Pfs25 mRNA-LNPs elicit high antibody titers in a dose-dependent fashion

To assess the antibody responses to Pfs25, female Balb/c mice were immunized with various doses (3, 10, and 30 μg) of Pfs25 mRNA-LNP and evaluated as shown in Fig. 1a. After the first immunization, the 3, 10, and 30 μg Pfs25 mRNA-LNPs elicited dose-dependent primary antibody responses with geometric mean titers of 43,528, 263,902, and 324,901, respectively (Fig. 2a). Administration of the second immunization of Pfs25 mRNA-LNP resulted in significant boosting of antibody responses with titers ~200-fold higher for the lowest dose (ELISA titer 8,444,851) and ~20-fold higher for the 10 μg (ELISA titer 11,942,822) and 30 μg (ELISA titer 12,800,000) Pfs25 mRNA-LNP doses (Fig. 2b). Additional immunization did not result in any further increase in antibody titers (Fig. 2c, d). While all doses elicited strong antibody production, the 30 μg Pfs25 mRNA-LNP elicited significantly higher titers compared with the 3 μg (p = 0.0079) and 10 μg (p = 0.0079) Pfs25 mRNA-LNP groups after three immunizations.

Fig. 1: Timelines for mRNA immunization experiment.

a Groups of female Balb/c mice were immunized (Im) with 3 μg, 10 μg, and 30 μg of mRNA-LNPs encoding either Pfs25 or PfCSP. Another group of mice received a combination of 10 μg of both Pfs25 and PfCSP mRNA-LNPs. In parallel, mice were also immunized with 50 μg of DNA plasmids encoding Pfs25 or PfCSP and a combination of 25 μg of both Pfs25 and PfCSP DNA plasmids. Immunizations were repeated as indicated, and test bleeds (B1-B4) and final bleeds (FB1 and FB2) were collected at indicated time points. Mice immunized with 3 μg and 10 μg Pfs25 mRNA-LNP were terminally bled at 12 weeks (FB1) while all other groups were terminally bled at 24 weeks (FB2). Mice immunized with PfCSP mRNA or DNA plasmids were challenged (Ch1 and Ch2) with sporozoites of PbPfCSP-GFPLuc to assess protection. 30 μg Pfs25 mRNA-LNP and Pfs25 DNA immunized mice were used as a negative control for sporozoite challenge experiments. b Table of immunization parameters corresponding to timeline in (a).

Fig. 2: Pfs25-specific antibody titers determined by ELISA.

mRNA-LNP immunization and blood collection schedules are shown in Fig. 1a. Sera (N = 5 per group) from bleeds at indicated time points [B1 in (a), B2 in (b), B3 in (c), and B4 in (d)] were analyzed using ELISA to determine antigen-specific antibody titers reported as the geometric mean endpoint titers with 95% confidence intervals. Endpoint titers were identified as the highest reciprocal serum dilution with an OD higher than a cutoff of the average OD plus three standard deviations of pre-immune sera replicates. Statistical analysis was performed using the Mann–Whitney U test (*p < 0.05; **p < 0.01).

PfCSP mRNA-LNPs elicit high antibody titers in a dose-dependent fashion

PfCSP mRNA-LNPs was evaluated at doses of 3, 10, and 30 μg, as shown in Fig. 1a. After one immunization, the 3, 10, and 30 μg PfCSP mRNA-LNP groups elicited PfCSP-specific antibody responses with geometric mean titers of 19,507, 15,658, and 46,976 (Fig. 3a). A second immunization resulted in 100 to 200-fold increases in antibody titers, with geometric means reaching 2,451,972, 4,740,109, and 11,415,247 for the 3 μg, 10 μg, and 30 μg PfCSP mRNA-LNP groups, respectively (Fig. 3b). Although there was a clear trend of a dose-dependent increase in the immunogenicity of PfCSP mRNA-LNP, the only statistically significant difference was between the 3 μg and 30 μg PfCSP mRNA-LNP groups (p = 0.0238). Any further immunization did not significantly alter antibody titers (Fig. 3c), with observed geometric mean titers of 1,968,300, 3,805,082, and 11,415,247 for the 3 μg, 10 μg, and 30 μg PfCSP mRNA groups, respectively.

