key: cord-0313007-5219hze9
authors: Bender, Nicole G.; Khare, Prachi; Martinez, Juan; Tweedell, Rebecca E.; Nyasembe, Vincent O.; López-Gutiérrez, Borja; Tripathi, Abhai; Miller, Dustin; Hamerly, Timothy; Vela, Eric M.; Howard, Randall F.; Nsango, Sandrine; Cobb, Ronald R.; Harbers, Matthias; Dinglasan, Rhoel R.
title: Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen
date: 2020-11-29
journal: bioRxiv
DOI: 10.1101/2020.11.29.402669
sha: 564cc55e7ee471da26f5f0a67e55bcf7f204adac
doc_id: 313007
cord_uid: 5219hze9

Malaria transmission-blocking vaccines (TBVs) are a critical tool for disease elimination. TBVs prevent completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen and structure-function studies have identified two Class II epitopes that induce potent transmission-blocking (T-B) antibodies. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ was immunogenic and immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity and establishing UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

constructs of AnAPN1 were developed based on structural and functional information for peptides 1, 7, and 9 (note: peptide locations were not assigned left to right). Location of a Mouse T Helper Epitope (brown) identified through the Epitope Identification Suite (Merck Research Labs) is indicated 7 The mosquito midgut surface protein Anopheline Alanyl aminopeptidase N (AnAPN1) is an advanced, pan-malaria mosquito-based TBV candidate 7 . Antibodies raised against the N-terminal domain of this protein were shown to prevent the Plasmodium falciparum ookinete from traversing the midgut wall and developing into oocysts, blocking further development and transmission of the parasite [7] [8] [9] [10] . Our previous studies demonstrated that anti-AnAPN1 antibodies can completely (100%) block transmission of naturally circulating P. falciparum in Cameroon [7] [8] [9] [10] . By solving the crystal structure of AnAPN1, we mapped two conformational and protective transmission-blocking (T-B) epitopes, peptides 7 and 9, and a non-protective, "decoy" peptide 1 epitope 7, 8 . Considering that a monoclonal antibody to Peptide 7 alone 8 , and polyclonal antibodies that target only Peptide 9 can both result in potent transmission-blocking activity 7 , a vaccine formulation that promotes a highly focused and functional humoral response in humans to either peptide epitope would be considered successful.

Peptide 1 includes a mouse CD4+ T cell epitope, allowing it to act as an "immune decoy" and elicit a significant humoral response with a robust but non-protective antibody titer 8 . Therefore, removing this immune-decoy epitope from the original 125 amino acid subdomain design of the AnAPN1 TBV (125 6 amino acid recombinant AnAPN1) 7, 8 may prevent the induction of an unintended and a predominant humoral response to peptide 1, and consequently improve T-B activity. We hypothesized that altering the immunogen design would focus ideally the humoral immune response to both of the critical peptide epitopes (peptide 7 and 9) and potentiate T-B activity, essentially requiring lower antibody titers to be elicited in the mammalian host while still achieving potent T-B activity. Here, we report the results of an immunological and functional screen in mice of new AnAPN1 constructs that do not contain the mouse immunodominant peptide 1 epitope, and are capable of immunofocusing the vertebrate humoral response to at least one of the critical T-B epitopes, peptide 9. In this study, we not only demonstrated the marked immunogenicity and T-B functionality of the selected second-generation AnAPN1 construct (UF6), but also identified a potent formulation of the preclinical product and purification process for a tag-free immunogen (UF6b) with the human-safe Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ) adjuvant 9, 11 . Overall, our results highlight UF6b:GLA-LSQ as a strong TBV candidate that may be a critical component in the fight to eliminate if not eradicate malaria.

