Replication-competent vaccinia virus-vectored oral vaccines provide complete protection against the SARS-CoV-2 challenge and effectively prevent viral transmission via respiratory droplets in mice and minks

doi:10.1016/j.jia.2025.11.025

Advanced Online Publication | Current Issue | Archive | Adv Search

Linya Feng¹*, Hong Huo¹*, Yunlei Wang¹*^†, Lei Shuai¹, Gongxun Zhong¹, Zhiyuan Wen¹, Liyan Peng¹, Jinying Ge¹, Jinliang Wang¹, Chong Wang¹, Weiye Chen¹, Xijun He², Xijun Wang¹^,³^#, Zhigao Bu^{1, 2, 3}^#

¹ State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

² National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

³ Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China

Highlights

l The rWR-S6P is orally immunogenic and provides complete protection against the SARS-CoV-2 challenge in mice and minks.

l Oral administration of rWR-S6P effectively prevents transmission of SARS-CoV-2 among minks via respiratory droplets.

l Combined vaccination with recombinant WRs is an effective administration strategy for preventing and controlling animal COVID-19.

Abstract
References

Download: PDF in ScienceDirect
Export: BibTeX | EndNote (RIS)

摘要

新型冠状病毒（Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）S蛋白是研制新型冠状病毒感染（COVID-19）载体疫苗的首选靶抗原，然而其免疫原性较低。为了优化SARS-CoV-2 S蛋白的免疫原性，本研究针对原始毒株、德尔塔（Delta）和奥密克戎（Omicron）BA.2变异株全长S蛋白基因，分别设计、合成了3种预融合构象稳定的S蛋白基因S6P、DS6P和BA2S6P。采用同源重组的方法，将3种修改的S基因分别插入到复制型痘苗病毒（Vaccinia virus, VACV）Western Reserve（WR）毒株病毒基因组中TK基因的EcoRⅠ位点，构建成分别表达S6P、DS6P和BA2S6P蛋白的3种重组病毒rWR-S6P、rWR-DS6P和rWR-BA2S6P。结果表明，3种重组病毒均保持了亲本毒株WR的在CV-1细胞上的生长特性。小鼠试验显示，亲本毒株WR接种的小鼠的体重显著下降12.77%，而重组病毒或PBS接种的小鼠体重仅出现轻微下降，很快恢复，表明修改的S基因的引入降低了载体病毒WR的毒力。利用重组病毒rWR-S6P口服或肌肉注射免疫小鼠和水貂，均能诱导强烈的SARS-CoV-2中和抗体反应和有效的攻毒保护效力。值得注意的是，相同剂量的rWR-S6P口服免疫比肌注免疫诱导的SARS-CoV-2中和抗体滴度更高，攻毒保护效果也更好。同时，rWR-S6P口服免疫能有效阻止SARS-CoV-2在水貂中的飞沫传播。此外，利用3种重组病毒联合免疫小鼠，能够诱导强烈、持久的SARS-CoV-2中和抗体反应，且不会影响各自的免疫原性。研究结果表明，基于复制型痘苗病毒弱毒株的载体疫苗是一种有希望的水貂用口服COVID-19候选疫苗，同时联合接种是此类疫苗的一种有效免疫策略。

Abstract

Here, we generated three recombinant replication-competent vaccinia virus (VACV) Western Reserve (WR) strains rWR-S6P, rWR-DS6P, and rWR-BA2S6P. These recombinant viruses express the prefusion-stabilized S proteins S6P, DS6P, and BA2S6P, which target the full-length S protein of the strain ancestor and variants Delta and Omicron BA.2 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. These recombinant viruses maintained the growth property of the parental virus WR in CV-1 cells. A mouse study indicated that the insertion of these modified S genes reduced the virulence of the vector virus WR. Oral or intramuscular vaccination with rWR-S6P elicited a robust neutralizing antibody (NA) response against live SARS-CoV-2 and provided complete protection against the SARS-CoV-2 challenge in mice and minks. Of note, oral vaccination with rWR-S6P induced significantly higher titers of SARS-CoV-2 NAs and superior protective efficacy compared to intramuscular vaccination at an equivalent dose. More importantly, oral administration of rWR-S6P effectively prevents transmission of SARS-CoV-2 among minks via respiratory droplets. Furthermore, combined oral vaccination with three recombinant WRs induced a strong and long-lasting NA response against homotypic SARS-CoV-2 pseudovirus in mice without compromising their immunogenicity profiles. These findings indicate that the attenuated replication-competent VACV-vectored vaccines hold promise as effective oral COVID-19 vaccines for minks while demonstrating that combined vaccination is an effective administration strategy for preventing and controlling COVID-19.