Fig. 3: PfCSP-specific antibody titers determined by ELISA.

mRNA-LNP immunization and blood collection schedules are shown in Fig. 1a. Sera from bleeds at indicated time points [B1 in (a), B2 in (b), B3 in (c), and B4 in (d)] were analyzed using ELISA to determine antigen-specific antibody titers reported as the geometric mean endpoint titers with 95% confidence intervals. Endpoint titers were identified as the highest reciprocal serum dilution with an OD higher than a cutoff of the average OD plus three standard deviations of pre-immune sera replicates. Statistical analysis was performed using the Mann–Whitney U test (*p < 0.05).

As discussed below, we initially sought to evaluate protection against sporozoite challenge after three immunizations, however, the purified sporozoites used for challenge were found to be largely non-viable thus necessitating another challenge after a period of drug treatment and a fourth immunization (Fig. 1a). Geometric mean antibody titers after a fourth immunization in the 3 μg and 10 μg PfCSP mRNA-LNPs groups increased to 7,355,917 and 11,415,247, with the 30 μg mRNA-LNP group remaining at similar titers to those prior to the fourth immunization (Fig. 3d).

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 to explore the immunogenicity of co-immunization of antigens targeting different parasite life cycle stages. Female Balb/c mice were co-immunized with 10 μg of Pfs25 and PfCSP mRNA-LNPs (Pfs25 + PfCSP mRNA-LNP), and the individual antigen-specific antibody titers were compared with antibody titers in mice immunized with Pfs25 mRNA-LNP or PfCSP mRNA-LNP alone. As shown in Figs. 2 and 3, Pfs25 and PfCSP-specific antibody titers after each immunization in the co-immunized group remained largely comparable to those obtained in mice immunized with either of the two immunogens individually. The only notable difference was higher Pfs25-specific titers after three immunizations in the combination group as compared to 10 μg Pfs25 mRNA-LNP (Fig. 2; p = 0.0079). Surprisingly, in the case of PfCSP-specific antibody responses, mice that had undergone experimental manipulations consisting of a challenge with largely non-viable sporozoites and drug treatment showed somewhat lower antibody titers after the fourth immunization in the combination group as compared to 10 μg PfCSP mRNA-LNP group (Fig. 3d). To rule out the possibility of any cross-reactivity between the two antigens, we tested sera from mice immunized with Pfs25 mRNA-LNPs for reactivity with PfCSP and vice versa using ELISAs. As shown (Supplementary Fig 3), immunized mice sera displayed antigen specific reactivity while the reactivity of Pfs25 immunized mice sera to PfCSP and the reactivity of PfCSP immunized mice sera to Pfs25 was comparable to that of pre-immune mouse sera used as a negative control for all ELISA.

mRNA-LNPs elicit superior antibody responses than electroporation-mediated DNA plasmids

Previously we have shown that 50 μg of Pfs25 DNA administered with electroporation (EP) is immunogenic and efficacious in mice26. Another goal of our studies was to compare the relative immunogenicity differences between mRNA-LNP and DNA with in vivo EP for both Pfs25 and PfCSP. After each immunization, the antibody titers elicited by Pfs25 and PfCSP mRNA-LNPs, regardless of the vaccine dose, were superior to those elicited by immunization with Pfs25 and PfCSP DNA vaccines administered using in vivo EP (Supplementary Fig. 4a-b). Likewise, Pfs25 and PfCSP mRNA-LNPs combination (10 μg each) vaccine-elicited antibody responses were also superior as compared to antibodies elicited in mice co-immunized with a combination of Pfs25 and PfCSP DNA (25 μg each) administered with EP (Supplementary Fig. 4).

