In a recent study posted to the bioRxiv* server, researchers developed a messenger ribonucleic acid (mRNA) technology-based vaccine candidate against the monkeypox virus (MPXV) and evaluated its immunogenicity in animal models.

Background

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The mRNA vaccines, mRNA-1273 and BNT162b2, attained unexpectedly high efficacies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), successfully preventing the progression to severe coronavirus disease 2019 (COVID-19). Indeed, this technology is promising and might also work against a variety of pathogens, including MPXV.

Due to its fast-spreading nature, the World Health Organization (WHO) declared MPXV a public health emergency in July 2022. Like any other fast-spreading viral infection, containing MPXV requires vaccination apart from widescale testing and quarantining. However, amid high demand, the MPXV vaccine supply is outstripping the supply in many countries.

Currently, two MPXV vaccines are available, viz., JYNNEOS and ACAM2000. Clinical trials evaluating their efficacy are ongoing, and data showing their efficacy in humans is inadequate. MPXV, like the Vaccinia virus, belongs to the Orthopoxvirus genus. Proteomic analysis of antibody responses to both viruses has uncovered several shared immunodominant envelope proteins, such as A27L, A33R, D8L, L1R, and B5R. In fact, deoxyribonucleic acid (DNA) vaccines using these antigens have shown promise in containing fatal MPXV infections in mice.

Unfortunately, these vaccines also elicit  >=3 grade adverse event rate of 7.7% in its recipients. Conversely, this adverse event rate of COVID mRNA vaccines is much lower, 1.5% vs. 1.3% in the vaccine and placebo groups.  Removing the undesired proteins from currently used MPXV vaccines could also improve their safety profile.

On the other hand, mRNA vaccines are more scalable and easily manufactured in vitro with no need for complex cell cultures compared to attenuated vaccines. Though it is an attractive proposition to design an mRNA-based MPXV vaccine, whether it would trigger an adequate immune response in vivo remains unknown.

About the study

In the present study, researchers designed a polyvalent, lipid nanoparticle (LNP) encapsulated mRNA vaccine candidate, MPXVac-097, against 2022 MPXV clade B.1 and tested its antibody response and T cell receptor repertoire in vaccinated mice.

They used five MPXV antigens as neutralizing antibody targets in this vaccine, viz., A29, E8L, A35R, M1R, and B6R, linked in tandem by 2A peptides. In parallel, the researchers designed a reporter construct with a green fluorescent protein (GFP) appended to the end, identical to the vaccine. They quantified GFP expression in transfected cells using flow cytometry (FC), which indicated successful translation of all residues from one mRNA transcript in the majority of 293T cells.

This vaccine adopted the circulating MPXV sequence, and its E8L antigen blocked the ligand binding site in host cells while accommodating the negatively charged chondroitin sulfate ligand of host cells. The researchers encapsulated the transcribed MPXVac-097 mRNA vaccine in LNP and used dynamic light scattering (DLS) to determine its size distribution.

The researchers tested the initial immunogenicity of MPXVac-097 in mice. They immunized mice with three doses, prime, boost, and booster of 8µg MPXVac-097 each at an interval of two weeks. Then, they collected the retro-orbital blood of test animals on days zero, 14, and 28 (before vaccination) and days 20 and 42 (six and 14 days post boost and booster, respectively). The team used an enzyme-linked immunosorbent assay (ELISA) to quantify antibody titers against all five MPXV antigens in isolated blood plasma samples at different time points.

Furthermore, the researchers investigated whether they could present full-length MPXV antigens on the cell surface. Additionally, they enquired whether MPXVac-097-triggered plasma antibodies recognized these antigens. The researchers used bulk T cell receptor-sequencing (TCR-seq)  to characterize the T cell response in vaccinated animals.

Study findings

The LNP-encapsulated MPXVac-097 mRNA transcript had a polydispersity index of 0.16. During in vivo experiments, the researchers noted an insignificant increase in antibody titers against A35R and E8L antigens 14 days after the prime dose in 50% of mice. However, these titers increased substantially in all mice following the second and third vaccine doses. Intriguingly, the antibody titer against the M1R antigen increased moderately only in one mouse after boost and booster doses, not post-prime.

The antibody responses to A29L or B6R antigens remained minimal post-booster doses, indicating the differences in immunogenicities of all five antigens used in this mRNA vaccine design. Perhaps these differences in antigen immunogenicities or surface display properties also delayed antibody titers against M1R in one mouse. Notably, the M1R antigen is nested between A35R and E8L antigens on the same mRNA transcript, so the researchers expected its expression would be similar to A35R and E8L.

The expression of A35R and E8L antigens led to a 12.6% and 1.2% increase in 293T cells bound to the MPXV plasma antibodies, respectively. This observation validated the findings in ELISA, which also showed an antibody response to the surface presentation of A35R and E8L antigens.

The authors noted similar TCR repertoire compositions between all mice before vaccination. However, these changed substantially after two-dose MPXVac-097 vaccination, specifically between day 0 and day 20. Moreover, two-dose MPXVac-097 vaccinated mice exhibited substantially diminished clonal diversity of samples compared to vaccine-naïve animals. The simultaneous reduction and increase in low-frequency and hyperexpanded clones indicated clonal expansion triggered by MPXVac-097 vaccine.

Conclusions

To summarize, the multivalent LNP-encapsulated mRNA vaccine candidate, MPXVac-097, elicited adequate antibody titers in mice against a subset of MPXV antigens, i.e., only two (A35R and E8L) of five antigen-tested but also T cell response. Together, the study data showed the initial feasibility of the MPXV mRNA vaccine design and preliminary evidence of its functionality, opening avenues for its optimization in the future.

*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.

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.