- Brault, A.C.; Domi, A.; McDonald, E.M.; Talmi-Frank, D.; McCurley, N.; Basu, R.; Robinson, H.L.; Hellerstein, M.; Duggal, N.K.; Bowen, R.A. A Zika vaccine targeting NS1 protein protects immunocompetent adult mice in a lethal challenge model. Sci. Rep. 2017, 7, 14769. [Google Scholar] [CrossRef]
- Malherbe, D.C.; Domi, A.; Hauser, M.J.; Meyer, M.; Gunn, B.M.; Alter, G.; Bukreyev, A.; Guirakhoo, F. Modified vaccinia Ankara vaccine expressing Marburg virus-like particles protects guinea pigs from lethal Marburg virus infection. npj Vaccines 2020, 5, 78. [Google Scholar] [CrossRef]
- Wyatt, L.S.; Earl, P.L.; Xiao, W.; Americo, J.L.; Cotter, C.A.; Vogt, J.; Moss, B. Elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection. J. Virol. 2009, 83, 7176–7184. [Google Scholar] [CrossRef]
- Kumari, P.; Rothan, H.A.; Natekar, J.P.; Stone, S.; Pathak, H.; Strate, P.G.; Arora, K.; Brinton, M.A.; Kumar, M. Neuroinvasion and Encephalitis Following Intranasal Inoculation of SARS-CoV-2 in K18-hACE2 Mice. Viruses 2021, 13, 132. [Google Scholar] [CrossRef]
- Kumar, M.; O’Connell, M.; Namekar, M.; Nerurkar, V.R. Infection with non-lethal West Nile virus Eg101 strain induces immunity that protects mice against the lethal West Nile virus NY99 strain. Viruses 2014, 6, 2328–2339. [Google Scholar] [CrossRef]
- Elsharkawy, A.; Stone, S.; Guglani, A.; Patterson, L.D.; Ge, C.; Dim, C.; Miano, J.M.; Kumar, M. Omicron XBB.1.5 subvariant causes severe pulmonary disease in K18-hACE-2 mice. Front. Microbiol. 2024, 15, 1466980. [Google Scholar] [CrossRef] [PubMed]
- Stone, S.; Rothan, H.A.; Natekar, J.P.; Kumari, P.; Sharma, S.; Pathak, H.; Arora, K.; Auroni, T.T.; Kumar, M. SARS-CoV-2 variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice. Viruses 2021, 14, 27. [Google Scholar] [CrossRef] [PubMed]
- Rothan, H.A.; Stone, S.; Natekar, J.; Kumari, P.; Arora, K.; Kumar, M. The FDA-approved gold drug auranofin inhibits novel coronavirus (SARS-CoV-2) replication and attenuates inflammation in human cells. Virology 2020, 547, 7–11. [Google Scholar] [CrossRef] [PubMed]
- Kumar, M.; Roe, K.; Orillo, B.; Muruve, D.A.; Nerurkar, V.R.; Gale, M., Jr.; Verma, S. Inflammasome adaptor protein Apoptosis-associated speck-like protein containing CARD (ASC) is critical for the immune response and survival in west Nile virus encephalitis. J. Virol. 2013, 87, 3655–3667. [Google Scholar] [CrossRef]
- Oh, S.-J.; Kumari, P.; Auroni, T.T.; Stone, S.; Pathak, H.; Elsharkawy, A.; Natekar, J.P.; Shin, O.S.; Kumar, M. Upregulation of Neuroinflammation-Associated Genes in the Brain of SARS-CoV-2-Infected Mice. Pathogens 2024, 13, 528. [Google Scholar] [CrossRef]
- Callaway, E. Remember Beta? New data reveal variant’s deadly powers. Nature 2021. [Google Scholar] [CrossRef]
- Soni, M.; Migliori, E.; Fu, J.; Assal, A.; Chan, H.T.; Pan, J.; Khatiwada, P.; Ciubotariu, R.; May, M.S.; Pereira, M.; et al. The prospect of universal coronavirus immunity: A characterization of reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses. bioRxiv 2023. [Google Scholar] [CrossRef]