Down-selection to the UF6 immunogen construct 7 construct (UF1), along with five new antigen candidates for the screening study (Fig. 1b, fig S1a,b) . A UF2 antigen was constructed based on UF1 without peptide 1, the mouse T helper epitope (RTEIHRNERTFT) and the flanking regions ( Fig. 1b) but produced insufficient yields in the wheatgerm system for further study. The four other constructs were as follows: UF3 deleted the decoy peptide (peptide 1) and consisted of peptides 2-9; UF4 consisted of a 13 amino acid, non-natural, pan-DRbinding human T helper epitope [PADRE: AKFVAAWTLKAAA] and the UF3 sequence; the UF5 antigen was a dimer construct of UF3 + PADRE + UF3; and finally the UF6 antigen, a dimer construct similar to UF5 but connected with a glycine linker (GS(GGGS)2G) (Fig. 1b, table S1 ). These antigens were tested in immunization studies with a prime and two boost regimen (boosting on days 28 and 42) using outbred CD1 mice formulated with either Alhydrogel™ or GLA-LSQ adjuvant. The ranking criteria for progressing forward were (1) induction of an antibody response to both peptide 7 and 9, (2) induction of an antibody response to either peptide 7 or 9, and (3) functional T-B activity. Our initial immunological screen demonstrated that wheat germ expressed AnAPN1 constructs UF5 and UF6 were more immunogenic than the original AnAPN1 construct (UF1) ( fig. S2 ). Based on serum endpoint titers alone, the immunogenicity of UF5 appeared to superior to that of other constructs. Antibodies generated using UF5:GLA-LSQ or UF6:GLA-LSQ were also found to be focused on the T-B epitope, peptide 9, and the recognition signals were significantly greater than that observed for UF1 ( fig. S2 ).

Serum samples from the immunized mice were then used to further determine the T-B activity of the different antigens in both the standard membrane feeding assay (SMFA) using laboratory strains of Plasmodium (NF54) and by direct membrane feeding assay (DMFA) using circulating parasite strains in Cameroon ( fig. S3) . Including the wild-type Plasmodium strains helped us to further demonstrate the high T-B potential of the two "down-selected" antigen candidates (UF5 and UF6) that had also shown Table 1 . UF6b-phoA-T7 Master Cell Bank (MCB) 1 :

Reference EVENT-2020-0039 the highest endpoint titers in the immunization studies ( fig. S2) . However, our results indicated that there was no clear correlation between the antibody titer and T-B activity. Although we did not initially observe any advantage in terms of immunogenicity of formulating UF5 with GLA-LSQ over 

Based on the finding that UF6 produced the most robust T-B antibodies, we developed a process to produce large scale CGMP quantities of the UF6 immunogen in preparation for First-In-Human clinical trials. In this process we made two changes to the antigen resulting in the "UF6b" immunogen: (i)

removal of the C-terminal His-tag that was used for purification and (ii) addition of an inert, nonimmunogenic peptide sequence, CGGSG at the C-terminus to protect from anticipated carboxyterminal protease clipping at higher fermentation scales ( (Fig. 2) . A Master Cell Bank was generated and used to produce UF6b ( expressing the UF6b, HIS-tag-free antigen. Using a lot from the MCB, pilot small-scale expression was followed by replicate 120-L scale runs with the goal of high yield production of UF6b in inclusion bodies to aid in the purification steps to reduce endotoxin. In-process UF6b was used for all immunological and functional assays. Bulk Drug Substance was further characterized and observed to yield low endotoxin levels. kDa, kilodalton. BSA, bovine serum albumin standards. See Table 1 and Table 2 for more details.

To test the immunogenicity of the selected new preclinical UF6b product, outbred CD1 mice were immunized intramuscularly (20 μg/dose/mouse) following a prime plus two-boost regimen, with boosting on Days 28 and 70 to reflect an anticipated dosing interval in a clinical trial (Fig. 3a, table S1 ).

UF6b alone (in the absence of adjuvant) is highly immunogenic in mice, achieving reciprocal serum endpoint titers of 10⁶ on Day 98 of the study (Fig. 3b) , establishing its baseline immunogenicity. Mice were also immunized with UF6b formulated with either GLA-LSQ ( Fig. 3c) or AddaVax™ (RUO version of the more potent MF-59 human-safe adjuvant to replace Alhydrogel™ as the adjuvanted UF6b positive control, Fig. 3d ). The formulation of UF6b:GLA-LSQ elicited a higher, more rapid, and more stable response than either UF6b alone or UF6b:AddaVax™ (Fig. 3e) .