Pfs25 mRNA-LNPs induce potent transmission-blocking antibodies

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 (FB1 in Fig. 1a) from mice immunized three times with 3 μg and 10 μg Pfs25 mRNA-LNP were evaluated, while the 30 μg Pfs25 mRNA-LNP and (Pfs25 + PfCSP) mRNA-LNP combination groups were evaluated after four immunizations (FB2 in Fig. 1a). Initially, purified IgG from all the groups of the 3 μg, 10 μg, and 30 μg Pfs25 mRNA-LNP and Pfs25+PfCSP mRNA-LNP groups were evaluated at three concentrations (2, 1, 0.5 mg/ml) and all revealed greater than 94% TRA at all three concentrations of IgG tested. In subsequent experiments, lower concentrations of IgG (0.25, 0.125, 0.0625, 0.03125 mg/ml) were evaluated. IgG purified from pooled pre-immune mouse sera was used at 1 mg/ml as a negative control. IgG purified from the 3 μg and 10 μg Pfs25 mRNA-LNP immunized mice achieved greater than 90 % oocyst reduction (TRA) at IgG concentrations as low as 0.0625 mg/ml. Surprisingly, IgG from the 30 μg Pfs25 mRNA-LNP and Pfs25+PfCSP combination groups revealed relatively lower reduction with TRA ≥ 90% at 0.125 to 0.25 mg/ml IgG concentrations (Fig. 4).

Fig. 4: Evaluation of transmission reducing activity of antibodies induced by Pfs25 mRNA-LNPs.

IgG was purified from pooled sera collected from mice immunized with 3 μg and 10 μg of Pfs25 mRNA-LNP (FB1) and 30 μg Pfs25 mRNA and 10 μg of both Pfs25+PfCSP mRNA-LNP combinations groups (FB2). Purified IgG were evaluated in mosquito standard membrane feeding assays (SMFA) at indicated final concentrations (0.25, 0.125, 0.63, and 0.031 mg/ml). Data points representing the oocyst counts from each mosquito, arithmetic means, and 95% confidence intervals are reported. N is the fraction of uninfected mosquitoes over the total number of mosquitoes dissected. %TRA is the mean reduction in oocysts in the presence of the test IgG compared with the IgG (1 mg/ml) from pre-immune mouse sera (Pre) used as a negative control. %TBA is the percent reduction in the proportion of infected mosquitoes in the presence of the test IgG compared with pre-immune IgG negative control (Pre). The inset shows IgG1/IgG2a isotype ratios and antibody avidity index for sera evaluated by SMFA. Statistical analysis was performed using the Mann–Whitney U test (ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

To further characterize the anti-Pfs25 antibodies of sera that have transmission-blocking activity, Pfs25-specific antibody avidity and isotypes were evaluated using pooled serum of terminal bleeds of each group by ELISA (Fig. 4). The avidity indices of 1.73, 1.48, and 1.69 were similar among the pooled sera of the 3 μg, 10 μg, and 30 μg Pfs25 RNA-LNP immunized mice, respectively. We also compared Pfs25-specific IgG isotypes (expressed as IgG1/IgG2a ratios) in these sera and the results revealed balanced IgG isotypes with IgG1/IgG2a ratios of 1.20, 1.07, and 1.17 for the 3 μg, 10 μg, and 30 μg Pfs25 mRNA-LNP groups, respectively.

