Safety and Immunogenicity of a Newcastle Disease Virus Vector-Based SARS-CoV-2 Vaccine Candidate, AVX/COVID-12-HEXAPRO (Patria), in Pigs
Jesús Horacio Lara-Puente, Juan Manuel Carreño, Weina Sun, Alejandro Suárez-Martínez, Luis Ramírez-Martínez, Francisco Quezada-Monroy, Georgina Paz-De la Rosa, Rosalía Vigueras-Moreno, Gagandeep Singh, Oscar Rojas-Martínez, Héctor Elías Chagoya-Cortés, David Sarfati-Mizrahi, Ernesto Soto-Priante, Constantino López-Macías, Florian Krammer, Felipa Castro-Peralta, Peter Palese, Adolfo García-Sastre, Bernardo Lozano-Dubernard, Jesús Horacio Lara-Puente, Juan Manuel Carreño, Weina Sun, Alejandro Suárez-Martínez, Luis Ramírez-Martínez, Francisco Quezada-Monroy, Georgina Paz-De la Rosa, Rosalía Vigueras-Moreno, Gagandeep Singh, Oscar Rojas-Martínez, Héctor Elías Chagoya-Cortés, David Sarfati-Mizrahi, Ernesto Soto-Priante, Constantino López-Macías, Florian Krammer, Felipa Castro-Peralta, Peter Palese, Adolfo García-Sastre, Bernardo Lozano-Dubernard
Abstract
Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were developed in record time and show excellent efficacy and effectiveness against coronavirus disease 2019 (COVID-19). However, currently approved vaccines cannot meet the global demand. In addition, none of the currently used vaccines is administered intranasally to potentially induce mucosal immunity. Here, we tested the safety and immunogenicity of a second-generation SARS-CoV-2 vaccine that includes a stabilized spike antigen and can be administered intranasally. The vaccine is based on a live Newcastle disease virus vector expressing a SARS-CoV-2 spike protein stabilized in a prefusion conformation with six beneficial proline substitutions (AVX/COVID-12-HEXAPRO; Patria). Immunogenicity testing in the pig model showed that both intranasal and intramuscular application of the vaccine as well as a combination of the two induced strong serum neutralizing antibody responses. Furthermore, substantial reactivity to B.1.1.7, B.1.351, and P.1 spike variants was detected. Finally, no adverse reactions were found in the experimental animals at any dose level or delivery route. These results indicate that the experimental vaccine AVX/COVID-12-HEXAPRO (Patria) is safe and highly immunogenic in the pig model. IMPORTANCE Several highly efficacious vaccines for SARS-CoV-2 have been developed and are used in the population. However, the current production capacity cannot meet the global demand. Therefore, additional vaccines-especially ones that can be produced locally and at low cost-are urgently needed. This work describes preclinical testing of a SARS-CoV-2 vaccine candidate which meets these criteria.
Keywords: COVID-19; HexaPro; NDV; Newcastle disease virus; SARS-CoV-2; coronavirus vaccine; pig model; pigs; prolines; spike; vaccine.
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References
- Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273. doi:10.1038/s41586-020-2012-7.
- Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W, China Novel Coronavirus Investigating and Research Team . 2020. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382:727–733. doi:10.1056/NEJMoa2001017.
- Krammer F. 2020. SARS-CoV-2 vaccines in development. Nature 586:516–527. doi:10.1038/s41586-020-2798-3.
- Pan-American Health Organization. 2021. PAHO Director warns that vaccines alone will not stop current COVID-19 surge. Pan-American Health Organization, Washington, DC. .
- Sun W, McCroskery S, Liu WC, Leist SR, Liu Y, Albrecht RA, Slamanig S, Oliva J, Amanat F, Schäfer A, Dinnon KH, Innis BL, García-Sastre A, Krammer F, Baric RS, Palese P. 2020. A Newcastle disease virus (NDV) expressing a membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine. Vaccines (Basel) 8:771. doi:10.3390/vaccines8040771.
- Sun W, Leist SR, McCroskery S, Liu Y, Slamanig S, Oliva J, Amanat F, Schäfer A, Dinnon KH, García-Sastre A, Krammer F, Baric RS, Palese P. 2020. Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate. EBioMedicine 62:103132. doi:10.1016/j.ebiom.2020.103132.
- DiNapoli JM, Kotelkin A, Yang L, Elankumaran S, Murphy BR, Samal SK, Collins PL, Bukreyev A. 2007. Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens. Proc Natl Acad Sci USA 104:9788–9793. doi:10.1073/pnas.0703584104.
