Babesia gibsoni, a unicellular eukaryotic parasite causing babesiosis in dog, is primarily transmitted through tick feeding. The intraerythrocytic stage, during which the parasite reproduce within the host's red blood cells, is a vital part of Babesia's life cycle. Continuous in vitro culture B. gibsoni provides an opportunity to study its biological processes. The establishment and development of gene editing systems for Babesia offer a powerful tool to investigate the functions of important genes in specific biological processes. This protocol expands on the existing techniques for in vitro culture and genes editing of B. gibsoni. Specifically, we describe a continuous in vitro culture method employing VP-SFM as a base medium, supplemented with Albumax I and small amount of canine serum (2.5 %), This method, designed for long-term culture, achieving high parasitemia and facilitates subclone culture. By employing homology-dependent repair pathways, the gene editing method utilizing introducing homologous fragments and electroporation can effectively manipulate the genetic of B. gibsoni. This protocol would contribute to the reproducibility of experiments and the overall reliability of research findings.
The objective of this study was to determine whether a new combination of immunogens could be more effective than the S–2P design in terms of eliciting an immune response. The study aimed to use a unified formulation standard to make a comparison between the new immunogen combination than the S–2P design.
The study analyzed the published immunogen mutation strategies of known COVID-19 vaccines and also Spike protein variants in the RCSB database to identify the most promising immunogen combination. By choosing different Spike protein variants, we prepared well characterized mRNA preparations and administered them to BALB/C mice using a commercial lipid for encapsulation.
The study found that our mRNA preparations stimulated strong humoral and cellular immunity, with a neutralizing antibody titer of >1*104 at 28 days and a Th1-biased cellular immune response. Furthermore, our results indicate that the S–6P-GSAS variant elicits superior immunogenicity at lower doses compared to the S–2P variant.
Our study suggests that the S–6P-GSAS variant may elicit a stronger immune response at lower doses compared to the S–2P design, indicating its potential as a promising immunogen candidate for COVID-19 vaccines. Further research is needed to explore the efficacy of this novel combination in addressing the challenges posed by emerging Spike protein variants.