A stabilized tandem antigen chimera that elicits potent malaria transmission-reducing activity

Domain: neuroscience
Published: 2026-01-24
Editorial outcome: minor_revision

Abstract
Malaria parasite transmission remains a barrier to elimination since asymptomatic individuals sustain the infectious reservoir. Transmission-blocking vaccine (TBV) candidates targeting Plasmodium falciparum (Pf) gametocyte surface proteins Pfs230 and Pfs48/45 have shown promise in clinical trials. Several vaccine candidates have been developed for these antigens, yet it is unclear which elicit the most robust and durable transmission-blocking responses. From structure-function relationships of monoclonal antibodies in complex with both antigens, we report the development of a stabilized tandem antigen chimera (STAC), which presents the most potent epitopes from Pfs230 domain 1 (Pfs230-D1) and Pfs48/45 domain 3 (Pfs48/45-D3) in a single construct, while masking non-functional epitopes using an engineered pseudo-native domain disposition. Iterative structure-guided optimization improved antigen yields and stability, while nanoparticle-based multimerization enhanced the functional transmission-reducing activity elicited by the immunogen in female mice. Immunizations with STAC genetically conjugated to self-assembling protein nanoparticles elicited antibodies with potent transmission-reducing activity comparable or superior to the multimerized Pfs230-D1 and Pfs48/45-D3. These findings establish STAC as a promising next-generation TBV candidate to disrupt malaria transmission and accelerate elimination efforts. More broadly, our results support the engineering of highly ordered and stable multi-domain antigens in a single protein as a strategy for the cost-efficient development of multi-component vaccines.

Introduction
Malaria remains a global health challenge, disproportionately affecting children in sub-Saharan Africa, with the disease causing 597,000 deaths in 2023. The World Health Organization has recommended two malaria vaccines, RTS,S and R21, for pediatric use against Plasmodium falciparum (Pf) malaria; together, they have the potential to significantly reduce morbidity and mortality in this vulnerable population. Despite the anticipated success of RTS,S and R21 in reducing pediatric morbidity by inhibiting the infectious pre-erythrocytic lifecycle stage of the Pf parasite, with preliminary data indicating RTS,S is associated with a 22% reduction (95% CI 3%-36%) in hospital admissions with severe malaria and a 13% reduction (95% CI 3%-23%) in all-cause mortality, these vaccines do not target the primary human reservoirs of transmission. Epidemiological studies have demonstrated that older age groups can harbor mature gametocytes, the transmissible form of the parasite. Variability in individual transmission potential is a feature of many infectious diseases, and for malaria, the human infectious reservoir is largely sustained by asymptomatic populations, particularly school-aged children and adults, and individuals with submicroscopic infections could transmit the malaria parasite. This sustained parasite circulation from human to mosquito represents a critical challenge in malaria control and elimination efforts. Yet, there is no approved vaccine to combat this parasite life-cycle stage, highlighting the need for interventions that target not just symptomatic malaria but also the hidden transmission reservoir. Given the parasite’s complex life cycle and the ability of asymptomatic carriers to sustain transmission, expanding vaccine strategies to include adolescents and adults could enhance the effectiveness of RTS,S, R21, and next-generation vaccines, ultimately strengthening malaria control and elimination efforts.

Epidemiological modeling has indicated transmission disruption may be an essential step to achieving malaria elimination. Transmission-blocking vaccines (TBVs) are a promising strategy to disrupt the malaria life cycle through the inhibition of parasite transmission from human to mosquito. By eliciting antibodies that target parasite proteins essential for gamete fertilization and parasite development within the mosquito midgut, TBVs could substantially reduce or eliminate the onward spread of malaria. Pfs230 and Pfs48/45 are sexual-stage surface proteins essential for Pf transmission and represent the two most advanced gametocyte/gamete TBV candidates in clinical development. Both proteins belong to the 6-Cysteine (6-Cys) family, categorized by the presence of Immunoglobulin-like folds containing up to three disulfide bonds and are essential for pre-fertilization gametocyte function. Pfs230 is comprised of fourteen 6-Cys domains and binds to membrane-bound Pfs48/45, comprising three 6-Cys domains and a GPI anchor, to enable presentation of the heterodimeric complex at the surfaces of gametocytes and gametes. While recombinant full-length Pfs230 protein is not an established TBV antigen, the full-length Pfs48/45 protein produced from insect cells is currently being evaluated in a phase I trial with Matrix-M adjuvant (NCT05400746).

