RESUMO
BACKGROUND: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). METHODS: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. RESULTS: The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. CONCLUSION: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.
RESUMO
Vaccines are the primary means of controlling and preventing pandemics and outbreaks of pathogens such as bacteria, viruses, and parasites. However, a major drawback of naked DNA-based vaccines is their low immunogenicity and the amount of plasmid DNA necessary to elicit a response. Nano-sized liposomes can overcome this limitation, enhancing both nucleic acid stability and targeting to cells after administration. We tested two different DNA vaccines in cationic liposomes to improve the immunogenic properties. For this, we cloned the coding sequences of the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) either alone or fused with small the small hepatitis virus (HBV) envelope antigen (HBsAg) encoding sequences, potentially resulting in HBsAg particles displaying PfRH5 on their outside. Instead of invasive intraperitoneal or intramuscular immunization, we employed intradermal immunization by tattooing nano-encapsulated DNA. Mice were immunized with 10 µg encapsulated DNA encoding PfRH5 alone or in fusion with HBsAg and this elicited antibodies against schizont extracts (titer of 104). Importantly, only IgG from animals immunized with PfRH5-HBs demonstrated sustained IgG-mediated inhibition in in vitro growth assays showing 58% and 39% blocking activity after 24 and 48 h, respectively. Intradermal tattoo-vaccination of encapsulated PfRH5-HBsAg coding plasmid DNA is effective and superior compared with an unfused PfRH5-DNA vaccine, suggesting that the HBsAg fusion may be advantageous with other vaccine antigens.