Human sterile immunity to Plasmodium vivax malaria correlates with specific antibody response elicited by immunization with radiation-attenuated sporozoites

doi:10.21203/rs.3.rs-3504086/v1

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Human sterile immunity to Plasmodium vivax malaria correlates with specific antibody response elicited by immunization with radiation-attenuated sporozoites

https://doi.org/10.21203/rs.3.rs-3504086/v1

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published 02 Feb, 2024

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Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S vaccine. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy+) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found that PvRAS group seroreactivity was lower in protected than non-protected volunteers. Nevertheless, protected volunteers showed higher reactivity to PvCSP and other antigens. In Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes, parasite reactivity increased throughout immunizations. Mock-vaccinated Fy + volunteers developed a vigorous response to CHMI. These findings allowed the identification of novel parasite antigens currently being pursued as vaccine candidates.

Biological sciences/Immunology/Infectious diseases

Biological sciences/Immunology/Vaccines

Malaria continues to be an important source of morbidity and mortality globally. In 2021, about 247 million clinical cases and 619,000 malaria-related deaths were estimated worldwide, with Plasmodium falciparum as the most prevalent parasite species ¹. Nevertheless, 4.9 million P. vivax cases were estimated in the same year, mainly in the Americas, Southeast Asia, and Oceania ¹. Significant effort has been invested in P. vivax malaria research, which despite multiple technical and financial constraints, has recently indicated the feasibility of an effective PvCS-based pre-erythrocytic vaccine ^2,3. Although emulating the immune mechanisms of protection is essential for vaccine development, they remain poorly understood ^4–6.

Naturally acquired clinical immunity to malaria is a slow process that occurs after repeated exposures to the parasite in endemic areas and rapidly wanes after individuals leave the endemic sites ⁷. Although sterile immunity is never achieved under natural conditions, it can be reproducibly induced by immunization via mosquito bites with radiation-attenuated sporozoites (RAS) ^8–15. This immunization approach induces immune responses that block sporozoite (spz) invasion of hepatocytes and subsequent schizogonic development in the liver. This prevents malaria disease caused by asexual parasite blood stages and further transmission mediated by sexual blood stages. Moreover, genetically (GAP) and chemically attenuated (CAP) P. falciparum and rodent malaria parasites have confirmed the protective efficacy of whole attenuated parasites ^16–24.

The high protective efficacy experimentally demonstrated using these whole attenuated parasites is probably due to the breadth of parasite antigens simultaneously exposed to the immune system. However, current methods to attenuate and deliver attenuated parasites remain challenging, indicating the need to further identify parasite antigens involved in protection that could be developed as subunit vaccines ^21,22. Antibodies to several P. falciparum antigens identified in RAS immunization as probably associated with protection have been the subject of intense research on developing subunit vaccines ^{21,22,25–27}. The recent approval of the P. falciparum RTS, S by WHO as well as the progress achieved by the Pf-R21/MM and, more recently, a PvCS formulation underscore the great value of subunit vaccines ^4,21,28–30

In contrast to P. falciparum, the overall progress in P. vivax vaccine development and the identification of antibodies against P. vivax has been limited to relatively few proteins made available through traditional cloning methods or peptide synthesis. Only a few of the ~ 5,500 genes encoded by the P. vivax genome have been studied as potential vaccine candidates. Currently, only three P. vivax antigens, the circumsporozoite protein (PvCSP) ^{12,28,31–33}, sexual-stage ookinete surface protein (Pvs25)^2,3,34, and P. vivax Duffy-binding protein (PvDBP) ³⁵ have reached vaccine clinical development phases (Phase Ia, Phase IIa/b). However, several others are in preclinical development and discovery phases ^{21,29,30,36,37}. Nevertheless, which of these are responsible for protection remains unclear.

To determine the feasibility of controlled vaccine clinical trials using whole attenuated P. vivax sporozoites, and their protective efficacy, a phase II trial was conducted ^10–12. The trial assessed the safety and protective efficacy of human immunization with PvRAS in Duffy-positive (Fy+) malaria-unexposed adult volunteers, using as controls mock-vaccinated Fy + individuals and Fy- exposed to non-attenuated sporozoites (Fig. 1). This study revealed that PvRAS inoculation was immunogenic, as volunteers developed antibodies and IFNγ specific to PvCSP and induced sterile immunity in 42% (5/12) of the Fy + volunteers ^10–12. This trial generated valuable reagents to investigate the mechanisms of immune protection and identify relevant parasite antigens. Here we report the breadth of the serologic response using a protein microarray displaying 515 P. vivax exon products tested with serum samples collected during this trial to characterize the antibody responses induced by PvRAS and their association with protection. The parasite proteins displaying the highest difference in reactivity between protected and non-protected volunteers are described and being further characterized ¹⁰, ³⁷.