- Nakaya T, Cros J, Park MS, Nakaya Y, Zheng H, Sagrera A, Villar E, Garcia-Sastre A, Palese P. 2001. Recombinant Newcastle disease virus as a vaccine vector. J Virol 75:11868–11873. doi:10.1128/JVI.75.23.11868-11873.2001.
- Park MS, Shaw ML, Munoz-Jordan J, Cros JF, Nakaya T, Bouvier N, Palese P, Garcia-Sastre A, Basler CF. 2003. Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins. J Virol 77:1501–1511. doi:10.1128/jvi.77.2.1501-1511.2003.
- Park MS, García-Sastre A, Cros JF, Basler CF, Palese P. 2003. Newcastle disease virus V protein is a determinant of host range restriction. J Virol 77:9522–9532. doi:10.1128/jvi.77.17.9522-9532.2003.
- Swayne DE, Suarez DL, Schultz-Cherry S, Tumpey TM, King DJ, Nakaya T, Palese P, Garcia-Sastre A. 2003. Recombinant paramyxovirus type 1-avian influenza-H7 virus as a vaccine for protection of chickens against influenza and Newcastle disease. Avian Dis 47:1047–1050. doi:10.1637/0005-2086-47.s3.1047.
- Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L, Greenbaum E, Rozenman-Yair S, Panet A, Libson E, Irving CS, Galun E, Siegal T. 2006. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther 13:221–228. doi:10.1016/j.ymthe.2005.08.016.
- Wagner S, Csatary CM, Gosztonyi G, Koch HC, Hartmann C, Peters O, Hernáiz-Driever P, Théallier-Janko A, Zintl F, Längler A, Wolff JE, Csatary LK. 2006. Combined treatment of pediatric high-grade glioma with the oncolytic viral strain MTH-68/H and oral valproic acid. APMIS 114:731–743. doi:10.1111/j.1600-0463.2006.apm_516.x.
- Hsieh CL, Goldsmith JA, Schaub JM, DiVenere AM, Kuo HC, Javanmardi K, Le KC, Wrapp D, Lee AG, Liu Y, Chou CW, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang N, Amengor D, Lavinder JJ, Ippolito GC, Maynard JA, Finkelstein IJ, McLellan JS. 2020. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369:1501–1505. doi:10.1126/science.abd0826.
- Lee KM. 1956. Propagation of transmissible gastroenteritis virus in tissue culture. Ann N Y Acad Sci 66:191–195. doi:10.1111/j.1749-6632.1956.tb40120.x.
- Bukreyev A, Huang Z, Yang L, Elankumaran S, St Claire M, Murphy BR, Samal SK, Collins PL. 2005. Recombinant Newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates. J Virol 79:13275–13284. doi:10.1128/JVI.79.21.13275-13284.2005.
- Amanat F, Stadlbauer D, Strohmeier S, Nguyen THO, Chromikova V, McMahon M, Jiang K, Arunkumar GA, Jurczyszak D, Polanco J, Bermudez-Gonzalez M, Kleiner G, Aydillo T, Miorin L, Fierer DS, Lugo LA, Kojic EM, Stoever J, Liu STH, Cunningham-Rundles C, Felgner PL, Moran T, García-Sastre A, Caplivski D, Cheng AC, Kedzierska K, Vapalahti O, Hepojoki JM, Simon V, Krammer F. 2020. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med 26:1033–1036. doi:10.1038/s41591-020-0913-5.
- Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, Tan J, Bhavsar D, Capuano C, Kirkpatrick E, Meade P, Brito RN, Teo C, McMahon M, Simon V, Krammer F. 2020. SARS-CoV-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup. Curr Protoc Microbiol 57:e100. doi:10.1002/cpmc.100.
- Tan CW, Chia WN, Qin X, Liu P, Chen MI, Tiu C, Hu Z, Chen VC, Young BE, Sia WR, Tan YJ, Foo R, Yi Y, Lye DC, Anderson DE, Wang LF. 2020. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat Biotechnol 38:1073–1078. doi:10.1038/s41587-020-0631-z.
- Amanat F, Thapa M, Lei T, Sayed Ahmed SM, Adelsberg DC, Carreno JM, Strohmeier S, Schmitz AJ, Zafar S, Zhou JQ, Rijnink W, Alshammary H, Borcherding N, Reiche AG, Srivastava K, Sordillo EM, van Bakel H, Personalized Virology Initiative, Turner JS, Bajic G, Simon V, Ellebedy AH, Krammer F. 2021. The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies and targets both the NTD and the RBD. medRxiv 2021.03.07.21253098. doi:10.1101/2021.03.07.21253098.