Keywords: vaccinia virus COVID-19 SARS-CoV-2 prefusion-stabilized S proteins oral vaccine

Online: 15 November 2025

Fund: This work was supported by the National Key Research and Development Program of China (2022YFC2604200).

About author: Linya Feng, E-mail: fenglinya@mail.kiz.ac.cn; #Correspondence Zhigao Bu, E-mail: buzhigao@caas.cn; Xijun Wang, E-mail: wangxijun@caas.cn. * These authors contributed equally to this work.

	Service
	E-mail this article
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

Linya Feng, Hong Huo, Yunlei Wang, Lei Shuai, Gongxun Zhong, Zhiyuan Wen, Liyan Peng, Jinying Ge, Jinliang Wang, Chong Wang, Weiye Chen, Xijun He, Xijun Wang, Zhigao Bu. 2025. Replication-competent vaccinia virus-vectored oral vaccines provide complete protection against the SARS-CoV-2 challenge and effectively prevent viral transmission via respiratory droplets in mice and minks. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.11.025

Anderson E J, Rouphael N G, Widge A T, Jackson L A, Roberts P C, Makhene M, Chappell J D, Denison M R, Stevens L J, Pruijssers A J, Mcdermott A B, Flach B, Lin B C, Doria-Rose N A, O'dell S, Schmidt S D, Corbett K S, Swanson P, Padilla M, Neuzil K M, Bennett H, Leav B, Makowski M, Albert J, Cross K, Edara V V, Floyd K, Suthar M S, Martinez D R, Baric R, Buchanan W, Luke C J, Phadke V K, Rostad C A, Ledgerwood J E, Graham B S, Beigel J H; mRNA-1273 Study Group. 2020. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. New England Journal of Medicine, 383, 2427-2438.

Arevalo-Romero J A, Chingaté-López S M, Camacho B A, Alméciga-Díaz C J, Ramirez-Segura C A. 2024. Next-generation treatments: Immunotherapy and advanced therapies for COVID-19. Heliyon, 10(5):e26423.

Bisht H, Roberts A, Vogel L, Bukreyev A, Collins P L, Murphy B R, Subbarao K, Moss B. 2004. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proceedings of the National Academy of Sciences of the United States of America, 101, 6641-6646.

Breitbach C J, Burke J, Jonker D, Stephenson J, Haas A R, Chow L Q M, Nieva J, Hwang T H, Moon A, Patt R, Pelusio A, Le Boeuf F, Burns J, Evgin L, De Silva N, Cvancic S, Robertson T, Je J E, Lee Y S, Parato K, Diallo J S, Fenster A, Daneshmand M, Bell J C, Kirn D H. 2011. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature, 477, 99-102.

Cliquet F, Aubert M. 2004. Elimination of terrestrial rabies in Western European countries. Developments in biologicals, 119, 185-204.

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, Dinnon K H, Elbashir S M, Shaw C A, Woods A, Fritch E J, Martinez D R, Bock K W, Minai M, Nagata B M, Hutchinson G B, Wu K, Henry C, Bahl K, Garcia-Dominguez D, Ma L, Renzi I, Kong W, Schmidt S D, Wang L, Zhang Y, Phung E, Chang L A, Loomis R J, Altaras N E, Narayanan E, Metkar M, Presnyak V, Liu C, Louder M K, Shi W, Leung K, Yang E S, West A, Gully K L, Stevens L J, Wang N, Wrapp D, Doria-Rose N A, Stewart-Jones G, Bennett H, Alvarado G S, Nason M C, Ruckwardt T R, McLellan J S, Denison M R, Chappell J D, Moore I N, Morabito K M, Mascola J R, BaricR S, Carfi A, Graham B S. 2020. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature, 586, 567-571.

Hassan A O, Kafai N M, Dmitriev I P, Fox J M, Smith B K, Harvey I B, Chen R E, Winkler E S, Wessel A W, Case J B, Kashentseva E, Mccune B T, Bailey A L, Zhao H Y, Vanblargan L A, Dai Y N, Ma M S, Adams L J, Shrihari S, Danis J E, Gralinski L E, Hou Y J, Schäfer A, Kim A S, Keeler S P, Weiskopf D, Baric R S, Holtzman M J, Fremont D H, Curiel D T, Diamond M S. 2020. A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2. Cell, 183, 169-184.

