In a recent study posted to the bioRxiv* preprint server, researchers developed two monkeypox (MPX) virus (MPXV) quadrivalent messenger ribonucleic acid (mRNA) vaccines, mRNA-A-lipid nanoparticle (LNP) and mRNA-B-LNP.
The vaccines were based on the intracellular mature virus (IMV)-specific proteins, i.e., M1R and A29L, and extracellular enveloped virus (EEV)-specific proteins, i.e., B6R and A35R.
The ongoing 2022 MPXV outbreak has affected several nations globally at a rapid pace, warranting the development of safe and efficient anti-MPXV vaccines. The unprecedented success of mRNA-based vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accelerated swift and extensive mRNA vaccine development to formulate vaccines effective against other viruses.
About the study
In the present study, researchers developed IMV-specific and EEV-specific quadrivalent mRNA-based mRNA-A-LNP vaccine and the mRNA-B-LNP vaccine against MPXV that generated potent immunoglobulin G (IgG) titers and vaccinia virus (VACV)-neutralizing antibodies.
As antigen targets, the vaccines were prepared using A35R, A29L, B6R, and M1R. The mRNA-A-mix group and the mRNA-B-mix group of mRNA sequences were constructed and verified by capillary electrophoresis. Western blot analysis confirmed successful protein expression by the mRNA sequence groups, following which the mRNAs formulated into LNPs.
In addition, cryo-TEM (cryo-transmission electron microscopy) analysis was performed, which showed that both the mRNA0LNP formulations displayed a uniform, solid, and spherical morphology. The findings indicated mRNA sequence-loaded LNPs stability and successful IMV and EEV protein expression by the mRNA preparations in vitro. Human embryonic kidney (HEK)293T, Huh-7, RD, Vero, and 143TK cells were used for the cell culture experiments.
Modified mRNA sequences were synthesized, capped, and purified. Further, the efficacy and immunogenicity of the two vaccine candidates were evaluated in BALB/c-type mice immunized with the vaccines twice intramuscularly. Subsequently, serum samples were obtained from the mice on day ten and day 24 of their initial immunization for humoral immunity assessments. Additionally, enzyme-linked immunosorbent assays (ELISA) were performed to assess antibody binding to MPXV antigen proteins A35R, A29L, B6R, and M1R.
Neutralizing antibodies were evaluated based on live-virus neutralization tests utilizing the Tian Tan strain of the vaccinia virus. Further, the induction of cell-based immunity by the vaccine candidates was assessed by measuring MPXV-specific GC (germinal center) B lymphocytes, Tfh (follicular helper T) lymphocytes, and the cluster of differentiation 4+ (CD4+) and CD8+ Tem (effector memory T) lymphocytes. Flow cytometry was performed to evaluate GC B lymphocytes [apoptosis antigen 1+ (Fas+)/GL7+] and Tfh lymphocytes [C-X-C chemokine receptor type 5+(CXCR5+)/programmed cell death protein 1+ (PD-1+)] in DLNs (draining lymph nodes) after 30 days of the initial immunization.
Subsequently, particular CD8+ and CD4+ Tem lymphocytes (CD44+/CD62L-) in the splenic tissues of immunized mice were evaluated. To evaluate in vitro sera protection, sera and VACV were mixed before the intravenous and intraperitoneal challenge of nude mice. To evaluate in vivo sera protection, serum was injected intravenously into nude mice, followed by a subcutaneous VACV challenge. Bioluminescence imaging (BLI) was performed to detect to measure viral load in the animals. Furthermore, the in vivo toxicity of the vaccines was assessed based on the changes in mice’s body weight, blood biochemical parameters, and histological assessments.
The vaccine candidates induced potent anti-MPXV IgG titers and anti-VACV neutralizing antibody titers in mice with the generation of durable anti-MPXV killer memory T-lymphocyte and memory B-lymphocyte immune responses in the animals. The passive serological transfer of sera from mice immunized with the two vaccines protected nude mice against VACV. Further, double-dose vaccinations of the two vaccine candidates also protected against VACV in BALB/c mice.
IgG titers against A29L, A35R, M1R, and B6R were detectable among all immunized mice and significantly increased over the frequency of vaccination, indicating that the two vaccines induced robust anti-MPXV humoral immunity. After initial vaccination, median values of neutralizing antibody titers for the mRNA-A-LNP vaccine and the mRNA-B-LNP vaccine were 73.0 and 49.0, respectively. The corresponding titers increased to 6,284 and 5,057, respectively, after subsequent vaccinations.
A significant increase in anti-MPXV GC B lymphocytes and Tfh lymphocytes was observed among vaccinated mice compared with control mice upon MPXV-specific antigen stimulation. Remarkably, the two vaccines induced anti-MPXV CD8+ Tem lymphocytes more effectively than CD4+ Tem lymphocytes. The vaccinated mice sera effectively neutralized VACV in vitro, and the vaccine-inoculated mice sera exhibited passive immune protection against the subcutaneous vaccinia virus challenge.
Bioluminescent signals were negligible among vaccinated mice, indicating that VACV was cleared rapidly cleared by the mRNA vaccine-induced antibodies after the VACV challenge. After the mRNA-LNP vaccinations, no loss of weight was observed. On further investigation, mRNA-A-LNP vaccinations did not harm the heart, liver, or kidneys of mice. In addition, no statistically significant histopathological changes were observed between the immune and control mice tissues, indicating that mRNA-A-LNP was safe for administration.
Overall, the study findings showed that the quadrivalent mRNA-A-LNP vaccine and the mRNA-B-LNP vaccine appeared to be effective and safe vaccine candidates against MPXV and other orthopoxviruses such as VACV.
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.