- McCallum M, De Marco A, Lempp FA, Tortorici MA, Pinto D, Walls AC, Beltramello M, Chen A, Liu Z, Zatta F, Zepeda S, di Iulio J, Bowen JE, Montiel-Ruiz M, Zhou J, Rosen LE, Bianchi S, Guarino B, Fregni CS, Abdelnabi R, Foo SC, Rothlauf PW, Bloyet LM, Benigni F, Cameroni E, Neyts J, Riva A, Snell G, Telenti A, Whelan SPJ, Virgin HW, Corti D, Pizzuto MS, Veesler D. 2021. N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell 184:2332–2347.e16. doi:10.1016/j.cell.2021.03.028.
- Amanat F, White KM, Miorin L, Strohmeier S, McMahon M, Meade P, Liu WC, Albrecht RA, Simon V, Martinez-Sobrido L, Moran T, García-Sastre A, Krammer F. 2020. An in vitro microneutralization assay for SARS-CoV-2 serology and drug screening. Curr Protoc Microbiol 58:e108. doi:10.1002/cpmc.108.
- Public Health England. 2021. Investigation of novel SARS-CoV-2 variant. Variant of concern 202012/01. Technical briefing 5. Public Health England, London, United Kingdom. .
- Davies NG, Abbott S, Barnard RC, Jarvis CI, Kucharski AJ, Munday JD, Pearson CAB, Russell TW, Tully DC, Washburne AD, Wenseleers T, Gimma A, Waites W, Wong KLM, van Zandvoort K, Silverman JD, CMMID COVID-19 Working Group, COVID-19 Genomics UK (COG-UK) Consortium, Diaz-Ordaz K, Keogh R, Eggo RM, Funk S, Jit M, Atkins KE, Edmunds WJ. 2021. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science 372:eabg3055. doi:10.1126/science.abg3055.
- Cele S, Gazy I, Jackson L, Hwa S-H, Tegally H, Lustig G, Giandhari J, Pillay S, Wilkinson E, Naidoo Y, Karim F, Ganga Y, Khan K, Balazs AB, Gosnell BI, Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A. 2021. Escape of SARS-CoV-2 501Y.V2 variants from neutralization by convalescent plasma. medRxiv 2021.01.26.21250224. .
- Tegally H, Wilkinson E, Giovanetti M, Iranzadeh A, Fonseca V, Giandhari J, Doolabh D, Pillay S, San EJ, Msomi N, Mlisana K, von Gottberg A, Walaza S, Allam M, Ismail A, Mohale T, Glass AJ, Engelbrecht S, Van Zyl G, Preiser W, Petruccione F, Sigal A, Hardie D, Marais G, Hsiao NY, Korsman S, Davies MA, Tyers L, Mudau I, York D, Maslo C, Goedhals D, Abrahams S, Laguda-Akingba O, Alisoltani-Dehkordi A, Godzik A, Wibmer CK, Sewell BT, Lourenço J, Alcantara LCJ, Kosakovsky Pond SL, Weaver S, Martin D, Lessells RJ, Bhiman JN, Williamson C, de Oliveira T. 2021. Detection of a SARS-CoV-2 variant of concern in South Africa. Nature 592:438–443. doi:10.1038/s41586-021-03402-9.
- Faria NR, Mellan TA, Whittaker C, Claro IM, Candido DDS, Mishra S, Crispim MAE, Sales FCS, Hawryluk I, McCrone JT, Hulswit RJG, Franco LAM, Ramundo MS, de Jesus JG, Andrade PS, Coletti TM, Ferreira GM, Silva CAM, Manuli ER, Pereira RHM, Peixoto PS, Kraemer MUG, Gaburo N, Camilo CDC, Hoeltgebaum H, Souza WM, Rocha EC, de Souza LM, de Pinho MC, Araujo LJT, Malta FSV, de Lima AB, Silva JDP, Zauli DAG, Ferreira ACS, Schnekenberg RP, Laydon DJ, Walker PGT, Schlüter HM, Dos Santos ALP, Vidal MS, Del Caro VS, Filho RMF, Dos Santos HM, Aguiar RS, Proença-Modena JL, Nelson B, Hay JA, Monod M, Miscouridou X, et al. . 2021. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science 372:815–821. doi:10.1126/science.abh2644.
- Csatary LK, Eckhardt S, Bukosza I, Czegledi F, Fenyvesi C, Gergely P, Bodey B, Csatary CM. 1993. Attenuated veterinary virus vaccine for the treatment of cancer. Cancer Detect Prev 17:619–627.
- Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, Flach B, O’Connell S, Bock KW, Minai M, Nagata BM, Andersen H, Martinez DR, Noe AT, Douek N, Donaldson MM, Nji NN, Alvarado GS, Edwards DK, Flebbe DR, Lamb E, Doria-Rose NA, Lin BC, Louder MK, O’Dell S, Schmidt SD, Phung E, Chang LA, Yap C, Todd JM, Pessaint L, Van Ry A, Browne S, Greenhouse J, Putman-Taylor T, Strasbaugh A, Campbell TA, Cook A, Dodson A, Steingrebe K, Shi W, Zhang Y, Abiona OM, Wang L, Pegu A, Yang ES, Leung K, Zhou T, Teng IT, Widge A, et al. . 2020. Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates. N Engl J Med 383:1544–1555. doi:10.1056/NEJMoa2024671.
- Corbett KS, Edwards D, Leist SR, Abiona OM, Boyoglu-Barnum S, Gillespie RA, Himansu S, Schäfer A, Ziwawo CT, DiPiazza AT, Dinnon KH, Elbashir SM, Shaw CA, Woods A, Fritch EJ, Martinez DR, Bock KW, Minai M, Nagata BM, Hutchinson GB, Bahl K, Garcia-Dominguez D, Ma L, Renzi I, Kong WP, Schmidt SD, Wang L, Zhang Y, Stevens LJ, Phung E, Chang LA, Loomis RJ, Altaras NE, Narayanan E, Metkar M, Presnyak V, Liu C, Louder MK, Shi W, Leung K, Yang ES, West A, Gully KL, Wang N, Wrapp D, Doria-Rose NA, Stewart-Jones G, Bennett H, Nason MC, Ruckwardt TJ, et al. . 2020. SARS-CoV-2 mRNA vaccine development enabled by prototype pathogen preparedness. bioRxiv doi:10.1101/2020.06.11.145920.
- Jackson LA, Anderson EJ, Rouphael NG, Roberts PC, Makhene M, Coler RN, McCullough MP, Chappell JD, Denison MR, Stevens LJ, Pruijssers AJ, McDermott A, Flach B, Doria-Rose NA, Corbett KS, Morabito KM, O’Dell S, Schmidt SD, Swanson PA, Padilla M, Mascola JR, Neuzil KM, Bennett H, Sun W, Peters E, Makowski M, Albert J, Cross K, Buchanan W, Pikaart-Tautges R, Ledgerwood JE, Graham BS, Beigel JH, mRNA-1273 Study Group . 2020. An mRNA vaccine against SARS-CoV-2 - preliminary report. N Engl J Med 383:1920–1931. doi:10.1056/NEJMoa2022483.
- Walsh EE, Frenck R, Falsey AR, Kitchin N, Absalon J, Gurtman A, Lockhart S, Neuzil K, Mulligan MJ, Bailey R, Swanson KA, Li P, Koury K, Kalina W, Cooper D, Fontes-Garfias C, Shi P-Y, Türeci Ö, Thompkins KR, Lyke KE, Raabe V, Dormitzer PR, Jansen KU, Sahin U, Gruber WC. 2020. RNA-based COVID-19 vaccine BNT162b2 selected for a pivotal efficacy study. medRxiv 2020.08.17.20176651. doi:10.1101/2020.08.17.20176651.
- Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, Liu J, Peter L, McMahan K, Tostanoski LH, He X, Martinez DR, Rutten L, Bos R, van Manen D, Vellinga J, Custers J, Langedijk JP, Kwaks T, Bakkers MJG, Zuijdgeest D, Huber SKR, Atyeo C, Fischinger S, Burke JS, Feldman J, Hauser BM, Caradonna TM, Bondzie EA, Dagotto G, Gebre MS, Hoffman E, Jacob-Dolan C, Kirilova M, Li Z, Lin Z, Mahrokhian SH, Maxfield LF, Nampanya F, Nityanandam R, Nkolola JP, Patel S, Ventura JD, Verrington K, Wan H, Pessaint L, Ry AV, Blade K, Strasbaugh A, Cabus M, et al. . 2020. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature 586:583–588. doi:10.1038/s41586-020-2607-z.
- Gerdts V, Wilson HL, Meurens F, van Drunen Littel-van den Hurk S, Wilson D, Walker S, Wheler C, Townsend H, Potter AA. 2015. Large animal models for vaccine development and testing. ILAR J 56:53–62. doi:10.1093/ilar/ilv009.
