Study finds chimeric mRNA induces potent and broadly acting neutralizing antibodies against SARS-CoV-2 variants

In a recent study posted to the bioRxiv* preprint server, researchers explored strategies to develop a messenger ribonucleic acid (mRNA)-based coronavirus disease 2019 (COVID-19) vaccine that could elicit safer and wider protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs).

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Background

Emerging SARS-CoV-2 VOCs threaten public health amid the ongoing COVID-19 pandemic. In a previous study, Puranik et al. showed that the effectiveness of existing mRNA vaccines decreased from ~95% for the original Washington (WA) strain to 76% and 42% against Delta VOC due to the mRNA-1273, and BNT162b2 vaccines, respectively.

Furthermore, there are safety concerns related to currently used mRNA vaccines. First, as demonstrated in a study by Peacock et al., 2021, mRNA vaccines encode a polybasic furin cleavage site (FCS) at the junction of S1 and S2 subunits that affects the stability of spike (S) protein and reduces the number of antigenic epitopes available to induce cellular and humoral immunity.

In another study by Ogata et al., 2022, they detected cleaved S1 in the blood of immunized subjects receiving an mRNA vaccine. Thus, raising concerns that this free moiety could mimick full-length S protein to activate angiotensin-converting enzyme 2 (ACE2) and trigger side effects, such as myocarditis.

Hence, there is an urgent need to develop more efficacious mRNA vaccines and optimize the existing ones to combat infection from current and potential future SARS-CoV-2 VOCs, including respiratory pathogens outside of the Coronaviridae family.

About the study

In the current study, researchers mutated FCS in the S protein of predominant VOCs, confirmed the expression and cleavage deficiency of furin mutants, and assessed the capacity of the resultant mRNAs to induce neutralizing antibodies (nAb) in mice following intramuscular administration.

To this end, they first screened five monovalent mRNA vaccines encoding the S protein of SARS-CoV-2 WA strain and VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2). They constructed two sets of mRNAs encoding the S protein of all the abovementioned SARS-CoV-2 VOCs, one with the wild-type (WT) FCS and the other with the mutated site, and examined the expression of these VOC mRNAs in 293T cells.

The flow cytometry results showed that removal of the FCS increased the surface expression of VOC S protein in transfected 293T cells. Western blot confirmed that all these vaccines harbored mutation that abolished the furin-mediated cleavage between S1 and S2 domains of S protein.

They formulated WT or furin mutant mRNAs with a lipid nanoparticle (LNP), intramuscularly injected each LNP-mRNA in six-week-old female BALB/c mice with two doses at three-week intervals.

They performed an enzyme-linked immunosorbent assay (ELISA) on the mice sera collected 14 days after boost (two-dose vaccine) to determine endpoint titer (EPT) of total binding antibody. Next, they performed the plaque reduction neutralization test (PRNT) and challenged VeroE6 cells with the live virus of five VOCs in the absence or presence of diluted serum collected from the immunized mice.

They also investigated the ability of individual SARS-CoV-2 VOC mRNA vaccines to generate nAb and profiled their coverage spectrum in vivo by immunizing six-week-old female BALB/c mice with LNP-encapsulated furin-mutant VOC mRNAs and compared the performance of individual mRNAs in vaccinated mice.

Past studies have extensively used K18-hACE2 transgenic mice to evaluate vaccine efficacy in preclinical trials. Following suit, the researchers used these mice to confirm the broad spectrum of generated nAb. These mice were immunized with 5μg of WA-Furin or Beta-Furin mRNA twice at three-week intervals; and five weeks after vaccination, challenged with 1 x 10⁵ half-maximal tissue culture infectious dose (TC_ID50) of the WA or Beta variants.

Lastly, the researchers also addressed the limitations of an Omicron VOC-specific mRNA vaccine. For this, they developed a bivalent vaccine having a chimeric mRNA by incorporating one additional receptor-binding domain (RBD) from the Delta variant into the intact S antigen of Omicron.

Study findings

In vaccinated mice, the individual VOC mRNAs induced the generation of nAbs in a VOC-specific manner, except the Gamma variant.

The nAbs produced in mice receiving the WA-Furin and Beta-Furin mRNAs cross-reacted with other VOCs, indicating that some VOC mRNA vaccines, especially Beta-Furin led to the production of antibodies capable of binding to S proteins of more than one VOCs. However, both these vaccines could not effectively neutralize Omicron.

ELISA results revealed that the mRNA carrying the furin cleavage mutation elicited a higher average EPT of total binding antibody than its WT version. PRNT results confirmed the improved neutralizing activity of sera from WA furin mutant-injected mice.

In the live virus challenge experiments, immunization with either WA-Furin or Beta-Furin mRNA almost totally inhibited the replication of the virus in the lungs, with virus titers falling below the limit of detection. None of the mice immunized with Beta- or WA-Furin vaccines showed any sign of weight loss, and some animals in these treatment groups even gained weight after infection up to five days.

Intriguingly, boosting mice with Omicron- and WA-Furin mRNA vaccines elicited almost similar protection against Omicron. Although the mRNA vaccine encoding the Omicron S induced the strongest protection against Omicron, it did not exhibit broad neutralizing capacity against other VOCs, such as Delta.

The chimeric mRNA vaccine elicited the most potent and broad-spectrum nAbs with significantly higher cross-neutralization activity against Delta; while retaining efficacy against Omicron.

Conclusions

Together, the study data indicated that while the VOC-specific strategy could provide SARS-CoV-2 strain-specific protection, some having relatively enhanced potential could trigger a broader and more potent immune response to the genetically divergent set of existing SARS-CoV-2 variants. Additionally, the in-house designed mRNA vaccine was much safer than existing vaccines.

More importantly, the chimeric vaccine design achieved the balance between effectiveness and coverage, not only for the variants of SARS-CoV-2 but also for other viruses. It remarkably restored the strong protection against Delta infection while retaining effective immunity against Omicron. This aspect of the chimeric mRNA vaccine candidate is also quite appealing as SARS-CoV-2 will keep mutating to annul the efficacy of existing mRNA vaccines.

Overall, chimeric mRNA-based vaccines pave the way to develop next-generation COVID-19 vaccine candidates that could target emerging SARS-CoV-2 variants and several other respiratory pathogens in the future.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources