A thermostable messenger RNA based vaccine against rabies

Abstract

Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

Fig 1. RABV-G mRNA vaccine stored at temperatures up to +40°C retains immunogenicity and protective for up to twelve months.

(A-B: storage for six months). Vaccine or control injections were done on day 0 and 21. Induction of rabies virus neutralizing antibodies was measured on day 35 in mice vaccinated with RABV-G mRNA stored from -80°C to +40°C or HDC stored at the recommended temperature of 4°C for six months (dashed line in A and C indicates the WHO recommended titer of 0.5 international units per ml [IU/ml]). (C, D: storage for twelve months) Mice were challenged with 40fold MLD₅₀ of rabies virus CVS-11, 71 (A, B) or 63 (C, D) days after the first immunization. Body weight was recorded after immunization using material stored for six (B) or (D) twelve months. For antibody titers, the mean is indicated, for body weight kinetics the mean and standard deviation is given (n = 5). Significance ** p < .001 to buffer control group was calculated using the Mann Whitney test.

Fig 2. RABV-G mRNA vaccine stored at temperatures up to 70°C retains immunogenicity and protection for up to three months.

mRNA vaccine or licensed vaccines were stored for one month (A-F) or three months (G-I). Storage temperature was 60°C (A-C), 70°C (D-F), or as indicated in the figure (G-I). Vaccine or control injections were done on day 0 and 21. Induction of rabies virus neutralizing antibodies was measured 35 days after first immunization (A, D, G; dashed line indicates the WHO standard titer of 0.5 IU/ml). Mice were challenged with a 40fold MLD₅₀ of rabies virus CVS-11 on day 47 (B, C, E, F) and day 51 (H-I), respectively. Survival (B, E, H) and body weight (C, F, I) was recorded. For antibody titers, the mean is indicated, for body weight kinetics the mean and standard deviation is given (n = 6 to 8). Significance *p<0.1, **p<0.01, ***p < .001 to HDC or buffer control group in A and D/G, respectively, was calculated using the Mann Whitney test. Groups included in A-F were performed simultaneously; therefore buffer control displayed in D-F is valid for all groups.

Fig 3. Oscillating temperatures and potential handling and storage errors tested do not compromise mRNA vaccine efficacy.

mRNA vaccine, Rabipur and HDC were reconstituted in buffer and subsequently stored at +40°C for one week (A-C). In a second experiment, RABV-G mRNA was stored as a lyophilisate at 56°C for 8–9 hours per day and then at 4°C for 15–16 hours with a total of 20 cycles of oscillating temperatures (D-F). Vaccine or control injections were done on day 0 and 21. Induction of rabies virus neutralization titer was measured on day 35 after first immunization (A, D: dashed line indicates the WHO standard titer of 0.5 IU/ml). Mice were challenged with a 40fold MLD₅₀ of rabies virus CVS-11 on day 47 (B-C) or day 51 (E-F) and survival (B, E) and body weight (C, F) was recorded. As groups in A-F were treated in parallel, the buffer control group in Fig 3D–3F is valid also for Fig 3A–3C. For antibody titers, the mean is indicated, for body weight kinetics the mean and standard deviation is shown (n = 6 to 8). Significance: ns = non-significant, **p<0.01, ***p < .001 to HDC or buffer control group in A and D, respectively, was calculated using the Mann Whitney test.