Novel mRNA-lipid nanoparticle vaccine encoding four mpox virus surface proteins generates robust immune responses against orthopoxviruses

In a recent study posted to the bioRxiv* preprint server, researchers described a messenger ribonucleic acid (mRNA)-lipid nanoparticle mpox vaccine encoding four conserved mpox virus surface proteins which induced mpox virus-specific immunity and elicited heterologous protection when challenged with lethal vaccinia virus.

Study: A monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge. Image Credit: MIA Studio/Shutterstock


The global mpox outbreak in 2022 led to widespread infections in close to 30 countries outside the endemic distribution of the mpox virus. This strain of mbox virus was different from those responsible for previous outbreaks because of its human-to-human transmission ability. The mpox virus belongs to the Orthopoxvirus genus, which also contains the etiological agent of smallpox — the variola virus.

While the smallpox vaccine, which uses live-attenuated modified vaccinia virus, has been effective against the mpox virus, breakthrough mpox infections have been observed after the first and second doses of JYNNEOS, the modified vaccinia Ankara (MVA)-based vaccine. Furthermore, since smallpox vaccinations were discontinued in most countries after the eradication of the disease, only stockpiled vaccines were available, and challenges in manufacturing the vaccine added to the concern. Therefore, there is a pressing need for vaccines that elicit mbox virus-specific immune responses and possibly protect against other Orthopoxvirus infections.

About the study

In the present study, the researchers targeted a set of mbox virus surface proteins that are highly conserved in orthopoxviruses and can induce type 1 T helper cell (Th-1) dependent immune responses. in vivo. The selected immunogens included surface proteins from both the infectious Orthopoxvirus forms — the extracellular enveloped virion and the intracellular mature virion. Mpox virus antigens involved in cellular entry (M1 and A29) and transmission (A35 and B6), which share over 94% similarity with vaccinia virus antigens, were selected for the vaccine.

Of the four, M1 and A29 are found on the mature virion, and A35 and B6 are found on the enveloped virion. The mRNA encoding the four antigens were selected from the mbox virus clade II, which is responsible for the 2022 outbreak. The mRNA sequences were modified to include a signal peptide from an influenza hemagglutinin 1 region (M1 and A29), an N-terminal transmembrane region from influenza neuraminidase 2 region (A35), or truncated cytoplasmic tail (B6).

The lipid nanoparticle comprised four lipids — cholesterol, a new ionizable lipid SM-102, PEG2000-DMG, and 1,2-distearoyl-snglycero-3-phosphocholine (DSPC). Expi 293 suspension cells were used to assess the expression of the antigens encoded by the selected mRNAs. Lipid nanoparticles containing individual antigens or various combinations of the four antigens were intramuscularly injected twice into mouse models with a three-week gap. The control group of mice was injected with the MVA vaccine following the same regimen. The serum samples from the mice were analyzed using flow cytometry to detect neutralizing antibodies against the mpox virus. The binding antibody titers were also analyzed.

Fragment crystallizable (Fc) gamma receptors (FcgR) binding profiles and mpox virus-specific antibody isotypes induced by the quadrivalent mRNA vaccine were determined and compared to those induced by the MVA vaccine. Additionally, the ability of the mpox virus mRNAs to elicit cross-reactive antibodies against the vaccinia virus orthologs was also tested. Serum samples from mice models vaccinated with an individual, and combination mpox virus antigens were also tested for cross-reactive immune responses against the vaccinia virus orthologs. The immunized mice were also intranasally challenged with lethal doses of the vaccinia virus.


The results indicated that the mRNA-lipid nanoparticle vaccine elicited greater neutralizing and spread-inhibiting activity than the MVA vaccine against the mpox virus and vaccinia virus. The mRNA vaccine also induced a higher Th-1-biased humoral immune response with greater FcgR binding.

Messenger RNA-lipid nanoparticle vaccines with individual antigens provided partial protection in mice challenged with lethal doses of vaccinia virus. In contrast, mRNA vaccines with combinations of two or three antigens or all four antigens protect the mice against weight loss and death during the lethal vaccine virus challenge.

The cross-protection elicited by the multivalent mbox virus mRNA vaccine was remarkably higher than the immune response induced by the homologous MVA vaccine. Even a quarter dose of the quadrivalent mpox virus mRNA vaccine protected against weight loss during the lethal vaccinia virus challenge. The bi- and tri-valent mRNA vaccines also conferred sterilizing immunity during the vaccinia virus challenge. The individual M1 antigen mRNA vaccine protected against the mature virion form of the vaccinia virus, and the B6 antigen mRNA vaccine prevented the spread of the enveloped virion form.


The results indicated that the mRNA-lipid nanoparticle mpox vaccine with individual and combinations of four surface protein antigens confer broad immunity against orthopoxviruses, with bi-, tri-, and quadrivalent vaccines eliciting significantly higher Th-1 biased humoral immune responses against vaccinia virus than the MVA vaccine.

*Important notice

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

Journal reference:

  • Alec W Freyn, Caroline Atyeo, Patricia L Earl, Jeffrey L Americo, Gwo-Yu Chuang, Harini Natarajan, Tiffany R Frey, Jason Gall, Juan I Moliva, Ruth Hunegnaw, Guha Asthagiri Arunkumar, Clinton Ogega, Arshan Nasir, Hamilton Bennett, Joshua Johnson, Michael A Durney, Guillaume Stewart-Jones, Jay W Hooper, Tonya Colpitts, Galit Alter, Nancy J Sullivan, Andrea Carfi, and Bernard Moss. (2022). monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge. bioRxiv. doi:


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