Hsieh C L, Goldsmith J A, Schaub J M, Divenere A M, Kuo H C, Javanmardi K, Le K C, Wrapp D, Lee A G, Liu Y T, Chou C W, Byrne P O, Hjorth C K, Johnson N, Ludes-Meyers J, Nguyen A W, Park J, Wang N S, Amengor D, Lavinder J J, Ippolito G C, Maynard J A, Finkelstein I J, McLellan J S. 2020. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science, 369, 1501-1505.

Kirn D H, Thorne S H. 2009. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer. Nature Reviews Cancer, 9, 64-71.

Kreijtz J H C M, Suezer Y, Van Amerongen G, De Mutsert G, Schnierle B S, Wood J M, Kuiken T, Fouchier R a M, Löwer J, Osterhaus A D M E, Sutter G, Rimmelzwaan G F. 2007. Recombinant modified vaccinia virus Ankara-based vaccine induces protective immunity in mice against infection with influenza virus H5N1. Journal of Infectious Diseases, 195, 1598-1606.

Li M C, Wang H, Tian L L, Pang Z H, Yang Q K, Huang T Q, Fan J F, Song L H, Tong Y G, Fan H H. 2022. COVID-19 vaccine development: milestones, lessons and prospects. Signal Transduction and Targeted Therapy, 7,146.

Liu R, Americo J L, Cotter C A, Earl P L, Erez N, Peng C, Moss B. 2021. One or two injections of MVA-vectored vaccine shields hACE2 transgenic mice from SARS-CoV-2 upper and lower respiratory tract infection. Proceedings of the National Academy of Sciences of the United States of America, 118, e2026785118.

Mcbride D S, Garushyants S K, Franks J, Magee A F, Overend S H, Huey D, Williams A M, Faith S A, Kandeil A, Trifkovic S, Miller L, Jeevan T, Patel A, Nolting J M, Tonkovich M J, Genders J T, Montoney A J, Kasnyik K, Linder T J, Bevins S N, Lenoch J B, Chandler J C, Deliberto T J, Koonin E V, Suchard M A, Lemey P, Webby R J, Nelson M N, Bowman A S. 2023. Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer. Nature Communications, 14, 5105.

Oreshkova N, Molenaar R J, Vreman S, Harders F, Munnink B B O, Hakze-Van Der Honing R W, Gerhards N, Tolsma P, Bouwstra R, Sikkema R S, Tacken M G J, De Roolj M M T, Weesendorp E, Engelsma M Y, Bruschke C J M, Smit L a M, Koopmans M, Van Der Poel W H M, Stegeman A. 2020. SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020. Eurosurveillance, 25, 2-8.

Panicali D, Davis S W, Weinberg R L, Paoletti E. 1983. Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proceedings of the National Academy of Sciences of the United States of America, 80, 5364-5368.

Perera L P, Waldmann T A, Mosca J D, Baldwin N, Berzofsky J A, Oh S K. 2007. Development of smallpox vaccine candidates with integrated interleukin-15 that demonstrate superior immunogenicity, efficacy, and safety in mice. Journal of Virology, 81, 8774-8783.

Rosatte R. 2011. Evolution of wildlife rabies control tactics. Advances in virus research, 79, 397-419.

Rupprecht C E, Hanlon C A, Slate D. 2004. Oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control. Developments in biologicals, 119, 173-184.

Schmidt C. 2011. Amgen spikes interest in live virus vaccines for hard-to-treat cancers. Nature Biotechnology, 29, 295-296.

Schudel A A, Lombard M. 2007. Recommendations of the OIE International Conference on the Control of Infectious Animal Diseases by Vaccination, Buenos Aires, Argentina, 13 to 16 April 2004. Revue scientifique et technique, 26, 519-521.

Shi J Z, Wen Z Y, Zhong G X, Yang H L, Wang C, Huang B Y, Liu R Q, He X J, Shuai L, Sun Z R, Zhao Y B, Liu P P, Liang L B, Cui P F, Wang J L, Zhang X F, Guan Y T, Tan W J, Wu G Z, Chen H L, Bu Z G. 2020. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science, 368, 1016-1020.