Considering that immunization regimens can influence the magnitude and durability of antibody responses to an immunogen, we also tested a Day 0 (prime) with a Day 28 and Day 56-boosting regimen to determine whether a more contracted 70-Day study would affect the overall humoral immune 12 response in mice. We found that the contracted 70-Day immunization regimen (Fig. 3a) produced a comparable, antibody response to the 98-Day study with serum endpoint titers at ~1:10 8 (Fig. 3f) . We hypothesized that deleting the N-terminal "decoy" peptide epitope (peptide 1) would focus the humoral response of immunized mice to the two key T-B epitopes, peptide 9 and peptide 7. We tested this hypothesis by conducting indirect peptide enzyme-linked immunosorbent assays (ELISAs) using only synthetic peptides for these two epitopes. Using sera collected from the 70-Day study as a representative humoral response for two different dosing regimens, we observed that the mice mounted an antibody response to peptide 9, with UF6b formulated with either adjuvant but failed to mount a response to peptide 7 (Fig. 3g) . Both the longer and contracted studies were repeated with new cohorts of mice and the pooled antibody responses generated against UF6b and both key peptides remained similar ( fig. S4 ). Importantly, a closer examination of individual mouse antibody titers from the 70-Day study indicated a high peptide 9 responder rate across all mice per group ( fig. S5-S6 ).

Previously, we have shown that immunizing mice with the N-terminal 125-aa recombinant AnAPN1 protein generates functional T-B antibodies [7] [8] [9] [10] . In the current study, we assessed whether total IgG purified from mice immunized with the new construct UF6b similarly blocked parasite transmission by both SMFA and DMFA. The total IgG concentration of 50 µg/mL, corresponding to about 3.3 µg/mL of antigen-specific IgG, effectively blocked transmission of P. falciparum in Anopheles gambiae mosquitoes ( Fig. 4) , leading to a significant reduction in oocyst intensity (i.e., the number of oocysts present per midgut). Antibodies raised in mice immunized with UF6b:GLA-LSQ were able to 14 significantly reduce mean oocyst intensity greater than 90% (P<0.001, GLMM, Kruskal-Wallis, α<0.05) ( Fig. 4a, c) . Additionally, higher concentrations of total IgG did not appear to enhance T-B activity at concentrations above 50 µg/mL (Fig. 4b) . This phenomenon has been described previously, wherein higher antibody concentrations have either resulted in plateauing of T-B activity or enhancement of parasite infection of the midgut; effects within the known intrinsic error of the assay 12-14 . We also tested the ability of purified total IgG from mice immunized with UF6b:GLA-LSQ to block naturally circulating P. falciparum parasites from infecting An. gambiae mosquitoes in Cameroon by DMFA. UF6b-specific IgG at 3.3 µg/mL resulted in reproducible reductions of 80-90% in mean oocyst intensity (P<0.001, GLMM, α<0.05) (Fig. 4d,e) .

To determine whether the observed differences in apparent T-B activity for antisera from UF6b:GLA-LSQ and UF6b:AddaVax immunizations can be explained by variances in antibody quality and quantity, we compared the immunoglobulin isotypes for these two groups as well as for UF6b alone and the contracted 70-Day dosing studies. In the absence of adjuvant, UF6b elicited primarily an IgG1 response with lower levels of IgG2a and IgG2b (Fig. 4f) . When given in conjunction with either GLA-LSQ or AddaVax™, the immunoglobulin profile is more equally divided between these three subtypes.

Also, between these three immunization groups no differences were noted for IgG3, IgA, and IgM, which were all comparably low and all groups had a higher proportion of kappa to lambda light chains ( Fig. 4f) . Very little variation in the immunoglobulin profile was seen in the D98 study after the D70 time point, although we did notice an increase in lambda light chain in the repeat D98 study (Fig. 4g) .

We noted that the contracted study of UF6b:GLA-LSQ produced a comparable immunoglobulin subclass profile (Fig. 4h ) as compared to the longer D98 study (Fig. 4g) , indicating flexibility in dose regimen schedules in future clinical studies. There was some variation in the amounts of IgG2a and lambda light chain between the two replicate 70-Day studies, which can be expected with outbred mice.