Protection against sporozoite challenge after immunization with PfCSP mRNA-LNP

The protection provided by the immunization with PfCSP was evaluated using an in vivo challenge model using sporozoites of transgenic P. berghei expressing PfCSP and luciferase (PbPfCSP-GFPLuc). For the first challenge (Ch1 in Fig. 1a), mice were inoculated with ~2000 PbPfCSP-GFPLuc sporozoites four weeks after the third immunization. Liver stage parasite burden was evaluated 42–44 h after the challenge by IVIS, quantifying the subsequent bioluminescence from the liver. None of the challenged mice, including negative controls, had any detectable liver stage burden suggesting that the sporozoites used for the challenge were not very viable or infectious. Additionally, only a fraction of Pfs25 immunized control mice (6/10) had any detectable blood-stage parasites by day 7 further corroborating that the viability of PbPfCSP-GFPLuc sporozoites was severely compromised during sporozoite isolation. To further evaluate the protective efficacy of the PfCSP mRNA LNP vaccine, mice were treated with chloroquine and sulfadiazine for six days to treat for any low-level parasitemia. Mice were given a fourth immunization after a rest period, and four weeks after the last dose of immunization mice were challenged again (Ch2 in Fig. 1a) and monitored for blood-stage parasitemia for 11 days. Five out of five mice of each 10 μg and 30 μg PfCSP mRNA-LNP group and four out of five mice of the Pfs25+PfCSP mRNA-LNP group were fully protected. In contrast, the 3 μg PfCSP mRNA-LNP group only had one out of five mice that achieved complete protection. Kaplan-Meier survival curves representing the percent of mice with detectable parasitemia each day following the challenge were used to assess any significant delays in blood-stage parasitemia. The survival curves of the 10 μg PfCSP mRNA-LNP (p = 0.004), 30 μg PfCSP mRNA-LNP (p = 0.004), and Pfs25+PfCSP mRNA- LNP groups (p = 0.0132), were significantly different to the Pfs25 mRNA-LNP immunized groups which served as a negative control for protection against infection by sporozoites. In contrast, the 3 μg PfCSP mRNA-LNP group showed no significant difference when compared to the Pfs25 mRNA-LNP control (p = 0.8319) (Fig. 5a).

Fig. 5: PfCSP mRNA-LNP induced responses in mice and protection against sporozoite challenge after four immunizations.

Mice (N = 5 per group) immunized with 3 μg, 10 μg, 30 μg PfCSP mRNA-LNPs, or co-immunized with 10 μg of both Pfs25 and PfCSP mRNA-LNPs (as per Fig. 1a) were challenged with PbPfCSP-GFPLuc sporozoites and progression of infection was monitored by microscope examination of giemsa-stained blood smears for 11 days following challenge. Pfs25 immunized mice were used as negative controls. a 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). Antibodies from sera, collected one-week prior to the second challenge, bleed B4 in Fig. 1a, were characterized for antibody isotype (b) by ELISA using IgG1, IgG2a, IgG2b and IgG3 specific secondary antibodies. The optical densities (OD) of each isotype were normalized to the OD of total anti-mouse IgG. c Avidity was characterized by antibody-antigen dissociation curves resulting from treatment with different concentrations of NaSCN from sera collected from the Bleeds, B3 and B4, in Fig. 1a. The avidity index was calculated as the concentration (M) of NaSCN that resulted in 50% dissociation of bound antibodies.

To further characterize antibody responses at the time of protection, PfCSP-specific antibody avidity and isotypes were evaluated on pooled sera collected three weeks after the fourth dose (Fig. 5). The PfCSP-specific antibody avidity indices were similar between 3 μg, 10 μg, and 30 μg PfCSP mRNA-LNP groups, with avidity indices of 2.18, 2.20, and 1.72, respectively. To rule out influence of any potential impact of the transgenic parasites used in the first non-viable challenge, avidity of sera collected one week prior to the first challenge (B3 in Fig. 1a) was also assessed. The avidity indices for the 3 μg, 10 μg, and 30 μg PfCSP mRNA-LNP groups were 1.77, 2.01 and 1.59, respectively, which are within the experimental variability of the assay and similar to the avidity of sera collected three weeks after the fourth dose (B4 in Fig. 1a). In addition, the immune sera revealed IgG2a biased antibody isotypes with significantly pronounced IgG2a skewed isotypes in the sera from mice immunized with 3 μg and 30 μg PfCSP mRNA-LNP and the combination of Pfs25+PfCSP mRNA-LNP, with IgG1/IgG2a ratios of 0.75, 0.54, and 0.73, respectively.