- Vergara-Alert J, Rodon J, Carrillo J, Te N, Izquierdo-Useros N, Rodríguez de la Concepción ML, Ávila-Nieto C, Guallar V, Valencia A, Cantero G, Blanco J, Clotet B, Bensaid A, Segalés J. 2021. Pigs are not susceptible to SARS-CoV-2 infection but are a model for viral immunogenicity studies. Transbound Emerg Dis 68:1721–1725. doi:10.1111/tbed.13861.
- Laude H, Van Reeth K, Pensaert M. 1993. Porcine respiratory coronavirus: molecular features and virus-host interactions. Vet Res 24:125–150.
- Pensaert MB, Martelli P. 2016. Porcine epidemic diarrhea: a retrospect from Europe and matters of debate. Virus Res 226:1–6. doi:10.1016/j.virusres.2016.05.030.
- Zhou YL, Ederveen J, Egberink H, Pensaert M, Horzinek MC. 1988. Porcine epidemic diarrhea virus (CV 777) and feline infectious peritonitis virus (FIPV) are antigenically related. Arch Virol 102:63–71. doi:10.1007/BF01315563.
- Zhou P, Fan H, Lan T, Yang XL, Shi WF, Zhang W, Zhu Y, Zhang YW, Xie QM, Mani S, Zheng XS, Li B, Li JM, Guo H, Pei GQ, An XP, Chen JW, Zhou L, Mai KJ, Wu ZX, Li D, Anderson DE, Zhang LB, Li SY, Mi ZQ, He TT, Cong F, Guo PJ, Huang R, Luo Y, Liu XL, Chen J, Huang Y, Sun Q, Zhang XL, Wang YY, Xing SZ, Chen YS, Sun Y, Li J, Daszak P, Wang LF, Shi ZL, Tong YG, Ma JY. 2018. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature 556:255–258. doi:10.1038/s41586-018-0010-9.
- Mora-Díaz JC, Piñeyro PE, Rauh R, Nelson W, Sankoh Z, Gregg E, Carrillo-Ávila JA, Shen H, Nelli RK, Zimmerman JJ, Giménez-Lirola LG. 2021. Porcine hemagglutinating encephalomyelitis virus infection. J Virol 95:e02335-20. doi:10.1128/JVI.02335-20.
- Jung K, Hu H, Eyerly B, Lu Z, Chepngeno J, Saif LJ. 2015. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs. Emerg Infect Dis 21:650–654. doi:10.3201/eid2104.141859.
- Li G, Chen Q, Harmon KM, Yoon KJ, Schwartz KJ, Hoogland MJ, Gauger PC, Main RG, Zhang J. 2014. Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734. Genome Announc 2:e00278-14. doi:10.1128/genomeA.00278-14.
- Kong D, Wen Z, Su H, Ge J, Chen W, Wang X, Wu C, Yang C, Chen H, Bu Z. 2012. Newcastle disease virus-vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs. Virology 432:327–335. doi:10.1016/j.virol.2012.06.001.
- Zhang H, Nan F, Li Z, Zhao G, Xie C, Ha Z, Zhang J, Han J, Xiao P, Zhuang X, Wang W, Ge J, Tian M, Lu H, Bu Z, Jin N. 2019. Construction and immunological evaluation of recombinant Newcastle disease virus vaccines expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP3/GP5 proteins in pigs. Vet Microbiol 239:108490. doi:10.1016/j.vetmic.2019.108490.
- Kumar R, Kumar V, Kekungu P, Barman NN, Kumar S. 2019. Evaluation of surface glycoproteins of classical swine fever virus as immunogens and reagents for serological diagnosis of infections in pigs: a recombinant Newcastle disease virus approach. Arch Virol 164:3007–3017. doi:10.1007/s00705-019-04425-4.
- Sparrow E, Wood JG, Chadwick C, Newall AT, Torvaldsen S, Moen A, Torelli G. 2021. Global production capacity of seasonal and pandemic influenza vaccines in 2019. Vaccine 39:512–520. doi:10.1016/j.vaccine.2020.12.018.
- US Department of Agriculture. 2020. 9 CFR. Ch. I (1–1–20 edition) 113.44. Swine safety test. Animal and Plant Health Inspection Service, US Department of Agriculture, Riverdale, MD.
- Ciprián A, Pijoan C, Cruz T, Camacho J, Tórtora J, Colmenares G, López-Revilla R, de la Garza M. 1988. Mycoplasma hyopneumoniae increases the susceptibility of pigs to experimental Pasteurella multocida pneumonia. Can J Vet Res 52:434–438.
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