Here we successfully optimized our mosquito-based AnAPN1 candidate for further development and evaluation as a potent malaria TBV antigen. Moreover, our studies further demonstrated the benefits of using structural information in antigen optimization as a crucial step in the development of any malaria vaccine candidate. UF6b was reliably immunogenic in mice even when inoculated alone, unformulated with an adjuvant. The UF6b construct is based on a structure-function understanding of the immunogenic and mosquito-specific domains of the full-length APN1 from An. gambiae 10 . UF6b generated a peptide 9-specific response, which we have demonstrated previously 8 confers potent T-B activity. While our original goal had been to target both peptides 9 and 7, which were predicted to strongly bind MHC II encoded by the HLA-DRB1 alleles common to East African populations 7,11 , we were unable to elicit a peptide response specific to peptide 7. This lack of a peptide 7-specific antibody response may result from an altered presentation of the peptide 7 epitope due to the flexibility of the glycine linker introduced in the new UF6b dimer construct. However, the lack of a peptide 7-specific response did not appear to affect the overall T-B activity of the antibodies, and achieves two priority goals: induction of antibodies to either peptide 7 or peptide 9 and demonstrated functional T-B activity at lower antibody concentrations.

In previous studies, between 0.8-1.6 mg/mL of purified total IgG from rabbits immunized with a near-full length recombinant AnAPN1 (i.e., UF1) was needed to block oocyst development in both SMFAs and DMFAs 7 . At this high total IgG concentration, it was estimated that only ~5-10 µg/mL were antigen-specific IgG. However, these studies used an immunogen containing the "immune decoy" epitope (peptide 1), causing a larger proportion of the murine IgG profile to target this malaria-irrelevant epitope 7, 8 . We observed that a comparable amount of UF6b-specific IgG (3.3 µg/mL) was present in the antisera of mice immunized with UF6b:GLA-LSQ. These data suggest that the UF6b:GLA-LSQ formulation was indeed able to focus or skew the vertebrate immune response towards the peptide 9 epitope, resulting in a 32-fold lower concentration of total IgG (50 µg/mL as opposed to 1.6 mg/mL) needed to confer marked T-B activity. It remains to be seen if antibodies with similar potency can be induced in non-human primates or humans. Maximal T-B activity for total IgG following UF6b:GLA-LSQ immunization was comparable to that observed for the potent monoclonal antibody (mAb) 4H5B7, which targets peptide 7 only 8 . The degree of blocking activity appears to be related to the intensity of the infection of the mosquito in SMFAs and in DMFAs, the variation in T-B activity may be related to the genetic complexity of the gametocytes in the blood obtained from infected volunteers 15 . This is not entirely unexpected since the membrane feeder (although the gold standard for measuring T-B activity in the field) is artificial and circulation of infected red blood cells and antibody within the small feeders is not currently possible. As such, the distribution of antibody throughout the bloodmeal may not be even. The intrinsic error of membrane feeding assays notwithstanding [12] [13] [14] , complete blockade can be observed when oocyst intensities are less than 50 oocysts/midgut, which is a level expected in natural infections based on observations from field studies examining the range of possible oocyst intensities 16 .

