According to the most recent fact sheet for malaria from the World Health Organization (WHO), in 2021 there were an estimated 247 million cases of malaria worldwide leading to 619,000 deaths, mostly young children. Although the WHO African Region accounted for 95% of these cases and deaths, the Centers for Disease Control and Prevention (CDC) reported cases of locally acquired malaria in Texas and Florida in early 2023. Approximately 2,000 cases of travel-related malaria were diagnosed in the United States each year; ~300 people with severe disease leading to 5 to 10 deaths.
Malaria is caused by single-celled Plasmodium parasites transmitted by female anopheline mosquitoes. As depicted here, in the malaria transmission cycle, the mosquito bite introduces the parasites from the mosquito’s saliva into a person’s blood, leading to maturation and reproduction in the liver. This leads to infection of red blood cells (erythrocytes) that are then ingested by blood-feeding mosquitoes, which can subsequently transmit the parasites to an uninfected person.
The WHO notes antimalarial drug-resistance continues to increase and overall progress in incidence-reduction has started to stagnate. Therefore, vaccines are needed to achieve further progress toward malaria elimination. The recombinant protein–based vaccine, termed “RTS,S” (Mosquirix™), was the first and only malaria vaccine approved and in wide use. As of April 2022, the vaccine has been given to 1 million infants living in areas with moderate-to-high malaria transmission; however, the vaccine reduces hospital admissions from malaria by only ~30%.
Next-Generation Malaria Vaccines
- Hayashi et al. (2022) reasoned next-generation vaccines will need to apply new strategies for improved protection, namely, target Plasmodium parasites at multiple life-cycle stages:
- Pre-erythrocytic vaccines target sporozoite and liver-stage parasites with the aim of eliciting immune responses to prevent infection.
- Blood-stage vaccines target the disease-causing parasites to elicit an immune response to limit parasite burden, thereby reducing disease severity.
- Transmission-blocking vaccines target the sexual stage parasites in the female mosquitoes, leading to the disruption of the sexual life cycle and cessation of parasite development, and reduction of transmission.
One of the primary targets for pre-erythrocytic vaccine development is directed at Plasmodium falciparum, the deadliest species of Plasmodium that causes malaria in humans. The particular immunogen is the circumsporozoite protein termed PfCSP, which is expressed on the infectious sporozoite. Another P. falciparum immunogenic protein termed Pfs25 is a leading target for transmission-blocking vaccines that is expressed on the surface of developing ookinetes crucial for transmission of malaria in a new host. Numerous vaccine platforms, such as virus-like particles, nanoparticles, live vectors, and DNA plasmids, have been evaluated for PfCSP (Molina-Franky et al.2020) and Pfs25 (Mulamba et al. 2022) but with limited success.
Experimental Outline to Test a Bivalent mRNA Vaccine for Malaria
Hayashi et al. therefore evaluated the immunogenicity of Pfs25 and PfCSP formulated as N1-methylpseudouridine- modified mRNA vaccines with the geometric mean titers. Administration of the second immunization with Pfs25 mRNA-LNP resulted in significant boosting of antibody responses with titers ~200-fold higher for the 3 μg dose and ~20-fold higher for both the 10 μg and 30 μg doses. The second immunization with PfCSP resulted in ~100- to 200-fold increases in antibody titers.
No significant increases in antibody titers were found after additional immunizations with either Pfs25 or PfCSP. Importantly, the antibody titers elicited by each dose of Pfs25 and PfCSP mRNA-LNPs was superior to those elicited by immunization with Pfs25 and PfCSP DNA vaccines.
Co-immunization with Pfs25 and PfCSP mRNA-LNP Does Not Compromise Antibody Responses
A group of mice was immunized with a combination of both Pfs25 and PfCSP mRNA-LNPs (Pfs25+PfCSP) to explore the immunogenicity of bivalent co-immunization of antigens targeting different parasite life-cycle stages. Co-immunization with 10 μg of each mRNA-LNP in Pfs25+PfCSP led to individual antigen-specific antibody titers that were largely comparable to those obtained in mice immunized with either of the two immunogens individually. As mentioned above for monovalent vaccinations, this Pfs25+PfCSP mRNA-LNP combination of elicited antibody responses that were superior as compared to antibodies elicited in mice co-immunized with a combination of Pfs25 DNA and PfCSP DNA (25 μg each).