Shuai L, Zhong G X, Yuan Q, Wen Z Y, Wang C, He X J, Liu R Q, Wang J L, Zhao Q J, Liu Y X, Huo N N, Deng J H, Bai J J, Wu H C, Guan Y T, Shi J Z, Tian K G, Xia N S, Chen H L, Bu Z G. 2021. Replication, pathogenicity, and transmission of SARS-CoV-2 in minks. National Science Review, 8, nwaa291.

Slate D, Algeo T P, Nelson K M, Chipman R B, Donovan D, Blanton J D, Niezgoda M, Rupprecht C E. 2009. Oral rabies vaccination in north america: opportunities, complexities, and challenges. PLoS neglected tropical diseases, 3, e549.

Smith G L, Benfield C T O, Maluquer de Motes C, Mazzon M, Ember S W J, Ferguson B J, Sumner R P. 2013. Vaccinia Virus Immune Evasion: Mechanisms, Virulence and Immunogenicity. Journal of general virology, 94(11): 2367-2392.

Song F, Fux R, Provacia L B, Volz A, Eickmann M, Becker S, Osterhaus A D M E, Haagmans B L, Sutter G. 2013. Middle East Respiratory Syndrome Coronavirus Spike Protein Delivered by Modified Vaccinia Virus Ankara Efficiently Induces Virus-Neutralizing Antibodies. Journal of Virology, 87, 11950-11954.

Steinberg A P, Silander O K, Kussell E. 2023. Correlated substitutions reveal SARS-like coronaviruses recombine frequently with a diverse set of structured gene pools. Proceedings of the National Academy of Sciences of the United States of America, 120(5):e2206945119.

Suryawanshi R K, Chen I P, Ma T C, Syed A M, Brazer N, Saldhi P, Simoneau C R, Ciling A, Khalid M M, Sreekumar B, Chen P Y, Kumar G R, Montano M, Gascon R, Tsou C L, Garcia-Knight M A, Sotomayor-Gonzalez A, Servellita V, Gliwa A, Nguyen J, et al. 2022. Limited cross-variant immunity from SARS-CoV-2 Omicron without vaccination. Nature Reviews Immunology, 22, 410-410.

Sutter G, Moss B. 1992. Nonreplicating Vaccinia Vector Efficiently Expresses Recombinant Genes. Proceedings of the National Academy of Sciences of the United States of America, 89(22): 10847-10851.

Vitasek J. 2004. A review of rabies elimination in Europe. Veterinarni Medicina, 49, 171-185.

Wang J, Shuai L, Wang C, Liu R, He X, Zhang X, Sun Z, Shan D, Ge J, Wang X, Hua R, Zhong G, Wen Z, Bu Z. 2020. Mouse-adapted SARS-CoV-2 replicates efficiently in the upper and lower respiratory tract of BALB/c and C57BL/6J mice. Protein & cell, 11, 776-782.

Wiktor T J, Macfarlan R I, Reagan K J, Dietzschold B, Curtis P J, Wunner W H, Kieny M P, Lathe R, Lecocq J P, Mackett M, Et Al. 1984. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proceedings of the National Academy of Sciences of the United States of America, 81, 7194-7198.

WOAH. 2023. SARS-CoV-2 in animals-situation report 22. 1-3.

Zhu F C, Li Y H, Guan X H, Hou L H, Wang W J, Li J X, Wu S P, Wang B S, Wang Z, Wang L, Jia S Y, Jiang H D C, Wang L, Jiang T, Hu Y, Gou J B, Xu S B, Xu J J, Wang X W, Wang W, Chen W. 2020. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet, 395, 1845-1854.

