Development of an anti-Pfs230 monoclonal antibody as a Plasmodium falciparum gametocyte blocker

Vector control is a crucial strategy for malaria elimination by preventing infection and reducing disease transmission. Most gains have been achieved through insecticide-treated nets (ITNs) and indoor residual spraying (IRS), but the emergence of insecticide resistance among Anopheles mosquitoes calls for new tools to be applied. Here, we present the development of a highly effective murine monoclonal antibody, targeting the N-terminal region of the Plasmodium falciparum gametocyte antigen Pfs230, that can decrease the infection prevalence by > 50% when fed to Anopheles mosquitoes with gametocytes in an artificial membrane feeding system. We used a standard mouse immunization protocol followed by protein interaction and parasite-blocking validation at three distinct stages of the monoclonal antibody development pipeline: post-immunization, post-hybridoma generation, and final validation of the monoclonal antibody. We evaluated twenty antibodies identifying one (mAb 13G9) with high Pfs230-affinity and parasite-blocking activity. This 13G9 monoclonal antibody could potentially be developed into a transmission-blocking single-chain antibody for expression in transgenic mosquitoes.


Introduction
The mosquito-transmitted protozoan parasite Plasmodium falciparum is the major etiological agent of human malaria, causing more than 200 million clinical cases and 500,000 deaths per year, especially in young children in sub-Saharan Africa (1).Vector control strategies such as insecticide-treated nets (ITNs) or indoor residual spraying (IRS) have been the most effective approaches for malaria control.The documented reduction in the e cacy of insecticides and anti-parasite drugs arising from the evolved resistance of mosquitoes and parasites, respectively, calls for the development of new malaria control interventions (2).The RTS, S/AS01 (RTS, S), the rst-ever approved malaria vaccine, was released with a pilot program in Ghana, Kenya, and Malawi in 2019 and only demonstrates modest protective e cacy against malaria (3).During its life cycle, Plasmodium progresses through multiple developmental stages within the mosquito vector (sexual stage), before being transmitted to the human host through blood feeding.The injected Plasmodium sporozoites will migrate to the liver and invade the hepatocytes where they develop into merozoites.
The merozoites are released into the bloodstream, where they have a natural tropism for invading the red blood cells (RBCs).Within the RBC, they multiply until the cell bursts and releases merozoites that can infect other RBCs, eventually causing the clinical symptoms of malaria.It is at this point that the transition into micro-(male) and macro-(female) gametocytes occurs.The gametocytes have ve distinct maturation stages: only stage V ( ve) gametocytes can progress through the sexual reproduction that occurs in the mosquito host (4).Once ingested by female mosquitoes with a blood meal, gametocytes undergo gametogenesis to produce male and female gametes that mate to form zygotes.The zygotes transform into the motile ookinetes which invade the mosquito midgut, all within 18-36 hours post ingestion of the infected blood meal.The ookinetes traverse the mosquito midgut epithelium and differentiate into oocysts at the midgut basal side.Upon maturation, one oocyst releases thousands of sporozoites into the mosquito's hemolymph, eventually invading the salivary glands to complete the malaria parasite transmission cycle upon a second blood meal (5).
The complexity of the malaria parasite's sporogonic cycle in the mosquito vector offers multiple opportunities for intervention to halt parasite transmission.Targeting parasite antigens within the mosquito serves as the basis for transmission-blocking vaccines (TBV) (6, 7) and the development of transgenic mosquitoes expressing anti-Plasmodium molecules (8).
While a plethora of anti-Plasmodium effectors have been developed to block the parasite while it invades the mosquito midgut epithelium or translocates from the midgut to the salivary glands, the molecular targets blocking the parasites at the earlier gametocyte stages remained to be fully identi ed (9)(10)(11)(12)(13).In this study, we focused on developing and producing a gametocyte-stage blocker to target the early infection stages.It has already been reported that antisera isolated from immunized mice and monoclonal antibodies targeting sexual-stage antigens could successfully inhibit Plasmodium infection(14-18).After gametocyte ingestion, Plasmodium's sporogonic development and malaria transmission proceed by gamete fusion, achieved by species-speci c male-female gamete recognition mediated by membrane proteins on their surface.According to previous studies, only three Plasmodium proteins have a demonstrated role in this recognition process: P48/45, P47, and P230 (19)(20)(21)(22).Pfs230 plays a role in male/female gamete fusion, male gamete ex agellation, and interaction with erythrocytes.
Due to its large size (> 230 kDa) and complex disul de-bonded structure, recombinant expression of fulllength Pfs230 has not yet been successful, however, polyclonal antisera raised against the cysteine-rich domain 1 of Pfs230 have shown Plasmodium-blocking activity (23).Domain 1 is relatively well conserved compared to other domains of Pfs230 and has therefore become a leading malaria transmission-blocking vaccine candidate (22,23).Here, we used a standard immunization protocol to produce monoclonal antibodies targeting Pfs230 and identi ed an effective transmission-blocking clone (13G9) based on co-feeding assays with P. falciparum gametocyte cultures through a standard arti cial membrane feeding assay (SMFA).The anti-Pfs230 monoclonal antibody 13G9 has shown the strongest anti-Plasmodium activity among twenty monoclonal antibody candidates tested in this study.