Pfs25 mRNA-LNP Induces Potent Transmission-Blocking Antibodies
An important aspect of the experimental plan by Hayashi et al. was to demonstrate that Pfs25 mRNA-elicited antibodies could block transmission of malaria. To do this, they used the standard membrane-feeding assay (SMFA), which is regarded as the “gold standard” for determining the impact of test factors on gametocyte infectivity of mosquitoes, and is measured by %-inhibition in mean oocyst intensity (%TRA) (Miura et al. 2016).
Briefly, the SMFA involves feeding Anopheles mosquitos a blood meal containing a mixture of cultured P. falciparum gametocytes and test (or control) antibodies through a membrane-feeding apparatus. One week later, mosquitoes from the test and control groups are examined to enumerate the oocyst-forms of parasites visualized in the epithelium of the mosquito’s midgut by mercury-bromide staining.
Due to the limited volume of sera collected, serum from final bleeds from each mouse was pooled to purify IgG for evaluation in SMFA. Sera from mice immunized three times with 3 μg and 10 μg Pfs25 mRNA-LNP were evaluated, while the 30 μg combination Pfs25 + PfCSP mRNA-LNP group was evaluated after four immunizations.
Purified IgG from all these groups, which was first evaluated at 0.5 mg/ml, led to >94% TRA. To look for differences in % TRA, subsequent assays used 2- to 16-fold lower concentrations of IgG, using purified IgG from pooled pre-immune mouse sera at 1 mg/ml as a negative control. While various % TRA results were obtained, 2- to 8-fold lower serum concentrations for all of the immunized groups led to ≥ 90% TRA. These findings demonstrated significant production of transmission-blocking antibodies by Pfs25 mRNA.
Protection Against Sporozoite Challenge after Immunization with PfCSP mRNA-LNP
Another important aspect of the experimental plan by Hayashi et al. was to investigate whether PfCSP mRNA-elicited antibodies could block malarial infection by sporozoites. Possible protection provided by immunization with PfCSP was evaluated using an in vivo challenge model with sporozoites previously reported by other researchers (Flores-Garcia et al. 2019) who employed genetically engineered P. falciparum expressing PfCSP and luciferase (Luc), for quantification.
Briefly, groups of mice (N = 5) were inoculated with ~2000 of these transgenic sporozoites four weeks after the third immunization with PfCSP mRNA-LNP. Liver-stage parasite burden was monitored in vivo for 11 days, during which time 5/5 mice of the 10 μg and 30 μg PfCSP mRNA-LNP groups, and 4/5 mice of the co-immunized 10 μg each Pfs25+PfCSP mRNA-LNP group, were fully protected. By contrast, the 3 μg PfCSP mRNA-LNP group led to infection of 4/5 mice, thus indicating a dose-response.
In addition, Kaplan-Meier survival curves (Figure 1) representing the percent of mice without apparent malarial disease (parasitemia) each day, following the challenge, were used to assess any significant delays in blood-stage parasitemia.
FIGURE 1. Kaplan–Meier survival curves represent the percent of mice without detectable parasitemia following sporozoite challenge. Mice were considered completely protected if no parasitemia was detected by day 11. Statistical analysis was performed using log rank (Mantel-Cox) test (*p < 0.05; **p < 0.01). Taken from Hayashi et al. (2022) and free to use under a Creative Commons Attribution 4.0 International License.
The survival curves of the 10 μg PfCSP mRNA-LNP (p = 0.004), 30 μg PfCSP mRNA-LNP (p = 0.004), and 10 μg each Pfs25+PfCSP mRNA- LNP groups (p = 0.0132), were significantly different from the Pfs25 mRNA-LNP immunized group that served as a negative control for protection against infection by sporozoites. By contrast, the 3 μg PfCSP mRNA-LNP group showed no significant difference when compared to the Pfs25 mRNA-LNP control (p = 0.8319), thus confirming the dose-response.
Apart from establishing the immunogenicity of the LNP-formulated monovalent mRNA vaccines encoding either Pfs25 or PfCSP, co-immunization with the bivalent Pfs25+PfCSP mRNA vaccine was shown to elicit comparable antigen-specific antibody responses. The functional activities after immunization with these mono- or bivalent vaccines were also comparable, thus providing the first evidence to support the strategy of using the mRNA-LNP platform to combine multiple vaccines without negative consequences.
Moreover, Hayashi et al. concluded that this “combination of vaccines targeting both the infection stage and sexual/midgut stages is expected to provide effective ways to interrupt malaria transmission, which is critical for achieving elimination goals.”
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