Establishment of an Antiplasmodial Vaccine Based on PfRH5-Encoding RNA Replicons Stabilized by Cationic Liposomes

Abstract

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.

Keywords: PfRH5; RNA replicons; cationic liposomes; intradermal immunization; malaria; nucleic acid vaccines; tattooing.

Figure 1

Plasmid constructs for RNA replicon synthesis. The proteins nsp1, nsp2, nsp3 and nsp4, after the origin of replication (ori), are nonstructural alphavirus proteins, while the other proteins represented are transgenes (GFP and nLuc). The nLuc and PfRH5-amplified PCR products were cloned in the multiple cloning site (MCS) of the basic replicon vector. GFP and nanoluciferase were reporters used to monitor the transfection efficiency. The GFP protein was located in the cytosol, and was detected in transfected cells using flow cytometry and immunofluorescence of the transfected tissue. The nanoluciferase protein was secreted from the cells using the tissue plasminogen activator (tPA) signal sequence, and its bioluminescence could be measured using a luminometer (in vitro assays), and also through an in vivo imaging system (IVIS^® Spectrum). The plasmids also included puromycin and ampicillin resistance genes (PuroR and AmpR).

Figure 2

NS-TEM (negative-stained transmission electron microscopy) images of the formulated liposomes showing the final size of packaged replicons/samRNAs. (A) Cationic liposomes alone; (B) cationic liposomes complexed with 1 μg of GFP replicon; and (C) cationic liposomes complexed with 1 μg of nLuc replicon. Data scales of 100 nm, 200 nm and 50 nm, respectively.

Figure 3

Vero cells were easily transfected with packaged samRNA encoding GFP. Comparison of Vero cells transfected with (A) 8 nM of cationic liposomes conjugated with 6 µg of samGFP RNA; (B) 8 nM of cationic liposomes conjugated with 2 µg of samGFP; (C) only liposomes. The nuclei were stained with DAPI, and the green cytoplasm of the transfected cells indicates GFP expression. Images were captured at 40× magnification.

Figure 4

Comparison of samRNA or lipid composition quantities in transgene expression in vitro. (A) Representative histograms of Vero cells expressing GFP 24 h after transfection with the GFP-expressing replicon in comparison to an untransfected control. (B) Dot plot comparing the populations described in (A). (C) Representative histograms of GFP expression 24 h and 48 h after transfection with samRNA encoding GFP encapsulated in liposomes in comparison to a control transfected with empty liposomes. (D) Representative histograms of GFP expression dependent on the amount of transfected replicon (2 μg, 4 μg, 6 μg, 8 μg or 10 μg of transfected replicons) in HEK293T cells 24 h after transfection. (E) Representative histograms of GFP expression in dependence on the concentration of liposome used (2 nM, 4 nM, 6 nM, 8 nM or 10 nM) to deliver 1 μg of GFP replicon to HEK293T cells 24 h after transfection.

Figure 5

Quantification of GFP and Nano-Luciferase expression demonstrated their strong production in Vero cells. In (A), pellets from Vero cells transfected with replicons expressing either GFP or nanoluc are shown under UV light (48 h after transfection). The third sample was transfected with cationic liposomes containing nLuc replicons under the same conditions. (B) Luminescence activity of Vero cells transfected with nLuc replicon complexed with 4 nM or 8 nM of cationic liposomes compared with non-transfected cells (control). The luminescence from each sample was normalized regarding the volume of the sample and protein concentration from each one. p-values are given for one-way ANOVA where * represents p < 0.005. (C) Relative luminescence of Vero cells transfected with nLuc replicon in relation to non-transfected cells. The difference in luminescence between the two samples (with a higher and lower concentration of liposomes) was not statistically significant. All experiments were performed in triplicate. Error bars show the deviations from three technical replicates.

Figure 6

Stronger GFP expression was observed after tattooing samRNA compared with plasmid DNA. Comparison of tattoo scars of animals immunized with nLuc expressing replicon (A, negative control) and GFP-expressing replicon (B), respectively, in cationic liposomes, observed under black light. Asterisks depict green spots on the skin of the animal tattooed with GFP replicons 48 h after the intradermal inoculation. Histological analyses of tissue sections (C) of BALB/c mice tattooed with liposomes encapsulating either 10 µg of DNA plasmid or RNA-replicon-encoding GFP 48 h after inoculation in the dermis. Control mice were not tattooed, and fluorescence in the control mice is due to hair in the tissue section. Images were acquired by confocal laser scanning microscopy. Data scales of 100 µm.

Figure 7

Sera from immunized mice recognized native PfRH5 in P. falciparum NF54 schizont extracts, and exerted an inhibitory effect in vitro. In (A), endpoint titers of anti-samPfRH5 and TEV mRNA PfRH5 sera were measured (titers of Log10, no statistical difference between the groups). Individual sera of 5 liposome-packaged RNA-immunized mice were tested in endpoint dilutions. For coating, ELISA plate schizont extracts were used instead of recombinant PfRH5 antigens. (B) Sera from samPfRH5- and TEV PfRH5 RNA-immunized mice were used to recognize extracts from P. falciparum NF54. Lane 1 is pooled sera from samPfRH5-immunized mice, lane 2 from TEV PfRH5-immunized mice, and Ctr, controls from pre-immune sera. In (C), the percentage of growth inhibition of parasite proliferation (measured by flow cytometry) is shown for two dilutions of purified IgGs from four mice immunized with encapsulated samPfRH5 or TEV PfRH5. The shown values were calculated by comparing parasitemias in parallel cultures supplemented with 300 µg/mL non-related IgG (=0% growth inhibition, for samPfRH5 is shown to average 26% ± 1.5 for 180 µg/mL and 43.1% ± 2.4 for 300 µg/mL in 24 h; average 214.5% ± 4.5 for 180 µg/mL and 24% ± 9.5 for 300 µg/mL in 48 h; in the case of TEV PfRH5, average of 29.4% ± 2 for 180 µg/mL and 49% ± 3.5 for 300 µg/mL). Analyses assuming equal variance and non-parametric values showed no difference between 24 h antisera from both RNA immunizations, and a large difference after 48 h of cultivation, Kruskal–Wallis test, *** = 0.0001) The values are shown for 24 h and 48 h of inhibition. See Supplementary Figure S2 for original Western blot X-ray film.