Study population characteristics

Study sera from adults without previous malaria exposure, who were exposed to seven immunization doses of either PvRAS, live sporozoites, or mock immunizations using mosquito bites were analyzed. Detailed information about the study participants, immunization schedule, and CHMI was previously reported¹¹. Briefly, none of the volunteers developed clinical malaria or microscopic parasitemia during the immunizations; however, low levels of parasite DNA were detected by qPCR in peripheral blood after immunizations in all Fy- volunteers, which resolved spontaneously. At day 60 post-CHMI, 5/12 volunteers of the PvRAS group (42%) were fully protected from the CHMI, while both mock-immunized and Fy + control individuals developed parasitemia determined by microscopy and confirmed by qPCR. As expected, none Fy- volunteers developed malaria infection after the CHMI.

Antibody response after PvRAS immunization

To identify the antibody responses induced by vaccination with PvRAS, serum samples taken ten days after each of seven immunizations were probed against a custom P. vivax protein microarray¹⁰. Although reactivity levels were low, the fluorescence intensity increased as immunizations continued (Fig. 2). The number of reactive antigens was variable among the volunteers, ranging from 0 to 14% ten days after the first immunization and from 1.4–38% after the complete immunization process and CHMI (Fig. 3).

Taking advantage of the fact that five of twelve volunteers immunized with PvRAS did not develop malaria after the CHMI and aiming to identify correlates of protection, we separately analyzed the data of protected and non-protected groups. Overall, the reactivity to the antigens on the P. vivax array was lower in protected volunteers (F = 43; p < 0.0001; Fig. 3a) and they recognized fewer antigens than non-protected individuals (Fig. 3b). However, a group of ten proteins, mostly hypothetical, displayed significantly higher reactivity in the protected volunteers (Table 1, Table S1). Reactivity against PvCSP (141.6 vs. 2170.3; p = 0.002) and one hypothetical protein (PVX_089630; 400.2 vs. 4523.1; p < 0.001) increased significantly at the seventh immunization but remained lower in the non-protected than in the protected volunteers during the immunization period (Table 1, Fig. 4). Likewise, we observed higher reactivity to several hypothetical proteins (n = 6) compared to PvCSP in protected volunteers, encouraging further characterization, with emphasis on their potential value as P. vivax pre-erythrocytic vaccine candidates ³⁷ as reported by other studies ^45,46 (Fig. S1). When tested by ELISA using a PvCSP-derived long synthetic peptide (PvCSP-NRC), all PvRAS volunteers displayed specific antibodies against it after the second immunization, which remained positive during the immunization period ¹⁰; more importantly, the specific IgG1 response to this peptide was significantly higher in protected than in non-protected individuals. A group of 13 antigens with higher reactivity was identified in the non-protected volunteers six months after CHMI (Table 2).

Table 1

Antigens significantly recognized by the protected PvRAS volunteers during the immunizations.
ORF PlasmoDB ID	Product description	Exon	Average normalized fluorescence intensity ^a		p value^b
ORF PlasmoDB ID	Product description	Exon	Non-protected	Protected	p value^b
			Post-1st	Post-1st
PVX_001000	Hypothetical protein, conserved	1 of 1	199.2	11871.9	< 0.0001
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	508.2	5051.2	< 0.0001
PVX_085025	Hypothetical protein, conserved	1 of 1 S2	303.9	2008.1	0.005
			Post-2nd	Post-2nd
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	864.4	2998.2	< 0.0001
PVX_089630	Hypothetical protein, conserved	1 of 5	477.4	1746.4	0.002
PVX_092570	Hypothetical protein, conserved	1 of 3	296.8	1341.4	0.009
PVX_094650	RAD protein (Pvfame)	2 of 2	180.7	1002.2	0.039
			Post-3rd	Post-3rd
PVX_085025	Hypothetical protein, conserved	1 of 1 S2	156.7	4651.9	< 0.0001
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	355.7	4186.5	< 0.0001
PVX_089630	Hypothetical protein, conserved	1 of 5	251.6	1652.1	0.008
			Post-4th	Post-4th
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	2477.5	3550.5	0.004
PVX_085025	Hypothetical protein, conserved	1 of 1 S2	148.7	1156.7	0.007
			Post-5th	Post-5th
PVX_089630	Hypothetical protein, conserved	1 of 5	1487.4	5860.7	< 0.0001
PVX_091105	Membrane associated calcium-binding protein, putative	1 of 1	93.2	4111.4	< 0.0001
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	675.8	3301.9	< 0.0001
PVX_119355	Circumsporozoite protein (CSP)	1 of 1	398.2	2301.1	< 0.0001
			Post-6th	Post-6th
PVX_089630	Hypothetical protein, conserved	1 of 5	1117.0	4837.5	< 0.0001
PVX_113825	Hypothetical protein, conserved	1 of 1 S3	403.4	2582.2	< 0.0001
PVX_119355	Circumsporozoite protein (CSP)	1 of 1	375.7	2379.1	< 0.0001
PVX_084580	Kinesin, putative	3 of 3 S1	414.5	1245.4	0.016
			Post-7th	Post-7th
PVX_089630	Hypothetical protein, conserved	1 of 5	2239.7	4523.1	< 0.0001
PVX_119355	Circumsporozoite protein (CSP)	1 of 1	538.1	2170.3	0.0007
			Post-CHMI	Post-CHMI
PVX_110950	Hypothetical protein, conserved	1 of 1	1301.1	12352.4	< 0.0001
^a the raw signal intensity was reduced by its corresponding median IVTT-control value; ^b multiple comparison test without p-value correction.