Mouse-antisera generated after immunization with recombinant Pfs230 D1M domain show high reactivity in vitro
Pfs230 is a 230 kDa cysteine-rich protein, originally present as a 360 kDa precursor on the gametocyte surface (24).It includes 14 cysteine-rich domains (CM) and a natural protease cleavage site at position 542 (25).Previous studies have reported that high transmission-blocking activity can be achieved using the CM1 domain as an immunogenic antigen (23).In addition, analyses of polymorphisms within that region revealed only two predominant amino-acid substitutions at positions G605S and K661N, with the G605S having the highest frequency (AF 0.94) (23).A low polymorphism frequency in the targeting epitope is a desirable trait that reduces the risk of escaper mutations arising in the parasite that would impair the e cacy of the antibody.Twelve new putative missense mutations have been recently identi ed in the same region; however, they are based on de novo variant call data that require further validation (Fig. S3) (26).
Our selected antigen for BALB/c mice immunization comprised a 195 amino acids region from the cleavage site in position 542 through the end of the cysteine-rich domain 1, which we refer to as the Pfs230 D1M domain according to previous publications (23,26) (Fig. 1A).Test bleeds were collected after the 3rd antigen boost to assess antibody titers elicited by immunization according to standard protocols.Indirect-ELISAs (enzyme-linked immunosorbent assays) with antiserum collected from each individual mouse were used to check the antibody titrations (as illustrated in Fig. 1B).All samples were found to be reactive at a 1:512,000 dilution with mice S4 and S5 showing the highest antibody titers (Fig. 2A), con rming the highly immunogenic properties of the Pfs230 D1M domain.

Mouse-antisera generated by immunization with the Pfs230 D1M domain signi cantly reduces oocyst loads
After assessing the immunogenic response elicited by the Pfs230 D1M domain antigen, we evaluated the anti-Plasmodium activities of all mouse-antisera through a standard membrane-feeding assay (SMFA).Immunized mice were boosted 3 times and 50 µL of antiserum from each mouse was kept after each boosting, pooled, and used to isolate the IgG fraction.The IgG fraction from each mouse was then supplied to Plasmodium falciparum gametocytes cultures blood mix (with RBC and human serum) to a nal concentration of 250 µg/ml and fed to the Anopheles female mosquitoes through a membrane feeder (Fig. 1B).The IgG fraction isolated from pre-immunized mice was used as a negative control (Fig. 2B, Ctl-IgG), together with the group of mosquitoes fed on the gametocytes blood mix supplied with PBS as the mock control (Fig. 2B, Pf-only).Since the transmission-blocking activity of previously characterized Pfs230-speci c antibodies was complement-dependent (27), the human serum in the blood meal was not heat-inactivated.The infectious blood meal was delivered with high gametocytaemia to achieve a strong infection prevalence and intensity that would facilitate the selection of the most effective anti-Plasmodium IgGs.
The in vitro reactivities from all immunized mice (S1-S5) were comparable (Fig. 2A), however, IgGs isolated from mouse S2 and S5 showed a signi cantly higher level of transmission-reducing activity (Fig. 2B).Similar to previous studies describing the anti-Plasmodium activity of Pfs230 (28), we found a prominent reduction in oocysts number in infected mosquito midguts (8-fold reduction of median oocyst load with S5 IgG, Mann-Whitney test, p < 0.0001; and a signi cant reduction of infection prevalence, Fisher's exact test, p < 0.01) (Fig. 2B, Fig. 2C).Taking these results together, we selected mouse S5 for hybridoma production to produce monoclonal antibodies.

