Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks
Fowl cholera is caused by the bacterium <i>Pasteurella multocida</i>, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-09-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/11/9/1498 |
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| author | Nisachon Apinda Yongxiu Yao Yaoyao Zhang Anucha Muenthaisong Kanokwan Sangkakam Boondarika Nambooppha Amarin Rittipornlertrak Pongpisid Koonyosying Venugopal Nair Nattawooti Sthitmatee |
| author_facet | Nisachon Apinda Yongxiu Yao Yaoyao Zhang Anucha Muenthaisong Kanokwan Sangkakam Boondarika Nambooppha Amarin Rittipornlertrak Pongpisid Koonyosying Venugopal Nair Nattawooti Sthitmatee |
| author_sort | Nisachon Apinda |
| collection | DOAJ |
| description | Fowl cholera is caused by the bacterium <i>Pasteurella multocida</i>, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from <i>P. multocida</i> integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of <i>P. multocida</i> OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent <i>P. multocida</i> X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases. |
| first_indexed | 2024-03-10T21:52:12Z |
| format | Article |
| id | doaj.art-181091d13d3f41568dc8ef4a1aa31b70 |
| institution | Directory Open Access Journal |
| issn | 2076-393X |
| language | English |
| last_indexed | 2024-03-10T21:52:12Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj.art-181091d13d3f41568dc8ef4a1aa31b702023-11-19T13:19:48ZengMDPI AGVaccines2076-393X2023-09-01119149810.3390/vaccines11091498Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in DucksNisachon Apinda0Yongxiu Yao1Yaoyao Zhang2Anucha Muenthaisong3Kanokwan Sangkakam4Boondarika Nambooppha5Amarin Rittipornlertrak6Pongpisid Koonyosying7Venugopal Nair8Nattawooti Sthitmatee9Laboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandThe Pirbright Institute, Woking GU24 0NF, UKThe Pirbright Institute, Woking GU24 0NF, UKLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandThe Pirbright Institute, Woking GU24 0NF, UKLaboratory of Veterinary Vaccine and Biological Products, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50200, ThailandFowl cholera is caused by the bacterium <i>Pasteurella multocida</i>, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from <i>P. multocida</i> integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of <i>P. multocida</i> OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent <i>P. multocida</i> X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.https://www.mdpi.com/2076-393X/11/9/1498Cre-LoxCRISPR-Cas9HVTfowl choleraNHEJ<i>Pasteurella multocida</i> |
| spellingShingle | Nisachon Apinda Yongxiu Yao Yaoyao Zhang Anucha Muenthaisong Kanokwan Sangkakam Boondarika Nambooppha Amarin Rittipornlertrak Pongpisid Koonyosying Venugopal Nair Nattawooti Sthitmatee Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks Vaccines Cre-Lox CRISPR-Cas9 HVT fowl cholera NHEJ <i>Pasteurella multocida</i> |
| title | Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks |
| title_full | Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks |
| title_fullStr | Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks |
| title_full_unstemmed | Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks |
| title_short | Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks |
| title_sort | efficiency of nhej crispr cas9 and cre loxp engineered recombinant turkey herpesvirus expressing i pasteurella multocida i omph protein for fowl cholera prevention in ducks |
| topic | Cre-Lox CRISPR-Cas9 HVT fowl cholera NHEJ <i>Pasteurella multocida</i> |
| url | https://www.mdpi.com/2076-393X/11/9/1498 |
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