Table 2

**Antigens significantly recognized six months after the CHMI by the non-protected** PvRAS volunteers.
ORF PlasmoDB ID	Product description	Exon	Average normalized fluorescence intensity ^a
ORF PlasmoDB ID	Product description	Exon	Non-protected	Protected
PVX_117680	Hypothetical protein	1 of 2	14796.7	3214.1	< 0.0001
PVX_111065	Early transcribed membrane protein (ETRAMP)	1 of 1	14321.2	1083.8	< 0.0001
PVX_083560	Hypothetical protein, conserved	2 of 2	13763.9	245.1	< 0.0001
PVX_085590	Hypothetical protein, conserved	1 of 1 S5	11158.4	1746.7	< 0.0001
PVX_097730	Hypothetical protein, conserved	1 of 1	11271.9	29.7	< 0.0001
PVX_084595	DNA replication licensing factor MCM8, putative	2 of 2 S2	11489.1	874.3	< 0.0001
PVX_000930	Sexual stage antigen s16, putative	1 of 1	10366.7	192.1	< 0.0001
PVX_099980	Merozoite surface protein 1 (MSP1)	1 of 1 S2	10753.2	1076.9	< 0.0001
PVX_118705	Hypothetical protein, conserved predicted Pf homolog liver stage antigen 3	1 of 1	10001.6	410.2	< 0.0001
PVX_090230	Early transcribed membrane protein (ETRAMP)	1 of 2	8731.8	193.1	< 0.0001
PVX_119535	Hypothetical protein, conserved	1 of 1	8661.8	110.4	< 0.0001
PVX_003830	Serine repeat antigen 5 (SERA5)	4 of 4	8302.4	518.4	< 0.0001
PVX_114060	Transport protein particle (TRAPP) component, Bet3, putative		8025.4	343.9	< 0.0001
^a the raw signal intensity was reduced by its corresponding median IVTT-control value; ^b multiple comparison test without p-value correction.

In contrast, in the mock-immunized control group, there was no significant difference in the average fluorescence intensity between immunizations (Fig. 2). However, significantly increased reactivity to 18 proteins, including two members of the MSP family (PvMSP1, PvMSP10), SERA and eight hypothetical proteins with unknown function was observed after CHMI (Table 3).

Table 3

**Antigens recognized by the mock-immunized (non-infected mosquitoes) Fy + control group before and after the CHMI.**
ORF PlasmoDB ID	Product description	Exon	Average normalized fluorescence intensity ^a		p value^b
ORF PlasmoDB ID	Product description	Exon	Post-7th	Post-CHMI	p value^b
PVX_118705	Hypothetical protein, conserved predicted Pf homolog liver stage antigen 3	1 of 1	0.0	28681.7	< 0.0001
PVX_083560	Hypothetical protein, conserved	2 of 2	0.0	26579.7	< 0.0001
PVX_001665	Hypothetical protein, conserved	1 of 1	243.9	25159.6	< 0.0001
PVX_097730	Hypothetical protein, conserved	1 of 1	0.0	22178.7	< 0.0001
PVX_114145	Merozoite surface protein 10 (MSP10)	1 of 1	0.0	19252.2	< 0.0001
PVX_084595	DNA replication licensing factor MCM8, putative	2 of 2 S2	0.0	18429.7	< 0.0001
PVX_085025	Hypothetical protein, conserved	1 of 1 S2	0.0	16739.6	< 0.0001
PVX_121930	Hypothetical protein, conserved	2 of 2	0.0	13723.7	< 0.0001
PVX_110880	DNA repair helicase, putative	2 of 3	0.0	11836.7	0.0002
PVX_121885	Cytoadherence linked asexual protein, CLAG, putative	5 of 8	0.0	11652.7	0.0002
PVX_099980	Merozoite surface protein 1 (MSP1)	1 of 1 S2	0.0	10592.7	0.0008
PVX_084305	Hypothetical protein, conserved	1 of 1 S1	0.0	10549.7	0.0008
PVX_084625	P-type ATPase4, putative	1 of 1 S1	0.0	8761.2	0.0053
PVX_085590	Hypothetical protein, conserved	1 of 1 S5	0.0	7942.2	0.012
PVX_091105	Membrane associated calcium binding protein, putative	1 of 1	0.0	7280.7	0.022
PVX_116915	Exported protein 2	3 of 3	0.0	6840.7	0.030
PVX_003805	Serine repeat antigen (SERA), putative	4 of 4	0.0	6552.7	0.037
PVX_084720	Hypothetical protein, conserved	1 of 1	0.0	6200.2	0.048
^a the raw signal intensity was reduced by its corresponding median IVTT-control value; ^b multiple comparison test without p-value correction.