The 13G9 monoclonal antibody has a potent Plasmodium-blocking activity
Next, in order to assess the e cacy of candidate monoclonal antibodies to suppress P. falciparum infection, we rst isolated IgG fractions from hybridoma's supernatants 13G9, 3F10, 14D2 as testing groups, and IgG from complete hybridoma cell media as the negative control (Ctl-IgG) and evaluated their anti-Plasmodium activities by SMFA (Fig. 1B).Their reactivity to the gametocytes was again con rmed by the same assays as described above (Fig. 1B).Both in vitro indirect-ELISA and immuno uorescence have con rmed the speci c activities of these three monoclonal antibodies (Fig. 4).
Co-feeding Anopheles females 30 µg of 13G9 monoclonal antibody with P. falciparum gametocytesinfected blood meal (with a nal concentration of 166 µg/ml) through a membrane feeder resulted in a prominent reduction of infection intensity (Fig. 5A) (2.5-fold reduction of mean oocyst load, Mann-Whitney test p = 0.0035) and infection prevalence (50% reduction, Fisher's exact test, p = 0.0011) (Fig. 5B) at 8 days post-infection.Monoclonal antibodies 3F10 and 14D2 did not show any Plasmodium-blocking activity despite exhibiting binding to both the Pfs230 D1M domain and the full-length protein in vitro (Fig. 4), highlighting the necessity for in vivo functional assays in addition to the in vitro reactivity assays.