Differences in T-B activity between the UF6b:GLA-LSQ and UF6b:AddaVax™ vaccine formulations cannot be explained by total antibody titers alone. Previous studies suggest that a predominantly Th2 response is elicited in mice following immunization with AnAPN1 (UF1) formulated with Alhydrogel™ [10] . This is generally observed as lower IgG2 titers relative to IgG1 titers in immune sera. However, higher IgG2a and IgG2b titers to UF6b were observed with UF6b:GLA-LSQ and UF6b:AddaVax™ than UF6b alone, indicative of a Th1-biased humoral response in these outbred CD1 mice. UF6b:GLA-LSQ elicited a slightly higher IgG2a titer as compared to UF6b:AddaVax™ and more than a log higher IgG2a titer than UF6b alone. Saponin-based adjuvants (GLA-LSQ) and emulsions such as AddaVax™ or MF-59 are designed to induce robust and balanced Th1/Th2 responses, and this was clearly observed in our study for UF6b:GLA-LSQ and UF6b:AddaVax™ [17] . The observation of a higher proportion of kappa light chains in antigen-specific IgG (all Ig subclasses) for the UF6b:GLA-LSQ has not been previously observed in studies using UF1:Alhydrogel™. In humans, variable light chains can influence antibody-binding specificity, serum half-life, and conformational flexibility in addition to other physicochemical properties, whereas subtle differences in these same properties have been described for variable heavy chains 18 . IgG3 responses have not been noted in our previous work with AnAPN1-targeted immunization, and although associated as a co-marker of a Th1 profile, the overall IgG3 titers in this study were low. Given that the potent mAb 4H5B7 is an IgG2b, kappa isotype, it is possible that the higher titers of IgG2a and IgG2b and light chain isotype ratio may have resulted in not only more antibody, but also a qualitatively better antibody repertoire. This hypothesis bears further study and could be relevantly and appropriately addressed in a Phase 1 clinical trial. It should be noted that mice are an imperfect model organism for predicting the success of malaria vaccines in humans, as mice generate different immune responses with distinct antibody characteristics as compared to non-human primates (NHPs) and humans 19 . As such, further testing of the tolerance, immunogenicity, and longevity of our UF6b:GLA-LSQ formulation are currently being completed in NHPs to allow a quicker transition to First-In-Human clinical trials.

The process development workflow (Fig. 2) established in this study positions the program well to move forward to CGMP production of UF6b. Based on a theoretical 0.1 mg/mL/subject as the highest dose in a Phase 1 study, the conservative estimated endotoxin level would be 0.35 EU/mL. This is already well below the <20 EU/mL recommendation for each dose of a recombinant protein vaccine and is 15-fold lower than the upper limit of endotoxin for Rubella vaccines 20 . In fact, the current process sets 

For both SMFAs and DMFAs, IgG was purified from each 0.8-1 mL cardiac puncture sample per mouse. Since a single mouse cannot yield sufficient purified IgG for an assay, using current best practices, immune serum from either ten or twenty mice were pooled before purification. The purified

IgG from the pooled immune sera permitted us to perform dose-response membrane feeding assays in triplicate.

Data collection was stopped at a predetermined timepoint of either Day 70 or 98. As per the animal care use committee approved protocol (protocol #201909359), data collection endpoints were also determined by the following humane endpoints for the test mice: if any of the test mice sustained greater than or equal to 15% weight loss from baseline weight or age-matched controls for those animals still maintained during the study; a body condition score of 2 or less; inability to reach food or water; impaired mobility; the presence of tumors; labored breathing, respiratory distress or cyanosis (blue tinged color); tremors, convulsions or seizures lasting more than 1 minute or occurring more than once a day; dehydration lasting over 24 hours that is unresponsive to treatment; or moribund -unable to right itself. 

Templates for the expression of proteins UF1 to UF6 were prepared by gene synthesis (GenScript, USA). Before synthesis, sequences were optimized for expression in wheat, and a His-tag with six histidine residues was directly added without spacer to the C-terminus of the proteins.

Templates were then cloned into the expression vector pEU-E01-MCS (CellFree Sciences, Japan) using the XhoI and BamHI restriction sites, and the DNA sequences of the templates confirmed before use in (Fig. 3a) . Every two weeks post-priming, mice were bled to collect sera for anti-AnAPN1 antibody titer determination via ELISA. Mice were sacrificed four weeks after the final boost and serum was collected via cardiac puncture. A single replicate study was completed with 10 mice immunized with the same UF6b:GLA-LSQ formulation in the same manner and timepoints.

Unfortunately, due to COVID-19 closures, Day 84 sera could not be collected.

Two replicate studies with a contracted dosing schedule were completed with 20 female CD1 mice (Charles River) and used at 6 to 8 weeks of age. Ten mice were immunized with the same UF6b:GLA-LSQ formulation described above, and ten mice received the UF6b:AddaVax™ formulation. All mice received a prime dose i.m. on day 0 and were subsequently boosted on Days 28

and 56 post priming (Fig. 3a) . Sera was collected every two weeks post-priming until the endpoint at Day 70 when the mice were sacrificed.

