Self-amplifying RNA and mRNA vaccines

As Pfizer-BioNTech and Moderna Inc. have recently demonstrated, synthetic RNA platforms allow for rapid, scalable, and cell-free manufacturing of vaccines based on optimized, modified mRNA. This approach utilizes in vitro transcription of antigen-encoding sequences or immunotherapies as synthetic RNA transcripts. In general, the formulation of the final mRNA vaccine products for delivery occurs in synthetic lipid nanoparticles.

A new type of future RNA based vaccines utilize self-amplifying RNA using genetically engineered replicons. These mRNA vaccines containing self-amplifying RNA sequences are expected to be the next generation vaccines for the treatment of viral infections and possibly cancer.

The new mRNA-based vaccines from Pfizer and Moderna against the coronavirus infection are nucleoside-modified messenger RNAs (modRNAs) encoding the viral spike glycoprotein (S) from the SARS-CoV-2 coronavirus. Both vaccines do not contain a live virus and also no self-amplifying RNA. The formulation for delivery of both vaccines occurred in nanoparticles (LNP). The Pfizer-BioNTech COVID-19 vaccine BNT162b2 is recommended for people aged 16 years and older. The Moderna COVID-19 vaccine mRNA-1273 is recommended for people 18 years and older.

Since DNA-based vaccines have performed poorly in human trials due to insufficient responses to elicit a significant clinical benefit, there is a renewed interest in RNA-based vaccines (Hobernik et al., 2018).

Currently, there are two types of synthetic vaccines in development: conventional or nonreplicating mRNA and self-amplifying RNA-based vaccines.

In vitro transcribed mRNAs encoding viral antigens have been explored as vaccines. mRNAs encoding therapeutic proteins are candidates for immunotherapy. The incorporation of chemically modified nucleotides, sequence optimization, and purification resulted in improved mRNA translation efficiency and reduced intrinsic immunogenic properties. Since antigen expression is proportional to the number of mRNA transcripts delivered during vaccination, the adequate expression of antigens for protection may need large doses or repeated administration of the vaccine.

Self-amplifying RNA vaccines are genetically engineered replicons, nucleic acid molecules replicating as units, derived from self-replicating single-stranded RNA viruses, address these limitations.

Figure 1: Example of the Semliki Forest virus-based expression systems (SFV). Schematic illustration of expression vectors. Red triangle, SP6 RNA polymerase promoter; orange triangle, Semliki Forest virus (SFV) 26S subgenomic promoter (Adapted from Lundstrom, 2020). An engineered expression vector carrying the SFV nonstructural protein genes (nsP1-4) and the gene of interest inserted downstream of the strong 26S sub-genomic promoter allows translation of in vitro transcribed RNA in cell lines and in vivo.

In 2013, Hekele et al. showed that it is possible to formulate self-amplifying RNA (saRNA) vaccines in LNPs within eight days. The so-called SAM® vaccine platform expressing seasonal influenza hemagglutinin utilizes a synthetic, self-amplifying mRNA that allowed mRNA delivery packaged in synthetic lipid nanoparticles (LNPs). 

Immunized mice showed measurable hemagglutinin inhibition and neutralizing antibody titers against the new virus within two weeks. After the second immunization, all mice had hemagglutinin inhibition titers considered as protective. These results are an indication that saRNA based vaccines are a new platform for designing vaccines useful as tools to stop newly emerging infectious viruses at the beginning of an outbreak.

Self-amplifying RNA vaccines encode 5′- and 3′- conserved sequence elements (CSEs), the nonstructural protein 1 to 4 (nsP1-4) genes, a subgenomic promoter, and the vaccine immunogen. After in situ translation, the nsP1-4 proteins form an RNA-dependent RNA polymerase (RdRP) complex that recognizes flanking CSE sequences and amplifies the vaccine-encoded transcript.

Biddlecome et al. recently reported a new gene delivery platform that illustrated the benefits of a self-amplifying (“replicon”) used in mRNA vaccines protected in a viral-protein capsid. The research group used a purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) for the in vitro assembly of monodispersed virus-like particles and tested it in mice. The researchers showed that immunization mice with this packaged mRNA activated dendritic cells and induced antigen-specific T-cell responses.

Single-stranded RNA viruses, such as alphaviruses, self-amplify their RNA in host cells. These characteristics make alphaviruses and flaviviruses attractive vehicles for vaccine development. Alphaviruses belonging to the family of Togaviruses such as Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE) have been used for the engineering of expression vector systems. Alphaviruses are positive-sense, single-stranded RNA viruses. The 32 known alphaviruses can infect humans, rodents, fish, birds, and larger mammals. Enveloped alphavirus particles have a diameter of approximately 70 nm and a spherical 40 nm isometric nucleocapsid. The genome of alphaviruses contains two open reading frames (ORFs), one nonstructural and one structural.  All alphaviruses share antigenic sites on the capsid and at least one envelope glycoprotein. However, the viruses can be differentiated by several serological tests, particularly neutralization assays.

Reference and Links

Alphavirus:  Medical Microbiology. 4th edition.   

Alphavirus Wiki https://en.wikipedia.org/wiki/Alphavirus 

Biddlecome A, Habte HH, McGrath KM, Sambanthamoorthy S, Wurm M, Sykora MM, Knobler CM, Lorenz IC, Lasaro M, Elbers K, Gelbart WM. Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles. PLoS One. 2019 Jun 4;14(6):e0215031. doi: 10.1371/journal.pone.0215031. PMID: 31163034; PMCID: PMC6548422. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548422/

COVID-19 vaccines and pregnancy:  https://www.verywellhealth.com/who-updates-guidance-pregnancy-covid-19-vaccine-5104967

FDA and COVID-19 https://www.fda.gov/news-events/press-announcements/fda-takes-additional-action-fight-against-covid-19-issuing-emergency-use-authorization-second-covid ]

Hekele A, Bertholet S, Archer J, Gibson DG, Palladino G, Brito LA, Otten GR, Brazzoli M, Buccato S, Bonci A, Casini D, Maione D, Qi ZQ, Gill JE, Caiazza NC, Urano J, Hubby B, Gao GF, Shu Y, De Gregorio E, Mandl CW, Mason PW, Settembre EC, Ulmer JB, Craig Venter J, Dormitzer PR, Rappuoli R, Geall AJ. Rapidly produced SAM(®) vaccine against H7N9 influenza is immunogenic in mice. Emerg Microbes Infect. 2013 Aug;2(8):e52. doi: 10.1038/emi.2013.54. Epub 2013 Aug 14. PMID: 26038486; PMCID: PMC3821287. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821287/

Hobernik D, Bros M. DNA Vaccines-How Far From Clinical Use? Int J Mol Sci. 2018 Nov 15;19(11):3605. doi: 10.3390/ijms19113605. PMID: 30445702; PMCID: PMC6274812. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274812/ 

Lundstrom K. Self-Amplifying RNA Viruses as RNA Vaccines. Int J Mol Sci. 2020 Jul 20;21(14):5130. doi: 10.3390/ijms21145130. PMID: 32698494; PMCID: PMC7404065.   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404065/

Lundstrom K. Application of Viral Vectors for Vaccine Development with a Special Emphasis on COVID-19. Viruses. 2020 Nov 18;12(11):1324. [PMC]

Moderna COVID-19 vaccine

Pfizer-BioNTech COVID-19 vaccine BNT162b2