Subunit vaccines are capable of focusing immune responses to the most potent regions of a target protein, and TBVs displaying either Pfs230 domain 1 (Pfs230-D1) or Pfs48/45 domain 3 (Pfs48/45-D3) are two such examples currently undergoing clinical evaluation. The clinical Pfs230-D1 candidate contains D1 and a segment of its N-terminal pro-domain (referred to as D1M, amino acids 542–736), which is chemically cross-linked to the carrier protein exoprotein A (EPA). This Pfs230D1M-EPA subunit vaccine is the most advanced TBV candidate with two completed phase I trials assessing adjuvants AS01 and Matrix-M (NCT02942277 and NCT05135273, respectively), and one phase II trial with adjuvant AS01 (NCT03917654). The challenging biochemical tractability of Pfs48/45-D3 (also known as 6C, amino acids 291–428) once hampered the development of Pfs48/45-D3-based vaccines; however, several protein engineering strategies have enabled recent clinical evaluations. One such example is the phase I trial evaluating a TBV candidate comprising Pfs48/45-D3 fused to a part of blood-stage GLURP antigen (R0.6C) with Alhydrogel or a combination of Alhydrogel and Matrix-M (NCT04862416). Despite their potential, emerging phase I/II clinical data for the current subunit-based immunogens indicate that these leading TBVs may face significant challenges in generating consistently high levels of functional antibody responses. In addition, seroprevalence of antibodies to Pfs230 and Pfs48/45 indicates that some individuals in endemic regions elicit detectable antibody titers exclusively to one individual antigen but not to the other, so it is conceivable that an immunogen presenting both Pfs230 and Pfs48/45 could have the advantage of boosting the naturally acquired immunity for a broader diversity of individuals. Therefore, the challenge lies in designing TBVs that provide broad and potent protection while also being viable for widespread deployment in endemic regions. Accordingly, the feasibility of manufacturing cost-effective, multi-antigen TBVs at scale remains a key consideration.

Strategies that fuse Pfs230 and Pfs48/45 antigenic domains are emerging. Studies have indicated that Pfs230-D1-elicited antibodies typically require complement activation for maximal transmission-reducing activity (TRA), whereas Pfs48/45-D3 can induce potent, complement-independent antibodies. These distinct antibody mechanisms of action suggest that a multi-antigen TBV formulation incorporating both targets could provide superior and more consistent efficacy. Such an emerging design is the Pfs48/45-D3 fused to the Pro-domain of Pfs230 connected by a linker composed of PfCSP major and minor repeats. This fusion, ProC6C, adjuvanted with Alhydrogel or a combination of Alhydrogel and Matrix-M, has entered clinical evaluation (ISRCTN13649456, PACTR202201848463189).

Advances in reverse vaccinology and structure-guided antigen design continue to enable the development of more stable and immunogenic TBV candidates. For Pfs48/45, two recent studies leveraged structural insights from D3 bound to the highly potent mAb TB31F to rationally design stabilizing mutations that conferred enhanced biochemical tractability, leading to increased potency of the antibody response in preclinical studies. Recent structure-activity relationship (SAR) studies examining the human monoclonal antibody responses induced by natural exposure to Pf parasites have detailed the molecular blueprints describing functional transmission-reducing activity against Pfs230-D1 and Pfs48/45-D3. Antibody responses of Pfs230D1M-EPA clinical vaccinees have uncovered strong antibody responses to non-functional epitopes on Pfs230-D1, and further SAR characterization has identified a molecular determinant for these non-functional antibodies, which are directed to epitopes buried in the full-length native proteins, but exposed in the Pfs230-D1 fragment vaccine candidate.

Recent advances in protein engineering are also enabling approaches that expand beyond natural proteins. As such, a TBV vaccine design approach that is not merely the fusion of distinct Pfs230 and Pfs48/45 protein domains, but rather the efficient display of potent epitopes and the masking of non-functional ones in newly engineered constructs, is conceivable.