Antibody response in Fy- individuals

None of the Fy- volunteers developed patent parasitemia upon CHMI, as determined by microscopic examination of thick blood smears. However, these volunteers developed fever and malaise after the first few (n = 3) immunizations, and parasite DNA was detected by real-time qPCR. As tested by ELISA using the PvCSP-NRC peptide, seroconversion was observed in all Fy- volunteers between the second and fifth immunizations. In addition, they all developed antibodies against PvMSP-1 after seven immunizations as tested by ELISA ¹⁰. Moreover, although the reactivity to the antigens on the P. vivax array was very low after the first immunization (Fig. 2), the second immunization significantly increased reactivity against 28 proteins (Table 4). Reactivity against antigens was maintained or increased throughout the immunization schedule, peaking at the seventh immunization (Fig. 5a). The third immunization induced reactivity against two new antigens (PVX_117680 and PVX_091785); one more after the fifth immunization (PVX_117150); and only one (PVX_091970) after CHMI (Table S2). Although the Fy- volunteers individuals did not develop malaria after CHMI, the number of reactive antigens was higher than in the PvRAS group, and as expected in the mock-immunized controls (Fig. 5b).

Table 4

Antigens differentially recognized by the Fy- volunteers between the first and second immunizations.
ORF PlasmoDB ID	Product description	Exon	Average normalized fluorescence intensity ^a		p value^b
ORF PlasmoDB ID	Product description	Exon	Post-1st	Post-2nd	p value^b
PVX_099980	Merozoite surface protein 1 (MSP1)	1 of 1 S2	122.6	28078.0	< 0.0001
PVX_097625	Merozoite surface protein 8 (MSP8)	1 of 1	0.0	22691.4	< 0.0001
PVX_090090	CW type zinc finger domain containing protein	1 of 1 S1	300.3	10973.7	< 0.0001
PVX_091970	Deoxyuridine 5'triphosphate nucleotidohydrolase, putative	1 of 1	28.4	9951.4	< 0.0001
PVX_000860	Hypothetical protein, conserved	2 of 6 S2	56.7	9722.3	< 0.0001
PVX_122810	Hypothetical protein, conserved	1 of 1 S1	177.9	8211.9	< 0.0001
PVX_003840	Serine repeat antigen 3 (SERA3)	4 of 4	125.9	7939.7	< 0.0001
PVX_000995	Transmission blocking target antigen Pfs230, putative	1 of 1	2328.3	7799.7	< 0.0001
PVX_082645	Merozoite surface protein 7 (MSP7)	1 of 1	50.1	7035.2	< 0.0001
PVX_085930	Rhoptry-associated protein 1, putative	2 of 2	1088.9	6142.4	0.0003
PVX_003770	Merozoite surface protein 5 (MSP5)	1 of 2	3.5	5928.3	< 0.0001
PVX_099520	Ubiquitin-like protein, putative	3 of 8	1136.4	5688.8	0.0009
PVX_093650	Mannose-6-phosphate isomerase, putative	1 of 1	309.5	5385.4	0.0002
PVX_119355	Circumsporozoite protein (CSP)	1 of 1	1677.7	5261.3	0.009
PVX_099315	78 kDa glucose-regulated protein precursor (GRP 78), putative	2 of 2	5.61	4693.0	0.0007
PVX_091910	kelch domain containing protein	1 of 4	285.0	4761.9	0.001
PVX_003565	Early transcribed membrane protein (ETRAMP)	1 of 1	0.0	4442.6	0.001
PVX_085960	RNA binding protein, putative		0.0	4353.6	0.001
PVX_000610	Hypothetical protein, conserved	1 of 2	0.0	4000.5	0.004
PVX_086015	Hypothetical protein, conserved	1 of 2 S6	57.3	3920.9	0.005
PVX_090215	Hypothetical membrane protein, conserved	1 of 2	96.6	3853.0	0.007
PVX_114365	Hypothetical protein, conserved	1 of 1	0.0	3832.1	0.006
PVX_118705	Hypothetical protein, predicted Pf homolog liver stage antigen 3 (LSA3)	1 of 1	169.3	3743.2	0.010
PVX_084625	P-type ATPase4, putative	1 of 1 S1	0.0	3295.4	0.017
PVX_091545	Heat shock protein 90, putative	1 of 1	0.0	3146.2	0.023
PVX_122425	M1-family amino peptidase, putative		182.3	3043.3	0.038
PVX_085590	Hypothetical protein, conserved	1 of 1 S5	0.0	2943.4	0.033
PVX_115070	Hypothetical protein, conserved	1 of 1	0.0	2855.7	0.039
^a the raw signal intensity was reduced by its corresponding median IVTT-control value; ^b multiple comparison test without p-value correction.