Evaluation of the minimum effective dose of Pfs25 mRNA-LNP and PfCSP mRNA-LNP

Due to the significant antibody responses elicited by the 3 μg mRNA-LNP group, we were interested in investigating whether lower doses of 1 μg or 0.1 μg mRNA-LNP would be immunogenic with effective antibody responses. Mice immunized three times (Fig. 6c) with 0.1 μg, 1 μg, and 10 μg Pfs25 mRNA-LNP revealed a dose-dependent antibody response with geometric mean titers of 606,287, 1,837,917, and 4,850,293, respectively (Fig. 7a inset). The 1 μg Pfs25 mRNA-LNP group had higher antibody titers than the 0.1 μg Pfs25 mRNA-LNP group, but the difference was not statistically significant. However, the antibody titers in the mice immunized with 10 μg Pfs25 mRNA-LNP were significantly higher than the 1 μg (p = 0.0476) and 0.1 μg (p = 0.0079) Pfs25 mRNA-LNP groups.

Fig. 6: Experimental details for immunization experiment to evaluate lower vaccine doses and repeat immunizations.

Groups of female Balb/c mice were immunized with 10 μg of either Pfs25 or PfCSP mRNA LNPs followed by a challenge with sporozoites after one (a), two (b), and three and four immunizations (c), as indicated. Two additional groups of mice for each mRNA-LNP were immunized with lower doses (0.1 μg and 1 μg) mRNA-LNPs to assess minimum immunogenic doses of Pfs25 or PfCSP mRNA-LNPs. B3 collected at 11 weeks from mice immunized with 0.1, 1.0 and 10.0 ug doses of Pfs25 mRNA-LNP were used in SMFA. Bleeds FB1 (a), FB2 (b) and B4 (c) were used in SMFA to evaluate transmission blocking activity in mice receiving one, two or three immunizations, respectively. d shows the immunization parameters corresponding to the timelines in (a–c).

Fig. 7: Transmission reducing activity of antibodies elicited by lower doses and varying immunization schedules of Pfs25 mRNA-LNP.

a Purified IgG from pooled sera (bleed B3) from Balb/c mice immunized with 0.1 μg, 1 μg, and 10 μg Pfs25 mRNA-LNPs (Fig. 6c) were evaluated at indicated final concentrations by SMFA. The inset shows the geometric mean antibody titers, avidity indices, and IgG1/IgG2a ratios for the serum evaluated by SMFA. b SMFA data for purified IgG in FB1, FB2, and B4 (Fig. 6a–c) from Balb/c mice immunized with one, two and three doses of 10 μg of Pfs25 mRNA-LNP, respectively. The inset shows the antibody avidity indices and IgG1/IgG2a ratios for the serum evaluated by SMFA. Data points representing the oocyst counts from each mosquito, arithmetic means, and 95% confidence intervals are reported. N is the fraction of uninfected mosquitoes over the total number of mosquitoes dissected. %TRA is the mean reduction in oocysts in the presence of the test IgG compared with the IgG (1 mg/ml) from pre-immune mouse sera (Pre) used as a negative control. %TBA is the percent reduction in the proportion of infected mosquitoes in the presence of the test IgG compared with pre-immune mouse IgG negative control (Pre). Statistical analysis was performed using the Mann–Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

SMFA was conducted to evaluate the functional efficacy of the antibodies elicited by lower doses of Pfs25 mRNA-LNP (Fig. 7a). Purified IgG was evaluated at 1.0, 0.5, 0.25, and 0.125 mg/ml final concentrations, with IgG purified from pre-immune sera used as a negative control at 1 mg/ml. The IgG purified from the 1 μg and 10 μg Pfs25 mRNA-LNP immunization groups elicited nearly 100% blocking at the lowest concentration of 0.125 mg/ml IgG tested. IgG from the 0.1 μg Pfs25 mRNA-LNP immunized mice, appeared to be relatively less potent, eliciting 98–99% TRA only at higher (0.5 to 1.0 mg/ml) IgG concentrations, with decreasing TRA at lower concentrations (<0.25 mg/ml) of IgG tested.

