Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a
Liang Yu1, Wang Yongbo1, Yang Shengjun1
1Department of Clinical Laboratory, The First People's Hospital of Yunnan Province, Kunming, China.
Background:
Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector.
Methods:
In this study, the major antigen genes P1a of MP adhesion factor P1(3862-4554 bases) and P30a of P30(49-822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS-P1a or NS-P30a. The recombinant pHW2000 plasmids containing NS-P1a or NS-P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU-P1a and rFLU-P30a were rescued. RT-PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU-P1a and rFLU-P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy.
Results:
P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT-PCR identification of the recombinant viruses rFLU-P1a and rFLU-P30a showed that P1a (693 bp), P30a (774 bp), NS-P1a (1992bp) and NS-P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins.
Conclusion:
The recombinant viruses rFLU-P1a and rFLU-P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU-P1a or rFLU-P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human.
Insights
Researchers developed a novel Mycoplasma pneumoniae (MP) vaccine using a genetically engineered influenza virus vector. This approach aims to overcome challenges with current MP vaccines and improve immunogenicity for better respiratory health outcomes.
Area of Science:
- Virology
- Vaccinology
- Molecular Biology
Background:
- Mycoplasma pneumoniae (MP) is a significant respiratory pathogen impacting vulnerable populations like the elderly and children.
- Existing inactivated or attenuated MP vaccines exhibit poor immunogenicity and potential side effects, hindering successful human vaccine development.
- There is a critical need for innovative MP vaccine strategies, such as genetic engineering approaches utilizing viral vectors.
Purpose of the Study:
- To construct recombinant influenza viruses expressing key Mycoplasma pneumoniae antigens for potential vaccine development.
- To assess the genetic stability and characteristics of these novel recombinant viral vectors.
Main Methods:
- Major MP antigen genes (P1a and P30a) were inserted into the nonstructural (NS) gene of Influenza A virus strain PR8, creating recombinant vectors.
- Recombinant viruses (rFLU-P1a and rFLU-P30a) were rescued in chicken embryos after plasmid transfection.
- RT-PCR, sequencing, hemagglutination assays, and electron microscopy were employed for identification, genetic stability assessment, and morphological analysis.
Main Results:
- Successful construction and identification of recombinant viruses rFLU-P1a and rFLU-P30a carrying MP antigen genes were confirmed by RT-PCR and sequencing.
- The recombinant viruses demonstrated relative genetic stability over five generations, maintaining hemagglutination titers.
- Electron microscopy revealed typical and complete influenza virus morphology for the recombinant strains.
Conclusions:
- Recombinant influenza viruses successfully expressing Mycoplasma pneumoniae immunogenic genes (P1a and P30a) were constructed and validated.
- The developed recombinant viruses exhibit promising genetic stability and characteristic viral morphology.
- This study provides a foundational platform for the future development of effective Mycoplasma pneumoniae vaccines for human use.