The findings of this study may aid in the discovery of novel P. vivax antigens with potential for vaccine development with capacity to prevent the infection ^34,47,48. To our knowledge, this is the first time that sera from a PvRAS clinical trial demonstrating significative sterile immunity have been screened for breadth of antibody response to identify parasite proteins associated with P. vivax malaria infection prevention. Although vaccines targeting other stages of the parasite cycle are important, in the case of P. vivax, due to the development of liver hypnozoites, and its consequent relapsing behavior pre-erythrocytic vaccines capable of inducing sterile immunity are of the utmost importance.

Although the original PvRAS aimed at delivering ten immunizations, the complex logistics imposed by the lack of P. vivax in vitro culture and the need for parasites from clinical infections reduced the immunization schedule to seven doses ¹¹. Clinical trials with PfRAS using in vitro adapted parasite clones are highly feasible and reproducible leading to > 77%- >90% sterile protection of volunteers ^49,50. Nevertheless, PvRAS vaccination led to 42% (5/12) protection, providing the opportunity to compare the seroreactivity of protected and non-protected volunteers. In both vaccination groups, the reactivity increased as immunization progressed, confirming the dose-response effect.

This study profited from the access to Fy + and Fy- volunteers which were included to attempt dissecting the early and late immune responses elicited during the P. vivax liver cycle. While Fy + individuals support the complete P. vivax cycle, both pre-erythrocytic and erythrocytic phases, Fy- individuals allow the complete parasite liver cycle but not the erythrocytic phase which is arrested upon parasite entry in the blood circulation, due to the lack of the Duffy Antigen Receptor for Chemokines (DARC/Fy receptor) on the erythrocyte surface, required for merozoite invasion ⁵¹; therefore, the immune response to P. vivax is expected to differ in these two populations. Additionally, including volunteers subjected to mock immunization allowed the dissection between parasite specific responses and the potential influence of mosquito saliva proteins in the specific immune response to the parasite antigens.

Regarding the human vaccination with Plasmodium RAS, it has been demonstrated that attenuated parasites get arrested early during the liver phase and that therefore, the immune responses elicited by RAS and responsible for sterile-protection presumably target parasite antigens expressed during the early hepatic schizogony. Indeed, the absence of microscopic or parasite DNA detection by qPCR during the immunization period in Fy + volunteers indirectly confirms the complete parasite radiation attenuation, and presumably the early parasite arrest ⁵²; in this context, no responses are expected to arise against blood parasite stages.

The comparison of antibody responses induced by PvRAS sterilely protected and non-protected volunteers revealed a notably lower reactivity in the protected volunteers. However, after probing serum from both groups in the custom P. vivax protein microarray, the antibody response profile associated with sterile immunity revealed ten proteins with the highest reactivity, notably for the PvCSP and the PVX_089630 proteins. Responses to these two antigens were significantly lower in the non-protected volunteers, suggesting them as potentially responsible for protection. Indeed, a PvCSP formulation recently evaluated in naïve and semi-immune volunteers conferred sterile protective efficacy of 35% and 40% and overall protection of 55% and 60%, respectively ⁵³. The extraordinary boost in the immune response induced by the CHMI was particularly intriguing, considering that it was performed with a limited number of parasites (2–4 mosquito bites). Again, this robust antibody boosting was more evident in non-protected volunteers, which might be explained by the parasite growth and multiplication in that group, which obviously did not occur in the sterile protected volunteers.