[1]	Niu Wang, Weidong Zhang, Zhenyu Zhong, Xiongbo Zhou, Xinran Shi, Xin Wang. FGF7 secreted from dermal papillae cell regulates the proliferation and differentiation of hair follicle stem cell[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3583-3597.
[2]	Lichao Zhai, Shijia Song, Lihua Zhang, Jinan Huang, Lihua Lv, Zhiqiang Dong, Yongzeng Cui, Mengjing Zheng, Wanbin Hou, Jingting Zhang, Yanrong Yao, Yanhong Cui, Xiuling Jia. Subsoiling before winter wheat alleviates the kernel position effect of densely grown summer maize by delaying post-silking root–shoot senescence[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3384-3402.
[3]	Tiantian Chen, Lei Li, Dan Liu, Yubing Tian, Lingli Li, Jianqi Zeng, Awais Rasheed, Shuanghe Cao, Xianchun Xia, Zhonghu He, Jindong Liu, Yong Zhang. Genome wide linkage mapping for black point resistance in a recombinant inbred line population of Zhongmai 578 and Jimai 22[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3311-3321.
[4]	Zuxian Chen, Bingbing Zhao, Yingying Wang, Yuqing Du, Siyu Feng, Junsheng Zhang, Luxiang Zhao, Weiqiang Li, Yangbao Ding, Peirong Jiao. H5N1 avian influenza virus PB2 antagonizes duck IFN-β signaling pathway by targeting mitochondrial antiviral signaling protein[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3614-3625.
[5]	Yang Sun, Yu Liu, Li Zhou, Xinyan Liu, Kun Wang, Xing Chen, Chuanqing Zhang, Yu Chen. Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC₂ and FfSdhD[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3511-3528.
[6]	Yufeng Xiao, Meiqi Dong, Xian Wu, Shuang Liang, Ranhong Li, Hongyu Pan, Hao Zhang. Enrichment, domestication, degradation, adaptive mechanism, and nicosulfuron bioremediation of bacteria consortium YM2[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3529-3545.
[7]	Yuxin He, Fei Deng, Chi Zhang, Qiuping Li, Xiaofan Huang, Chenyan He, Xiaofeng Ai, Yujie Yuan, Li Wang, Hong Cheng, Tao Wang, Youfeng Tao. Wei Zhou, Xiaolong Lei, Yong Chen, Wanjun Ren. Can a delayed sowing date improve the eating and cooking quality of mechanically transplanted rice in the Sichuan Basin, China?[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3368-3383.
[8]	Dili Lai, Md. Nurul Huda, Yawen Xiao, Tanzim Jahan, Wei Li, Yuqi He, Kaixuan Zhang, Jianping Cheng, Jingjun Ruan, Meiliang Zhou. *Evolutionary and expression analysis of sugar transporters from Tartary buckwheat revealed the potential function of FtERD23* in drought stress**[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3334-3350.
[9]	Zishuai Wang, Wangchang Li, Zhonglin Tang. Enhancing the genomic prediction accuracy of swine agricultural economic traits using an expanded one-hot encoding in CNN models[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3574-3582.
[10]	Yunji Xu, Xuelian Weng, Shupeng Tang, Weiyang Zhang, Kuanyu Zhu, Guanglong Zhu, Hao Zhang, Zhiqin Wang, Jianchang Yang. Untargeted lipidomic analysis of milled rice under different alternate wetting and soil drying irrigation regimes[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3351-3367.
[11]	Minghui Li, Yilan Chen, Siqiao Wang, Xueke Sun, Yongkun Du, Siyuan Liu, Ruiqi Li, Zejie Chang, Peiyang Ding, Gaiping Zhang. Plug-and-display nanoparticle immunization of the core epitope domain induces potent neutralizing antibody and cellular immune responses against PEDV[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3598-3613.
[12]	Jing Zhou, Bingshuai Du, Yibo Cao, Kui Liu, Zhihua Ye, Yiming Huang, Lingyun Zhang. Genome-wide identification of sucrose transporter genes in Camellia oleifera and characterization of CoSUT4[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3494-3510.
[13]	Yuheng Wang, Furong Kang, Bo Yu, Quan Long, Huaye Xiong, Jiawei Xie, Dong Li, Xiaojun Shi, Prakash Lakshmanan, Yueqiang Zhang, Fusuo Zhang. Magnesium supply is vital for improving fruit yield, fruit quality and magnesium balance in citrus orchards with increasingly acidic soil[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3641-3655.
[14]	Mingxin Feng, Ying Hu, Xin Yang, Jingwen Li, Haochen Wang, Yujia Liu, Haijun Ma, Kai Li, Jiayin Shang, Yulin Fang, Jiangfei Meng. Uncovering the miRNA-mediated regulatory network involved in postharvest senescence of grape berries[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3465-3483.
[15]	Li Liu, Yifeng Feng, Ziqi Han, Yaxiao Song, Jianhua Guo, Jing Yu, Zidun Wang, Hui Wang, Hua Gao, Yazhou Yang, Yuanji Wang, Zhengyang Zhao. *Functional analysis of the xyloglucan endotransglycosylase/hydrolase gene MdXTH2* in apple fruit firmness formation**[J]. >Journal of Integrative Agriculture, 2025, 24(9): 3418-3434.

No Suggested Reading articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed

Cite this article:

About JIA

Editorial board

For authors