We also characterized the antibody isotype and avidity in the sera used for the SMFA (Fig. 7a, inset). The avidity indices for the 0.1 μg, 1 μg, and 10 μg groups were not different from those seen earlier when higher immunizing doses up to 30 μg were evaluated (Fig. 4). Strikingly, the antibody isotype analysis did reveal rather unexpected IgG1 skewed responses with IgG1/IgG2a of 3.04, and 2.02 in the mice immunized with 0.1 μg and 1.0 μg Pfs25 mRNA-LNP, respectively. Consistent with the previous analysis, the 10 μg Pfs25 mRNA-LNP group elicited a balanced response, with an IgG1/IgG2a ratio of 1.16.

As above, we also re-examined the immunogenicity of the lower doses of the PfCSP mRNA-LNP vaccine. Mice were immunized with 0.1 μg, 1 μg, or 10 μg PfCSP mRNA-LNP (schedule in Fig. 6c), followed by an evaluation of antibody responses in bleeds (B3 and B5) and protective efficacy. There was a sharp increase in anti-PfCSP antibody response in bleed B3 from 0.1 μg to 1.0 μg, with geometric mean titers of 218,000, 1, 268,600, and 1,968,300 in mice immunized with three doses of PfCSP mRNA-LNP, respectively. Immunization with another dose did not appreciably boost antibody responses in the bleed B5 in the various groups, except for the 10 μg PfCSP mRNA-LNP group, which showed a ~50% increase in antibody titers (Fig. 8a).

Fig. 8: Evaluation of varying vaccine doses and immunization schedules of PfCSP mRNA-LNPs.

Details of immunization and challenge are described schematically in Fig. 6a–c. a Anti-PfCSP antibody responses from B3 and B5 (Fig. 6c) were analyzed by ELISA and are reported as the geometric mean endpoint titers with 95% confidence intervals. b Kaplan-Meier survival curves representing outcome of sporozoite challenge after three immunizations with PfCSP mRNA-LNP (N = 5 per groups). Mice after challenge were observed for 12 days by microscopic examination of giemsa-stained blood smears. Following the first challenge, mice were treated and immunized a fourth time and challenged as shown in Fig. 6c. c Kaplan-Meier survival curves after repeat challenge (N = 5 per group). Panels d shows antibody isotypes from the bleed, B5, and (e) shows antibody-antigen avidity index from the bleeds, B3 and B5 (Fig. 6c). Panels f, g, and h show Kaplan-Meier survival curves for mice immunized with 10 μg PfCSP mRNA-LNP (N = 5 per group except mice receiving 2 immunizations, N = 4) and challenged after each immunization (Fig. 6a–c), anti-PfCSP antibody isotypes and anti-PfCSP avidity index, respectively. Mann–Whitney U test was used for statistical analysis of antibody titers, while log rank (Mantel Cox) test was used for the analysis of survival curves (*p < 0.05; **p < 0.01).