As expected, in the case of the Fy- control group, despite the massive inoculation of live sporozoites during immunization, did not develop patent microscopic parasitemia. However, parasite DNA was detected in blood by qPCR during the first three vaccination doses, but not after; this observation indicated that these three sporozoite immunization doses were sufficient to induce sterile immunity¹¹. In this group, seroconversion was observed between the second and fifth immunizations, notably to PvCSP and PvMSP-1, but also to other 28 proteins. Overall, the number of reactive antigens was higher than in the PvRAS group, which is reasonable as volunteers were exposed to seven doses of live sporozoites. Surprisingly, a few antigens were only recognized once during immunization or after CHMI (PVX_117680, PVX_091785, PVX_117150, PVX_091970). This group displayed an important antibody boosting upon CHMI despite the significantly lower number of sporozoites inoculated during the 2–4 mosquitoes used for the CHMI.

It has been reported that Fy- individuals from Madagascar, Cameroon, and Ethiopia may develop parasitemias when infected by P. vivax ^54,55. However, all Fy- volunteers in this study were refractory to blood infection by P. vivax. Samples from those volunteers allowed the evaluation of antibody response elicited specifically against pre-erythrocytic stages. Notably, after the second immunization, the reactivity against a group of antigens was maintained or increased through the immunization schedule, suggesting that immunization with heterologous parasites induces a similar antibody response. Due to the need to use P. vivax parasites from natural clinical infections, each immunization is potentially a genetically different parasite, despite the limited number of circulating antigenic variants from which the blood samples to infect mosquitoes were obtained ¹². In addition, the presence of antibodies against several members of the MSP family and other antigens in the asexual blood stages indicates the presence of asexual parasites in peripheral blood. Although these stages cannot invade and replicate in Fy- erythrocytes, the number of parasites (merozoites) released from the liver is likely enough to elicit the production of detectable antibodies. This agrees with minor symptoms and parasite DNA found in the peripheral blood 8 to 16 days after the initial immunizations (second to fourth). Since several of the antigens identified in this study are hypothetical proteins, further studies should be carried out to better characterize them.

Although counter-intuitive, observing lower reactivity in the PvRAS protected group is not unusual. Recent studies on the response of individuals from malaria-endemic and non-endemic areas to Pf-RTS,S and PvCS showed a similar hypo-responsiveness^5,42,43,53 suggesting that individuals from malaria-endemic regions, either actively infected or not, display an altered basal immune status with a paucity of regulatory mechanisms and altered memory cell function leading to lower responsiveness to vaccines. It appears to correspond to an immunological imbalance caused by permanent exposure to malaria parasites, mosquito bites, and potentially other host and environmental factors that may influence the host’s immune response and immunity to malaria ^5,6,41,56. A similar trend was present in volunteers subjected to a P. vivax CHMI, in which individuals naturally exposed to P. vivax malaria with parasitemia and no fever (i.e., clinically protected) had lower reactivity compared to those with fever (i.e., clinically not protected) ¹⁰. Moreover, this finding is also consistent with those from P. falciparum vaccination studies in humans where protected individuals did not mount a significant antibody response to CHMI, whereas non-protected individuals had elevated signals to many blood-stage antigens ^34,47 perhaps indicating a decrease in strength and variety of exposure to antigens due to earlier control and arrest of parasitic development in protected individuals. This may also hint at the importance of the cellular immune response in achieving protection, which was not assessed in this study.

Despite most of the reactive proteins being hypothetical, a high response was observed against PvCSP, a protein initially discovered by induction of the circumsporozoite precipitation reaction by sera from mice immunized with P. berghei-RAS. Since its discovery, PfCSP has been the most extensively studied malaria antigen, leading to the only vaccine approved by the WHO for mass use ⁵⁷. Moreover, PvCSP has been the subject of extensive studies ^29,32,58 and likely represents the most advanced P. vivax vaccine candidate, with important protective efficacy ⁵³. Notably, we observed higher reactivity to several hypothetical proteins compared to PvCSP in protected volunteers, encouraging further characterization, with emphasis on their potential value as P. vivax pre-erythrocytic vaccine candidates as reported by other studies ^45,46.

Although most antibodies are short-lived, those found six months after CHMI can be used as markers of recent malaria infection. Indeed, one of those antigens (PVX_083560) was found previously in semi-immune individuals and was higher in those without fever at day 45 after CHMI¹⁰, thus it might be related to clinical protection. Nevertheless, whether the antigens found here remain for a longer period or whether they protect against clinical symptoms in future episodes is unknown.