Discussion
Targeting the malaria parasite in the mosquito vector using transmission-blocking vaccines is a disease control strategy that has been gaining increasing interest over the past decades due to the di culties in eliminating malaria for the lack of an effective vaccine (30).Transmission-blocking vaccine's (TBV) mechanism of action is based on vaccination-based immunization of the human host with a parasite or mosquito antigen which is essential for Plasmodium' sporogonic development in the mosquito.While such vaccines do not protect the vaccinated individual from disease, they could contribute to disease suppression at the population level since the infected individuals cannot transmit the pathogen.Three primary P. falciparum-encoded proteins, Pf s48/45, Pf s230, and Pf s25 are currently considered lead candidates for TBV development.Pfs230 is a member of the six-cysteine (6-Cys) family and is composed of fourteen 6-Cys domains forming a complex intra-domain disul de bond structure, the site of recognition for antibodies binding to conformational epitopes.Accordingly, polyclonal antibodies raised through immunization with the whole gametocyte did not recognize the antigen in its reduced form (27).
Previous studies aiming to identify the most suitable region of Pfs230 to use as an antigen showed that the N-terminal Prodomain, which lacks CM cysteine-rich domains, could also achieve this goal, suggesting that Transmission-blocking antibodies may also be directed against non-conformational epitopes (31,32) Extending these prior studies focused on the research of the best antigen to select and utilize in a vaccine development context (33,34), here we directed our attention to generating and isolating an effective monoclonal antibody targeting the early stages of parasite development within the mosquito, using the Pfs230 D1M domain previously demonstrated to elicit strong immuno-response (33,34).We introduced one additional level of testing to the standard pipeline of monoclonal antibody production by selecting the monoclonal antibody that displayed the highest functional parasite-blocking activity.
In a cohort of 5 mice immunized with the Pfs230 D1M domain in identical conditions, the IgG fraction isolated from the mouse S5 led to a signi cantly lower oocyst count and prevalence when fed with an infectious meal to Anopheles mosquitoes, con rming his selection as the best candidate in terms of both potency and effectiveness of the immune response elicited.
Alternatively to transmission blocking vaccines, new therapeutic approaches developed for malaria prevention and therapy include the use of recombinant transmission-blocking antibodies.The ability to target antigens expressed in the early sexual stages potentially allows to reduce the infection intensity within the mosquito host.Cocktails of different antibodies or bispeci c molecules could serve this speci c purpose and render this strategy more effective and long-lasting (35).In the recent past, panels of monoclonal antibodies targeting Pfs230 with a different range of a nity have been developed by various research groups, con rming the need to generate new reagents towards this antigen (36).
Finally, the same monoclonal antibodies developed as transmission-blocking molecules could be used in the context of malaria eradication strategies based on population modi cation of the vector host.Since Pfs230's essential biological function for malaria transmission takes place in the mosquito midgut lumen after ingestion of gametocytes through an infected blood meal, the 13G9 monoclonal antibody could potentially be developed into a single-chain antibody that could be expressed and secreted into the midgut lumen through an appropriate promoter.
Transgenic mosquitoes expressing transmission-blocking molecules and able to transmit the desired traits with a super mendelian inheritance are already a reality and proven to reduce the parasite burden below the transmission level in cage trial experimental settings (37).
A combination of multiple effectors targeting Plasmodium at different developmental stages is most likely the most effective strategy to overcome the insurgence of parasite resistance due to selective pressure during host-pathogen coevolution (38).In this light, the generation and validation of a new transmission-blocking agent targeting the early stage of the parasite in addition to already established ones is useful for the goal of malaria eradication.

Antigen production, immunization, and monoclonal antibody production
The Pfs230 D1M domain (SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSY DKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIE YVPKKSPYVVLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTE DDNKKGNRGIVEVYVEPYGNKING) was codon optimized according to the expression in E. coli (Fig. S1).The synthesized sequence with a 6x HIS tag on the C-terminus was cloned into a pET30a vector (EDM Millipore) and expressed in E. coli BL21 Star (DE3).Induction of recombinant protein was achieved with IPTG at 15°C for 16 hours as per standard protocols (GenScript).Cell pellets were resuspended with lysis buffer followed by sonication.The supernatant resulting from centrifugation was kept for future puri cation.Target proteins were dialyzed and sterilized by a 0.22µm lter before being stored in aliquots.The concentration was determined by BCA protein assay with BSA as a standard.The protein purity and molecular weight were determined by standard SDS-PAGE along with western blot con rmation (GenScript, Fig. S2).

IgGs and monoclonal antibody isolation
IgG fractions from antisera and monoclonal antibodies from hybridoma supernatants clone 13G9, 3F10, or 14D2 were isolated using NAb™ Protein G kit (Thermo Scienti c™) and stored in Phosphate Buffered Saline, pH 7.4 after buffer exchange with Zeba Desalt Spin columns 4 MWCO (Thermo Scienti c™).Antibody stocks were concentrated at the desired volume with an Amicon® ultra -centrifugal lter unit (30kDa lter Millipore-Sigma) and stored at -20 ℃.