To assess the protective efficacy of immunization with low doses of PfCSP mRNA-LNP, mice were challenged with sporozoites of PbPfCSP-GFPLuc. Even though significant levels of antibodies were detected, mice in the various dose groups immunized three times were not protected against the challenge infection. Blood-stage parasitemia was detected beginning on day 3 post-challenge in all the mice, however, mice immunized with 1 μg and 10 μg PfCSP mRNA-LNP revealed a one-day difference in the detection of parasites when compared with the Pfs25 mRNA-LNP control group, resulting in significant differences in survival curves between the Pfs25 control group and the 1 μg (p = 0.0145) and 10 μg (p = 0.0145) PfCSP mRNA-LNP groups (Fig. 8b). Given these results, we drug cured the challenged mice and immunized one more time, and subsequently challenged as per the schedule in Fig. 6c. As shown in Fig. 8c, 4 out of 5 mice in the 10 μg PfCSP mRNA-LNP group and 3 out of 5 mice in the 1 μg PfCSP mRNA-LNP group showed complete protection over 14 days period of monitoring blood-stage parasitemia. However, mice immunized with the 0.1 μg PfCSP mRNA-LNP were not protected against the challenge infection. The survival curves of uninfected mice of the 1 μg PfCSP mRNA-LNP group (p = 0.0031) and the 10 μg PfCSP mRNA-LNP group (p = 0.0031) were statistically different compared to the Pfs25 mRNA-LNP control group (Fig. 8c). There was no evidence that the survival curve of the 0.1 μg group was different from the Pfs25 mRNA-LNP negative control group (p = 0.0951). Results in Fig. 8d present evidence for a shift in antibody isotype profile from a balanced isotype response in the mice immunized with 0.1 and 1.0 μg dose which becomes IgG2a skewed in mice immunized with the higher 10 μg dose. Apart from this isotype profile shift, no significant difference was observed in the avidity index among the 0.1 μg, 1 μg and 10 μg PfCSP mRNA-LNP groups with indices of 1.72, 2.33 and 2.40. The avidity indices of sera collected prior to any challenge were 1.52, 1.91 and 1.80 (Fig. 8e) and the difference between bleeds are within the experimental variability of the assay, thus ruling out any modulation as a result of the exposure to the transgenic parasites used in the first challenge.

During the course of these studies, we also evaluated immunogenicity parameters and functional protective efficacy in mice immunized with different immunization regimens (1, 2, and 3 immunizations) of 10 μg of Pfs25 or PfCSP mRNA-LNPs (Fig. 6a–c). Figure 7b shows the results of transmission-blocking SMFA in the presence of purified IgG from mice immunized with 10 μg Pfs25 mRNA-LNP. IgG from sera after a single immunization (FB1) revealed only modest TRA. However, IgG from mice immunized two (FB2) or three times (B4) exhibited robust TRA. The Fig. 7b inset also shows data on antibody avidity and isotype for each bleed with results similar to those in earlier studies (Fig. 4). Results of protection against sporozoite challenge in groups of mice immunized with various regimens of 10 μg PfCSP mRNA-LNP are shown in Fig. 8f. Consistent with previous results showing the importance of a four-dose regimen (Figs. 5 and 8c), one, two, or three immunizations were not sufficient in giving any significant protection against sporozoite challenge (Fig. 8f). Further analysis of immune sera did reveal an antigen-specific effect of improved avidity with subsequent immunization. We first tested pooled sera after each immunization and then reconfirmed by testing individual mouse serum. As shown in Fig. 8h, the mean avidity indices increased from 0.908 to 1.25, and 1.886 in mice receiving one, two, or three immunizations of 10 μg PfCSP mRNA-LNP, respectively. A significant difference was detected between the mice that received one immunization and mice that received three immunizations (p = 0.0090). The isotype analysis revealed once again IgG2a skewed antibodies after three immunizations (Fig. 8g).

Analysis of splenic T cell and B cell responses in mice immunized with Pfs25 and PfCSP mRNA-LNPs

The cellular responses of each mRNA-LNP were investigated by the immunization of female Balb/c and C57Bl/6 mice. The C57Bl/6 mice were immunized with either one or two doses of either Pfs25 or PfCSP mRNA-LNP (1 μg or 3 μg) while the Balb/c mice were immunized with a single 3 μg dose of Pfs25 or PfCSP mRNA-LNP. Additionally, mice immunized with either recombinant Pfs25 or PfCSP proteins administered with the adjuvant, Alhydrogel, were used as a positive control. To confirm the immunogenicity of the immunization, antigen-specific antibody responses were determined from sera collected two weeks following each immunization (Fig. 9a and b). The C57Bl/6 mice immunized with 1 μg of Pfs25 mRNA-LNP were unresponsive two weeks following the first immunization while the C57Bl/6 mice immunized with 3 μg Pfs25 mRNA-LNP elicited antibody titers comparable to antibody responses of Balb/c mice. After a second immunization, antibody titers for all groups increased as expected.

Fig. 9: Evaluation of cellular responses in mice immunized with Pfs25 and PfCSP mRNA-LNPs.