Another interesting finding was the reactivity of sera from the mock-immunized control group, which was only exposed to mosquito saliva during immunization. Despite a low reactivity and no significant difference in the average fluorescence intensity between immunizations, all volunteers presented a robust reactivity with 18 parasite proteins, after volunteers’ exposure to parasite antigens upon CHMI. Whether this corresponds to a primary response to parasite antigens or a secondary (boosting) response to cross-reacting mosquito-parasite antigens remains to be determined. It is known that mosquito saliva activates innate and adaptative immune host immune responses at the biting site by activating neutrophils, monocytes, and dendritic cells and increasing both Th1/Th2 and T cell regulatory responses ^39,56. However, little is known about parasite antigens that could be expressed in the mosquito and released in the saliva ⁵⁹. Therefore, these findings open new avenues to studying the mosquito-parasite interaction.

Apart from the studies reported here on the identification of pre-erythrocytic antigens recognized by vaccinated volunteers, previous studies have focused on the analyses of parasite antigens associated with clinical protection induced by natural exposure to the parasite in endemic areas of Papua New Guinea (PNG) ^45,46. In those studies, authors selected the 20 most likely associated with protection from a total of 342 P. vivax antigens, from which four were merozoite surface antigens which were also recognized by sera from our PvRAS trial upon challenge and by Fy- volunteers vaccinated with live sporozoites. In addition, the PvCSP that was consistently recognized from early after sporozoite immunization was also reactive by clinical samples from PNG^45,46.

In summary, we identified a group of P. vivax antigens whose antibody responses are elevated after RAS exposure and appear to contribute to sterile immunity. We also identified candidate proteins to detect previous malaria exposure due to the more durable humoral response they elicit. Taken together, these findings contribute to understanding the antibody response to P. vivax infection, particularly to the correlates of protection. Deeper analyses are required for the identification of potential surrogate markers or signatures of immune protection using systems biology ^5,30,60,61

Ethics statement

The trial from which the samples used here were obtained was approved by the Institutional Review Boards of Centro Médico Imbanaco and the Malaria Vaccine and Drug Development Center (MVD/CECIV, No 0104 of 2009) (Cali, Colombia). Written informed consent (IC) was obtained from all volunteers, and only samples from volunteers who authorized IC their use in further studies were included. The methods involving human subjects were conducted with the applicable guidelines, regulations and in accordance with the Declaration of Helsinki. The clinical trial was registered at clinicaltrials.gov (registry number NCT01082341).

Study participants and sample collection

We used serum samples stored at the MVDC/CIV cryobank collected from volunteers participating in a former Phase II clinical trial conducted to assess the protective efficacy of PvRAS immunization in adults, healthy, malaria-unexposed volunteers ¹¹. We analyzed samples from volunteers with a complete immunization scheme followed by a CHMI, including 12/14 Fy + experimental volunteers, known to be susceptible to P. vivax infection, who were immunized with PvRAS delivered by mosquito bites, and 3/7 Fy + control volunteers exposed to the bites of non-infected mosquitoes (mock-vaccination). In addition, 3/7 Fy- volunteers, known to be refractory to the erythrocyte infection by P. vivax asexual blood forms (merozoites) but susceptible to sporozoite liver invasion and exposed to the bite of sporozoite infected non-irradiated mosquitoes ¹¹. All volunteers were subjected to seven immunizations with a mean of ~ 65 mosquito bites/dose, as shown in Fig. 1, and eight weeks after the last immunization, volunteers were subjected to a P. vivax CHMI as previously described ¹¹. The CHMI was carried out by exposing volunteers to 2–4 non-irradiated, P. vivax-infected mosquito bites until mosquitoes were blood-engorged. Starting on day five post-infection, volunteers were followed up for the development of patent infection using microscopic examination of Giemsa-stained thick-blood smears; curative antimalarial treatment was provided immediately to volunteers who developed patent parasitemia. Protected volunteers were also antimalarial drug-treated when the study was completed, at day 60 post-CHMI. Serum samples collected ten days after each immunization and six months after the CHMI were analyzed.

Protein microarray

A custom protein microarray (Pf/Pv500) displaying 515 P. vivax and 500 P. falciparum reactive exon products expressed on pre-erythrocytic and asexual parasite blood stages was purchased from Antigen Discovery Inc. (Irvine, CA) ³⁸. Although volunteers’ samples were hybridized to the whole array, we only present data for P. vivax antigens. Microarray information is publicly available on the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and is accessible through accession number GPL18316. Annotation of proteins presented in this study follows gene accession numbers published on PlasmoDB (www.plasmodb.org). Of 515 P. vivax features on the array, 444 mapped to unique P. vivax proteins, of which the majority (247; 56%) were classified as hypothetical or hypothetical conserved proteins. The P. vivax content for the array used was down-selected from the P. vivax 4,506-protein microarray by probing with highly reactive sera representative of different malaria-endemic populations worldwide, as detailed previously ^{31,36,39–43}. Arrays were produced as previously described ^43,44. Briefly, the proteins were produced in an Escherichia coli-based cell-free in vitro transcription/translation system (IVTT RTS 100 E. coli HY kits; 5-Prime). Each array contained multiple (n = 24) negative reaction spots lacking plasmid template expression (IVTT-control), providing a donor-specific ‘background’ signals to normalize data between individuals. Arrays also included anti-IgG and IgG spots that served as controls for the presence of primary and secondary antibodies, respectively.

