Study: Simultaneous and sequential multi-species coronavirus vaccination. Image Credit: LookerStudio/Shutterstock

Exploring a concurrent and sequencing COVID-19 candidate vaccine

In a recent study published in bioRxiv* Prepress server, researchers have developed new vaccine formulations of lipophilic nanoparticles (LNP) ribonucleic acid (mRNA) against three pathogenic coronaviruses (CoVs), including SARS, MERS-CoV (MERS-CoV), and acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Study: Simultaneous and sequential vaccination against a multispecies coronavirus. Image Credit: LookerStudio / Shutterstock

It is critical to develop comprehensive virus vaccines to prevent re-emergence of pre-existing pathogens and prepare for future outbreaks of the Coronavirus. Previous studies demonstrated the preliminary feasibility of mock RNA vaccine formulations against other virus families, such as influenza, herpes simplex virus and cytomegalovirus. However, chimeric vaccine candidates usually miss out on critical antigenic regions for one of the viral species they target.

Therefore, it is important to test the multiplex of placebo RNA vaccines for MERS-CoV species and to examine their immunogenic potential. Importantly, ensuring the optimal vaccination schedule for a multiplexed vaccine is critical. In other words, it is important to understand whether giving all of the vaccines simultaneously or spacing out different shots of the mRNA vaccine would perform better.

about studying

In this study, researchers systematically examined immune responses to species-specific LNP-mRNA vaccines in combination (or mixture) and sequenced in animal models. Vaccines encode the full-length (S) spikes of the delta variant SARS-CoV-2, SARS, and MERS.

The team prepared and encapsulated an isoelectric mixture of S mRNA for Delta, SARS, and MERS into LNPs to engineer triple CoV, with an average radius of 71 ± 3.6 137 nm and a multiple dispersion index of 0.157. Tri-CoV produced functional proteins in mammalian cells. It also successfully binds human angiotensin-converting enzyme 2 (hACE2) for SARS-CoV-2 and human dipeptide 4 (hDPP4) for MERS. In parallel, they tested the combination of a single LNP-mRNA delta vaccine.

The researchers used dynamic light scattering and transmission electron microscopy to assess the size and homogeneity of the aggregated LNPs. The team inoculated C57BL mice intramuscularly with initial and booster doses of one microgram of Delta LNP mRNA and one or three micrograms of triplex-CoV for three weeks. After two weeks of boosting, they collected peripheral blood mononuclear cells (PBMCs) and plasma from test animals to assess the antibody response against the S antigens present in these vaccine formulations.

Furthermore, the researchers performed single-cell RNA sequencing (scRNA-seq) to investigate the systemic immune repertoire of vaccinated animals. The researchers visualized the Uniform Forked Adduction and Projection (UMAP) of 12 test animals in four immunized groups. Animals in the control group received phosphate-buffered saline, and test animals received doses of one μg delta LNP-mRNA and one and three μg doses of the DPT vaccine.

Furthermore, they sequenced transcriptomes from 91,526 single cells to identify distinct immune cell populations in the vaccinated animals. More specifically, the team quantified the portions of each type of immune cell in all of the vaccination groups.

Finally, the authors performed a differential expression gene analysis (DEG) to examine the transcriptional changes in the matched immune cell subsets after vaccination, in particular all selected active B cells, differentiation group (CD4), and CD8 T cell subsets.


The LNP-mRNA triple vaccine significantly increased the level of active B-cell populations at low and high doses, resulting in adaptive immunity against SARS-CoV-2. Indeed, activated B cells, non-transformed memory B cells, and natural killer (NK) cells showed significant differences between groups at the level of total cell number. A series of gene cluster and pathway analyzes revealed several altered pathways related to B cells, CD4 T cells and CD8 T cells across three immune populations.

Indeed, the strong B-cell activation pathways induced by triple vaccination in B cells, immune cell differentiation and T-cell metabolic activity genes in all three vaccination groups. These data indicated extensive changes in gene expression signatures at the pathway and mass levels across B and T cell transcription of animals receiving multiplex vaccination.

Sequential vaccination separated the vaccinations against SARS-CoV-2 Delta, MERS-CoV, and SARS for three weeks. However, the vaccine dose remained the same, i.e., 1 μg of prime LNP-mRNA and 1 μg of LNP-mRNA.

Compared to the mixture vaccination group, animals in the sequential vaccination group showed significantly higher antibody responses across all antigens of SARS-CoV-2 Delta, MERS-CoV and SARS. They also showed significantly higher neutralization activities across the three viral pathogens. These data clearly demonstrated the superiority of sequential vaccination in obtaining stronger antibody responses than vaccination with a simultaneous mixture.


Pathogenic viruses should continue to infect humans in the future. Moreover, unfortunately, the immunity caused by the 2019 coronavirus vaccines currently in use is waning rapidly. Hence, multiplex vaccination against two or more species is critical in the future.

The current study demonstrated a direct comparison of simultaneous and sequential vaccination schedules of LNP-mRNA vaccine formulations and showed that they provide protective antibody immunity against all three pathogenic viruses. The results also provided invaluable insights into optimizing the vaccination schedule. Next-generation coronavirus vaccines must strike the right balance between the breadth and depth of immune protection that is obtained.

*Important note

bioRxiv publishes preliminary scientific reports that are not subject to peer review, and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.

Journal reference:

  • Lei Peng, Zhenhao Fang, Paul A Renauer, Andrew McNamara, Jonathan J Park, Qianqian Lin, Xiaoyu Zhou, Matthew B Dong, Biqing Zhu, Hongyu Zhao, Craig B Willen and Sidi Chen. (2022). Simultaneous and sequential vaccination of multispecies MERS-CoV. bioRxiv. bang:

2022-05-10 15:58:00

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