Maxisorp 96-well ELISA plates (Nunc, Fisher Scientific) were incubated overnight at 4°C with 1 μg/mL antigen in 0.1 M PBS (pH 7.2). After three washes with PBS-Tween 20 (0.1%) (PBST20), the plates were blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) in 1xPBS.

Serum samples were diluted to 1:50 and 1:100, then diluted ten-fold to 1:10 8 in 0.5% BSA in 1xPBS.

After discarding the BSA and drying the plates, 100 μL of each of the eight serum dilutions were added to each well in triplicate and incubated 1 h at room temperature. Plates were again washed three times with PBST20. For detection, 100 μL of a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG(H+L) (KPL) diluted 1:5,000 in 0.5% BSA was added to each well and incubated for 1 h at room temperature. Plates were washed three times with PBST20 and developed by adding 100 μL of (3,3′,5,5′-tetramethylbenzidine) microwell peroxidase substrate (KPL) to each well. Development was 

Maxisorp 96-well ELISA plates (Nunc, Fisher Scientific) were coated with 100 μL of Poly-L-Lysine (PLL) at a concentration of 50 μg/mL in 0.05 M sodium bicarbonate buffer (pH 9.6) and

incubated covered for one h at room temperature. The plates were washed once with PBST20, then coated in 100 μL 1% (v/v) glutaraldehyde in PBS and incubated covered for 15 minutes at room temperature. The plates were washed again once with PBST20, and then coated with 100 μL of either peptide 1, peptide 7, or peptide 9 (7, 8) and incubated covered overnight at 4°C. The next day the plates were washed twice with PBST20, then 200 μL 1M glycine was added to each well and plates were incubated covered for one h. The plates were washed twice with PBST20 and 300 μL of a 1:1 solution of 5% milk and 1% gelatin was added to the plates and incubated covered for one h. The serum samples were diluted in PBS using serial ten-fold dilutions from 1:10 2 to 1:10 9 . After washing the plates once with PBST20, each of the eight sample dilutions was added to each well in triplicate and incubated covered for one h. The plates were washed three times with PBST20 and 100 μL (HRP)-conjugated goat gametocytemia and 30% hematocrit. Infective blood was mixed with control (IgG purified from naïve mouse serum) or total IgG purified from pooled sera of mice immunized with UF6b prior to delivery directly into water-jacketed membrane feeders maintained at 37°C via a circulating water bath. The final concentration of total IgG in 300 µL total volume of infective blood was 50 µg/mL for each technical replicate. Female An. gambiae were starved for 6 h, placed in paper cups with tops covered with netting (n = 40 per cup) and allowed to feed from each feeder for 20 min. Fully engorged female mosquitoes were maintained for 8 d when they were dissected for midgut oocyst enumeration. The dissected midguts were stained with 0.1% mercurochrome to visualize oocysts. A total of three biological replicate experiments were conducted.

Mosquito infections were conducted during the rainy seasons in 2019. Procedures for gametocyte carrier detection, blood collection and mosquito infections were performed as previously described 24 . Briefly, P. falciparum gametocyte carriers were identified among asymptomatic children (ages 5 to 11) in primary schools in the Mfou district, 30 km from Yaoundé, Cameroon. Venous blood was drawn from gametocyte-positive individuals in heparinized Vacutainer tubes from the antecubital fossa. Membrane feedings were set using donor's blood with replacement of the serum by a nonimmune AB serum. The local laboratory strain of An. coluzzii, named Ngousso, was used for mosquito feedings. Following infection, mosquitoes were maintained at the laboratory under standard insectary conditions (27 ± 2°C, 85 ± 5% RH, 12h light/dark) for 8 days until dissections.

