Self-replicating intradermal RNA vaccine against SARS-CoV-2

In a recent study posted to the bioRxiv* preprint server, researchers assessed the efficiency of a controllable self-replicating ribonucleic acid (c-srRNA) vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The present SARS-CoV-2 pandemic encouraged the development of new vaccine types as well as the adaptation of existing vaccination platforms to SARS-CoV-2. The messenger RNA (mRNA)-based vaccines created by Pfizer/BioNTech and Moderna, in particular, were among the first to be made public and were used extensively. srRNA has also been employed as an alternative RNA vaccine technique. In contrast to mRNA, srRNA encodes both a gene or genes of interest and an RNA-dependent RNA polymerase, offering further benefits, including prolonged expression and minimizing the usage of modified nucleotides.

Study: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity. Image Credit: NIAIDStudy: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity. Image Credit: NIAID

About the study

Using c-srRNA as a platform, the team created a vaccine against SARS-CoV-2 and demonstrated that when applied intradermally, c-srRNA had the desirable properties required in a T-cell-inducing vaccine.

The team developed an initial RNA vector according to the widely used TRD strain along with a few minor sequence alterations, including A551D and P1308S, which are also present in other srRNA vectors, and a 3'-UTR truncation that is shorter than that in conventional srRNA vectors. A srRNA that can optimally function at approximately 33°C was produced by mutating the nonstructural proteins of the first RNA vector and testing the expression levels noted at 33°C and 37°C corresponding to the gene of interest encoded in the sub-genomic region. This in vitro transcribed mRNA was termed c-srRNA, particularly c-srRNA1.

The team intradermally injected mice with c-srRNA1 that encoded the luciferase gene (c-srRNA1-LUC) to determine whether c-srRNA1 functioned in vivo in the skin. 5-methoxyuridine (5moU)-modified synthetic mRNA encoding LUC (mRNA-LUC) was employed as a control. Using a bioluminescent imaging device, luciferase activity was detected and measured. Furthermore, the researchers created a c-srRNA1 that expressed the SARS-CoV-2 spike protein receptor-binding domain (RBD) to assess the viability of c-srRNA1 vectors for vaccination.

Mice were initially intradermally immunized with protein to determine if a c-srRNA vaccine could be employed as a booster dose. The mice were then administered intradermal injections of the RBD protein plus adjuvant, EXG-5003, EXG-5003o (c-srRNA3 encoding the SARS-CoV-2 Omicron variant RBD), or a placebo after fourteen days.


The expression of luciferase in vivo was demonstrated by luciferase imaging due to the intradermal injection of naked RNA encoding luciferase. However, the c-srRNA1-LUC-driven luciferase expression persisted in vivo for almost a month, presumably supported by its self-replication. However, the in vivo expression of LUC induced by mRNA-LUC was only present for one week. In addition, the expression level of LUC was 10- to 100-fold higher in recipients of c-srRNA1-LUC than it was in recipients of mRNA-LUC.

Notably, luciferase expression was not found in mouse body parts, like the internal organ or in the uninjected areas of the receivers' skin. Direct injection into skeletal muscle also failed to produce any luciferase activity. This finding suggested that the temperature-sensitive c-srRNA1-LUC did not show any replication or expression of luciferase under non-permissive conditions.

IFN-secreting cells, which are indicative of type 1 CD4+ T helper (Th) cells and CD8+ cytotoxic T cells, were produced by the EXG-5003 RNA vaccine and induced cellular immunity against the SARS-CoV-2 RBD. The EXG-5003 vaccination, in contrast, very faintly stimulated IL4-secreting cells, which are typical of type 2 CD4+ (Th cells. The findings demonstrated that intradermal delivery of EXG-5003 induced a Th1 dominant cellular immune response against SARS-CoV-2 RBD, a positive characteristic for a vaccine designed to prevent viral disease.

Contrary to the RBD (1st) + PBO (2nd) group, the RBD (1st) + RBD (2nd) group was able to elicit cellular immunity. This showed that while a single intradermal injection of the c-srRNA vaccination is adequate to elicit cellular immunity, a single dose of the intradermal protein vaccine generated little to no cellular immunity. Furthermore, cellular immunity was induced by the RBD (1st) + EXG-5003 (2nd) and EXG-5003o (2nd) groups. Only a very mild induction of antibodies was observed with the first immunization with the RBD protein and adjuvant. On the other hand, the ability of c-srRNA vaccinations to generate antibodies was comparable to that of a second immunization with the adjuvanted protein. Therefore, c-srRNA vaccines can serve as adjuvants for cellular immunity as well as humoral immunity.


The study findings demonstrated the development of a SARS-CoV-2 booster vaccine using c-srRNA that incorporated spike RBDs as viral surface proteins and conserved nucleoproteins as viral non-surface proteins. The researchers believe that this vaccine can potentially immunize recipients against SARS-CoV-2 and its variants while providing an important approach toward activating cellular immunity against several pathogens.

*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:
Bhavana Kunkalikar

Written by

Bhavana Kunkalikar

Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.


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