Groups of C57Bl/6 and Balb/c mice (N = 5) were immunized with 1 μg and 3 μg of either Pfs25 or PfCSP mRNA-LNPs following one or two immunizations. Additional groups were immunized with two doses of 10 μg of rPfs25 and rPfCSP formulated with Alhydrogel, as positive controls. a Anti-Pfs25 antibody responses, and b Anti-PfCSP antibody responses evaluated by ELISA are reported as the geometric mean endpoint titers with 95% confidence intervals. Germinal center B (GCB) cells and antigen specific B cells were quantified by flow cytometry. The reagents used are found in Supplementary Table 1. and the gating strategy is reported in Supplementary Fig. 1. Panels c and d show quantification of GCB cells and Pfs25-specific B cells respectively in Pfs25 mRNA-LNP immunized mice. Panels e and f show the quantification of GCB cells and PfCSP-specific B cells, respectively in PfCSP immunized mice. Additionally for the Pfs25 immunized mice, the harvested splenocytes were stimulated with a pool of overlapping peptides of Pfs25 for 6 h and assessed by flow cytometry for T cell responses. The frequency of stimulated cytokine-producing T cell subpopulations is reported. Panel g shows CD4 T cell responses and panel h shows the CD8 T cell responses. Error bars for panels (c-h) represent the standard error of the mean.

The GC B cells and antigen-specific B cells were measured via flow cytometry of splenocytes collected from the immunized C57Bl/6 mice. Spleens collected from unimmunized mice were used as a control. The number of GC B cells was very low following one immunization and increased significantly following the second dose. In comparison, the amount of GC B cells of mRNA-LNP immunized mice were lower than GC B cells of rPfs25 immunized mice (Fig. 9c). The same trend occurred with Pfs25-specific B cells, with very low number of cells observed after the first immunization and higher amounts observed following the second immunization (Fig. 9d). For PfCSP immunized mice, modest amount of GC B cells was observed in mice immunized with one dose of PfCSP mRNA-LNP and increased significantly following the second dose (Fig. 9e). Two doses of PfCSP mRNA-LNPs elicited higher GC B cells than two doses of rPfCSP with Alhydrogel. Interestingly PfCSP-specific B cells were very low for all groups, yet were similar between all PfCSP mRNA-LNP immunized mice and did not increase following a booster dose (Fig. 9f). Additionally, all PfCSP mRNA-LNP immunized mice had higher PfCSP-specific B cells than those of rPfCSP immunized mice.

For Pfs25 mRNA-LNPs, T cell responses were evaluated. Splenocytes were harvested and cultured with or without stimulation with Pfs25 overlapping peptide pools and the frequency of proliferated splenic T cells producing key cytokines as a result of stimulation was determined. The C57Bl/6 mice immunized with one dose of Pfs25 mRNA-LNP elicited extremely low IL4 and IL5 producing CD4 T cells, while IFNγ, IL2, and TNFα producing CD4 T cells were elevated, showing a Th1 skewed response. C57Bl/6 mice immunized with two doses of Pfs25 mRNA-LNP elicited elevated IL2 and TNFα producing CD4 T cells compared with mice those receiving one dose. In contrast IFNγ producing CD4 T cells were similar between the one dose and two dose regimens (Fig. 9g). In C57Bl/6 mice, the CD8 T cells producing IFNγ, IL2, and TNFα were elevated in mice that received one dose but were low in mice that received two doses (Fig. 9h).

Additionally, strain-specific T cells responses were observed. The Balb/c mice which received one 3 μg dose had IFNγ producing CD4 T cells that were similar to the C57Bl/6 mice which received one 3 μg dose while IL2 and TNFα producing CD4 T cells were slightly lower (Fig. 9g). In contrast, IFNγ and IL2 producing CD8 T cells were significantly higher in Balb/c mice compared with C57Bl/6 mice (Fig. 9h). Thus a clear difference in T cell responses was observed between the two mouse strains.