ELISAs (enzyme-linked immunosorbent assay)
The in vitro binding activity of antisera, hybridoma supernatants, and monoclonals was evaluated by ELISA.96 well microtiter plates (Immulon 4HBX -Thermo Scienti c™) were coated with 1µg/ml of the Pfs230 D1M domain in Carbonate -Bicarbonate buffer pH 9.6 and kept overnight at 4℃ in a humidi ed chamber.Unbound target protein was removed by four rinses with PBST buffer and wells were blocked for 1 hr at room temperature (RT) with superblock blocking buffer PBS (Thermo Scienti c™).Following three rinses with PBS tween 20 0.01% buffer (PBST), plates were incubated overnight in a humidi ed chamber with serial dilutions of antiserum from each immunized or naïve mouse, 100 µl of Hybridoma supernatant, or increasing concentrations of 13G9, 3F10, or 14D2 monoclonal antibodies in superblock blocking buffer PBS under gentle rocking.Wells lacking primary antibodies were used as a negative control.Following four rinses with PBST, wells were incubated for 1 hr at RT with 100 µL of peroxidasea nipure goat anti-mouse IgG, Fcy fragment speci c secondary antibody (Jackson Immunoresearch Laboratories) diluted 1:5000 in superblock blocking buffer PBS (Thermo Scienti c™).Following four rinses with PBST buffer, wells were incubated with TMB substrate (Sera care) at room temperature in the dark with gentle rocking.The reaction was stopped after 20 minutes with 50 µl of stop solution (Thermo Scienti c™) per well.Absorbance reads were immediately taken in duplicates at 450 nm with a microplate reader (Azure).Each sample was tested in at least 2 independent experiments.

Immunohistochemical staining and microscopy
Blood smears of the P. falciparum gametocytes were air-dried after methanol xation and evaluated by IFA.After membrane permeabilization and blocking of nonspeci c binding (3% BSA, 0.1% saponin in PBS for 1hr at RT) fallowed by 3 PBS washes, the preparations were then incubated individually with the hybridoma supernatants, or 10 µg/mL monoclonal antibodies from 13G9, 3F10, 14D2 clones, or IgG enriched fraction isolated from complete Hybridoma cell media in 1%BSA in PBS at room temperature for 1 hr.AlexaFluor ™ 488 anti-mouse IgG Fc antibody (1:1000 dilution; Invitrogen) in PBS was used for secondary staining and detection.After 3 washes with PBS, slides were let dry and mounted with Prolong™ Gold Antifade (Invitrogen).Microscopic examination was performed the fallowing day with a Zeiss AXIO uorescence microscope system.Each sample was tested in at least 2 independent experiments.

Mosquito rearing, Plasmodium falciparum infection and statistical analysis
Anopheles mosquitoes were maintained on a 10% sugar solution at 27°C and 70%-80% humidity and a 12-hours light/dark cycle according to standard rearing procedures.Anti-Plasmodium activity was determined by SMFA.The infectious blood meal was prepared with NF54 P. falciparum gametocyte cultures, active serum, and RBCs (provided by the Hopkins Malaria Research Institute Core Facility) (40,41) complemented with our experimental samples (IgG fractions from antisera or hybridoma supernatants) or PBS.After starving the adult mosquitoes for 3 to 6 hours, they were allowed to feed for 1hr on arti cial membrane feeders at 37°C.Only the cohort of fully engorged blood-fed mosquitoes was selected and kept until 8 dpi for oocyst counting and infection prevalence assessment.Midguts were dissected out in phosphate-buffered saline (PBS) and stained in 0.02% PBS-buffered mercurochrome (Millipore Sigma).Oocysts were examined using a light-contrast microscope (Olympus).All experiments were repeated at least 2 times.Each biological replicate corresponds to a different mosquito population cage, and each population corresponds to a different generation.All graphs were generated using GraphPad Prism8 software, and the statistical methods used for each experiment are indicated in the respective gure legends.

Declarations Ethics statement
The mosquito rearing and the functional studies (SMFA) were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH).The Johns Hopkins University Animal Care and Use Committee has approved this protocol, with permit number MO18H82.Commercial anonymous human blood was used for parasite cultures and mosquito feeding, and informed consent was therefore not applicable.The Johns Hopkins School of Public Health Ethics Committee has approved this protocol.

Authors contribution
Conceived and designed the experiments: E.C.C., G.D., and E.B.; performed the experiments: E.C.C., Y.D., and M.L.S.; analyzed the data: all authors; First draft: E.C.C.All authors have read and agreed to the published version of the manuscript.FundingThe studies in the Bier lab were supported by The Tata Institutes for Genetics and Society -UCSD and by NIH grants R01GM117321, R01AI162911.

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