Exploring simultaneous and sequential COVID-19 vaccine candidate

In a recent study posted to the bioRxiv* pre-print server, researchers developed novel lipid nanoparticle (LNP)-based messenger ribonucleic acid (mRNA) vaccine formulations against three pathogenic coronaviruses (CoVs), including SARS, Middle Eastern respiratory syndrome-CoV (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

It is crucial to develop pan CoV vaccines to prevent the reemergence of pre-existing pathogens and prepare for future CoV outbreaks. Previous studies have demonstrated the initial feasibility of chimeric mRNA vaccine constructs against other virus families, such as influenza, herpes simplex virus, and cytomegalovirus. However, the chimeric vaccine candidates usually lose critical antigenic regions for one or the other viral species they target.

Therefore, it is important to test the multiplexing of chimeric mRNA vaccines for CoV species and examine their immunogenicity. More importantly, ascertain an optimal vaccination schedule for a multiplexed vaccine is critical. In other words, it is important to understand whether administration of all vaccines simultaneously or spacing out different mRNA vaccine shots would perform better.

About the study

In the present study, researchers systematically examined immune responses of species-specific LNP-mRNA vaccines in combination (or mixture) and sequence in animal models. The vaccines encoded full-length spikes (S) of SARS-CoV-2 Delta variant, SARS, and MERS.

The team prepared and encapsulated an equal-mass mixture of S mRNA of Delta, SARS, and MERS in LNPs to engineer triplex-CoV, with an averaged radius of 71 ± 3.6 137 nm and a polydispersity index of 0.157. The triplex-CoV generated functional proteins in mammalian cells. It also successfully bound human angiotensin-converting enzyme 2 (hACE2) for SARS-CoV-2 and human dipeptidyl peptidase 4 (hDPP4) for MERS. Parallelly, they tested a Delta singlet LNP-mRNA vaccine formulation.

The researchers used dynamic light scatter and transmission electron microscope to evaluate the size and homogeneity of the assembled LNPs. The team intramuscularly vaccinated C57BL mice with primary and booster doses of one µg Delta LNP mRNA vaccine and one or three µg of triplex-CoV three weeks apart. Two weeks after boosting, they collected the peripheral blood mononuclear cells (PBMCs) and plasma from test animals to evaluate antibody response against S antigens present in these vaccine formulations.

Furthermore, the researchers performed single-cell RNA-sequencing (scRNA-seq) to investigate the systemic immune repertoires of the vaccinated animals. The researchers visualized the uniform manifold approximation and projection (UMAP) for 12 test animals in four vaccinated groups. The animals in the control group received phosphate buffer saline, and test animals received dosages of one µg Delta LNP-mRNA vaccine and one and three µg dosage of the triplex-CoV vaccine.

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

Lastly, the researchers performed differential expression gene (DEG) analysis to examine the transcriptomic changes in the matched immune cell sub-populations post-vaccination, particularly all identified activated B cells, a cluster of differentiation (CD4), and CD8 T cell subsets.

Study findings

The triplex LNP-mRNA vaccine induced a significantly increased level of activated B cell populations at low and high dosages, which elicit adaptive immunity against SARS-CoV-2. In fact, activated B cells, unswitched memory B cells, and natural killer (NK) cells showed significant differences between groups at a gross cell population level. A series of the gene set and pathway analyses revealed several altered pathways related to B cells, CD4 T cells, and CD8 T cells across three vaccinated groups.

Indeed, the triplex vaccination-induced strong B cell activation pathways in B cells and immune cell differentiation and metabolic activity gene sets in T cells across all three vaccination groups. This data indicated broad changes in gene expression signatures at the pathway and cluster levels across the B and T cells' transcriptomes of the animals receiving multiplexed vaccination.

Sequential vaccination separated vaccinations of SARS-CoV-2 Delta, MERS-CoV, and SARS by three weeks. However, the vaccine dosage remained the same, i.e., one μg LNP-mRNA prime and one μg LNP-mRNA boost.

Compared to the mixture vaccination group, animals in the sequential vaccination group showed significantly higher antibody responses across all antigens from SARS-CoV-2 Delta, MERS-CoV, and SARS. They also exhibited substantially higher neutralization activities across all three viral pathogens. This data clearly showed the superiority of sequential vaccination in eliciting more potent antibody responses than vaccination with a mixture simultaneously.


Pathogenic CoVs shall continue to infect humans in the future. Moreover, unfortunately, immunity induced by the currently used coronavirus disease 2019 vaccines wanes quickly. Hence, the multiplexed vaccination against two or more species is critical in the future.

The present study demonstrated a direct comparison between simultaneous and sequential vaccination schedules of LNP-mRNA vaccine formulations and showed it provided broad protective antibody immunity against all three highly pathogenic CoVs. The findings also offered invaluable insights regarding vaccination schedule optimization. The next-generation pan-CoV vaccines should seek the right balance between the breadth and depth of elicited immune protection.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.


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