Total IgG was purified from pooled serum using Protein A/G Magnetic Agarose Beads (Thermo Fisher Scientific) according to manufacturer's instructions. The antibodies were quantified using Peirce™ BCA protein assay kit. Antigen-specific IgG was purified from pooled serum with UF6bconjugated Dynabeads™ M-270 Epoxy (Thermo Fisher Scientific) following manufacturer's instructions. The antibody titer of the purified UF6b-specific IgG and total IgG (serum) was determined with the Easy-Titer™ Mouse IgG assay kit (Thermo Fisher Scientific).

Anti-UF6b immunoglobulin isotypes were determined with the Mouse Typer Isotyping Panel (Bio-Rad). Plates were coated and blocked, as described above, and mouse sera diluted 1:1,000 was added. After washing five times the isotyping panel was added, followed by five washes, then (HRP)conjugated goat anti-rabbit IgG(H+L) (KPL). Absorbances were detected as described above.

To determine differences in oocyst intensity (defined by either the mean oocyst count) between pre-immune sera and test sera in SMFAs the data was first cleaned of outliers using a robust nonlinear regression test (ROUT) and a Q coefficient of 1% for all treatment and control groups 27 using GraphPad Prism 6. After comparing the raw and cleaned data and considering that assumptions and overall significance results were not affected, outliers were removed to account for the intrinsic noise in the feeding assays, contributed in part by the uneven distribution of spiked antibody and pooling of gametocytes in the membrane feeder as well as variability in mosquito feeding voracity. The cleaned data was then analyzed by either a zero-inflated or normal Generalized Linear Mixed Model statistical test (GLMM) using the R package 28 , and when neither was appropriate, a non-parametric Kruskal-Wallis test followed by Dunn's post hoc test using GraphPad Prism 6 7,8,10,29 .

All materials generated through this study are available following a request with the corresponding author.

Geneva: World Health Organization

Flipping the paradigm on malaria transmission-blocking vaccines

A research agenda to underpin malaria eradication

Epidemiology and Infectivity of Plasmodium Falciparum and Plasmodium Vivax Gametocytes in Relation to Malaria Control and Elimination

The malERA Consultative Group on Vaccines. A Research Agenda for Malaria Eradication: Vaccines

Drug-resistant malaria: Molecular mechanisms and implications for public health

Antibodies to a Single

The Anopheles-midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope

Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen

Expression, Immunogenicity, Histopathology, and Potency of a Mosquito-Based Malaria Transmission-Blocking Recombinant Vaccine

Single locus polymorphisms of classical HLA genes

Two-Faced Immunity? The Evidence for Antibody Enhancement of Malaria Transmission

Reduction and enhancement of Plasmodium falciparum transmission by endemic human sera

Measuring the blockade of malaria transmission--an analysis of the Standard Membrane Feeding Assay

Impact of exposure to mosquito transmission-blocking antibodies on Plasmodium falciparum population genetic structure

High Plasmodium infection intensity in naturally infected malaria vectors in Africa

Design and evaluation of a safe and potent adjuvant for human vaccines

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions

Rhesus macaque and mouse models for down-selecting circumsporozoite protein-based malaria vaccines differ significantly in immunogenicity and functional outcomes

Acceptable levels of endotoxin in vaccine formulations during preclinical research

Circulating Th1-Cell-type Tfh Cells that Exhibit Impaired B Cell Help Are Preferentially Activated during Acute Malaria in Children

Adjuvant and carrier protein-dependent T-cell priming promotes a robust antibody response against the Plasmodium falciparum Pfs25 vaccine candidate

Fusion proteins: applications and challenges

Removal of endotoxin from protein solutions by phase separation using Triton X-114

NPC1161B, an 8-Aminoquinoline Analog, Is Metabolized in the Mosquito and Inhibits Plasmodium falciparum Oocyst Maturation

Plasmodium falciparum produce lower infection intensities in local versus foreign Anopheles gambiae populations

Detecting outliers when fitting data with nonlinear regressiona new method based on robust nonlinear regression and the false discovery rate

R: A language and environment for statistical computing. R Foundation for Statistical Computing

Measuring the blockade of malaria transmission -An analysis of the Standard Membrane Feeding Assay

wrote the draft and final manuscript with support from all authors.

The authors do not declare any competing interests.