- Guo, L.; Zhang, Q.; Gu, X.; Ren, L.; Huang, T.; Li, Y.; Zhang, H.; Liu, Y.; Zhong, J.; Wang, X.; et al. Durability and cross-reactive immune memory to SARS-CoV-2 in individuals 2 years after recovery from COVID-19: A longitudinal cohort study. Lancet Microbe 2024, 5, e24–e33. [Google Scholar] [CrossRef]
- Arieta, C.M.; Xie, Y.J.; Rothenberg, D.A.; Diao, H.; Harjanto, D.; Meda, S.; Marquart, K.; Koenitzer, B.; Sciuto, T.E.; Lobo, A.; et al. The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection. Cell 2023, 186, 2392–2409.E21. [Google Scholar] [CrossRef] [PubMed]
- Buchholz, U.J.; Bukreyev, A.; Yang, L.; Lamirande, E.W.; Murphy, B.R.; Subbarao, K.; Collins, P.L. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc. Natl. Acad. Sci. USA 2004, 101, 9804–9809. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.; Deng, Y.; Huang, B.; Han, D.; Wang, W.; Huang, M.; Zhai, C.; Zhao, Z.; Yang, R.; Zhao, Y.; et al. DNA Vaccines Expressing the Envelope and Membrane Proteins Provide Partial Protection Against SARS-CoV-2 in Mice. Front. Immunol. 2022, 13, 827605. [Google Scholar] [CrossRef] [PubMed]
- Boson, B.; Legros, V.; Zhou, B.; Siret, E.; Mathieu, C.; Cosset, F.L.; Lavillette, D.; Denolly, S. The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles. J. Biol. Chem. 2021, 296, 100111. [Google Scholar] [CrossRef]
- Miura, K.; Suzuki, Y.; Ishida, K.; Arakawa, M.; Wu, H.; Fujioka, Y.; Emi, A.; Maeda, K.; Hamajima, R.; Nakano, T.; et al. Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells. J. Virol. 2023, 97, e0042623. [Google Scholar] [CrossRef]
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Stone, S.; Elsharkawy, A.; Burleson, J.D.; Hauser, M.; Domi, A.; Kumari, P.; Nabi, Z.; Natekar, J.P.; Porto, M.; Backstedt, B.; et al. Multi-Antigen Viral-Vectored Vaccine Protects Against SARS-CoV-2 and Variants in a Lethal hACE2 Transgenic Mouse Model. Vaccines 2025, 13, 411. https://doi.org/10.3390/vaccines13040411
Stone S, Elsharkawy A, Burleson JD, Hauser M, Domi A, Kumari P, Nabi Z, Natekar JP, Porto M, Backstedt B, et al. Multi-Antigen Viral-Vectored Vaccine Protects Against SARS-CoV-2 and Variants in a Lethal hACE2 Transgenic Mouse Model. Vaccines. 2025; 13(4):411. https://doi.org/10.3390/vaccines13040411
Chicago/Turabian StyleStone, Shannon, Amany Elsharkawy, J. D. Burleson, Mary Hauser, Arban Domi, Pratima Kumari, Zainab Nabi, Janhavi P. Natekar, Maciel Porto, Brian Backstedt, and et al. 2025. "Multi-Antigen Viral-Vectored Vaccine Protects Against SARS-CoV-2 and Variants in a Lethal hACE2 Transgenic Mouse Model" Vaccines 13, no. 4: 411. https://doi.org/10.3390/vaccines13040411
APA StyleStone, S., Elsharkawy, A., Burleson, J. D., Hauser, M., Domi, A., Kumari, P., Nabi, Z., Natekar, J. P., Porto, M., Backstedt, B., Newman, M., Oruganti, S. R., & Kumar, M. (2025). Multi-Antigen Viral-Vectored Vaccine Protects Against SARS-CoV-2 and Variants in a Lethal hACE2 Transgenic Mouse Model. Vaccines, 13(4), 411. https://doi.org/10.3390/vaccines13040411