For probing, serum samples were diluted 1:100 in protein array blocking buffer (Maine Manufacturing, Sanford, ME) supplemented with E. coli lysate (GenScript, Piscataway, NJ) to reach a final concentration of 10 mg/mL, and pre-incubated at room temperature (RT) for 30 min. In parallel, arrays were rehydrated in a blocking buffer (without lysate) for 30 min. Arrays were probed with pre-incubated serum samples overnight at 4°C with gentle agitation and then washed at RT five times with TBS-0.05% Tween 20 (TBST), followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:200 in blocking buffer for one hour at RT. After incubation with secondary antibodies, arrays were washed three times in TBST, and bound IgG was visualized using streptavidin-conjugated SureLight P-3 (Columbia Biosciences, Frederick, MD) diluted 1:1000 in blocking buffer for 45 min at RT in the dark. Arrays were washed three times with TBST, and once with water. Chips were air-dried by brief centrifugation and scanned in a GenePix 4200AL laser scanner (Molecular Devices, Sunnyvale, CA). All samples in this study were probed simultaneously on the same batch of arrays.

Data analysis

The protein microarray data was accomplished following our previously published computational methods ^10,34,43,44. Briefly, microarray spot intensities (median fluorescence intensity, MFI) were quantified using ScanArray Express software (Perkin Elmer, Waltham, MA). The antibody reactivity analysis was carried out as follows: (i) the median background signal of IVTT-control spots was calculated for each sample; (ii) the raw signal intensity was reduced by its corresponding median IVTT-control value and defined as normalized fluorescence intensity. Samples with negative values were treated as zero fluorescence intensity. Normalized data were used for statistical analyses and figures. To identify differentially reactive proteins between groups and time points, multiple comparison tests without p-value correction were used (Prism v9.5, GraphPad Software Inc., La Jolla CA). A p-value < 0.05 was considered statistically significant. Antigens were considered reactive if the fluorescence intensity of an individual (or the average for a group of individuals) was higher than a cut-off defined as the average plus two standard deviations of the reactivity to all P. vivax antigens in the mock-immunized control group.

Data availability

All relevant data are within the paper and its Supplementary Information files. Microarray information is publicly available on the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and is accessible through accession number GPL18316. The clinical trial is registered with ClinicalTrials.gov, number NCT01082341.

Acknowledgments

The authors express their sincere gratitude to the volunteers who participated in this study. We wish to give special acknowledgment to the MVDC team that provided the logistical support for this study and the staff at Instituto de Salud del Pacífico (INSALPA) in Buenaventura (Colombia) for their superb technical support. We thank Imbanaco - QuironSalud; (clinical support), Hospital Universitario del Valle (for mosquito irradiation).

Funding

This trial was supported by the National Heart, Lung and Blood Institute at the National Institutes of Health (5R01HL086488; SH), the Colombian National Research Council, COLCIENCIAS (527-2009; SH), and the National Institute of Allergy and Infectious Diseases through grants (AI089686) International Centers of Excellence for Malaria Research, and (5U01AI155363-03) PSMaV program. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

PLF, SH, MAH, conceived and designed the experiments. MLP, AJ, DHD, JMVJ performed the experiments. MLP, AJ, DHD analyzed data. PLF, DHD contributed reagents/materials/analysis tools. MLP drafted the manuscript. MLP, AJ, DHD, JMVJ, SH, JO, PLF, SMH, MAH reviewed, edited, and approved the final manuscript.

Additional Information

Competing of interest

PLF and DHD have patent applications pertaining to this work and PLF owns stock in Antigen Discovery Inc. that has licensed the protein microarray technology described here. The other authors declare that they have no competing interests.

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No competing interests reported.

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Human sterile immunity to Plasmodium vivax malaria correlates with specific antibody response elicited by immunization with radiation-attenuated sporozoites

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Abstract

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Introduction

Results

Study population characteristics

Antibody response after PvRAS immunization

Antibody response in Fy- individuals

Discussion

Methods

Ethics statement

Study participants and sample collection

Protein microarray

Data analysis

Declarations

Data availability

Acknowledgments

Funding

Author contributions

Additional Information

Competing of interest

References

Additional Declarations

Supplementary Files

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