In this study, we report the development of a novel transmission-blocking vaccine candidate utilizing a stabilized tandem antigen chimera (STAC) comprising the primary potent epitopes of two leading vaccine antigens into a single polypeptide that masks non-functional epitopes exposed on isolated domains of subunit immunogens. Through a progressive design series, we incorporate interdomain linker optimization and interface stabilization, resulting in improved expression, thermostability, and potent immunogenicity, as determined by immunization using liposomal displayed nanoparticle immunogens. When formulated on protein nanoparticles, STAC elicited robust antibody responses and superior functional activity compared to individual or mixed antigens. Together, these data exemplify a strategy for engineering an optimized multi-antigen presentation in a single protein to enhance antibody responses.

Results
Mixing Pfs230-D1 and Pfs48/45-D3 on adjuvanted liposomes elicits strong transmission-reducing activity
To investigate the functional immunogenicity of Pfs230-D1 and Pfs48/45-D3 as individual antigens and assess the potential benefit of their combination, we utilized the cobalt porphyrin-phospholipid (CoPoP) multimerization platform to display His-tagged antigens. Immunogenic CoPoP liposomes, co-incorporating synthetic monophosphoryl lipid A (PHAD) and saponin QS-21 adjuvants (abbreviated as CPQ) were loaded with antigens and used to immunize CD-1 mice in a prime-boost regimen by intramuscular injection. For the Pfs230-D1, we selected the Pfs230 D1+ construct (comprising amino acids 552–731), which has been optimized for increased expression and minimal sample heterogeneity by removal of post-translational modification sites, while also presenting less of the N-terminal Pro-domain compared to the clinical construct D1M (amino acids 542–736). For Pfs48/45-D3, we selected 6C.mAgE1 (amino acids 291–428), which shares the same domain boundaries as the clinical stage Pfs48/45-D3 antigens and possesses three stabilizing mutations that have been demonstrated to induce superior potency in mouse immunizations compared to the wild-type antigen. Both antigens were expressed in HEK293 cells with C terminal 6x Histidine tags and formulated with CPQ liposomes individually (5.0 µg each) and in mosaic combination (2.5 + 2.5 µg) for immunizations following a 21-day prime-boost regimen. By combining Pfs230-D1 and Pfs48/45-D3 on liposomes in this way, we note that the equal mass amounts used for each antigen resulted in a sub-stoichiometric amount of the larger antigen, Pfs230-D1, to a ratio of 0.74-to-1. At 21-days following the boost immunization, sera were harvested, from which IgGs were purified for evaluation in ELISA and standard membrane feeding assays (SMFA).

As expected, sera from mice immunized with the individual antigens showed strong and specific antibody responses against each antigen. Mosaic combination of both antigens showed slightly lower ELISA titers than individual antigens, likely due to the half the dosage of the individual antigens. SMFAs using purified IgGs from the mouse immunizations showed that all three antigen groups exhibited nearly complete TRA at 225 µg/ml IgG. At a dilution of 75 µg/ml IgG, while all antigen groups exceeded 80% TRA, Pfs230-D1 group showed significantly higher inhibition than the other two groups. These data confirmed the compatibility of both Pfs230-D1 and Pfs48/45-D3 antigens to induce biologically active responses when adjuvanted on a multimerizing platform, but also provided a motivation for improved antigen co-display.

Stacking antigen domains presents epitopes in a pseudo-native arrangement
To reduce complexity, we sought to develop a single antigen capable of presenting potent epitopes from both Pfs230-D1 and Pfs48/45-D3, while simultaneously reducing exposure of non-inhibitory epitopes. Structures of Pfs230-D1D2 and full-length Pfs48/45 have been elucidated, allowing for a structure-based analysis of antigen presentation. For both Pfs230 and Pfs48/45, D1 and D2 appear to be rigidly coupled, while flexible linkage of Pfs48/45-D3 appears to allow for a greater degree of conformational freedom. We hypothesized that the presentation of the antigens in a disposition matching the native arrangement of 6-Cys domains may facilitate antigen co-display with structural integrity. Template modeling (TM)-scores were used to cross-compare Pfs230 domains 1 and 2 with Pfs48/45 domains 1, 2, and 3. Domain 1 of each protein gave the highest TM-score (0.71), but intriguingly, Pfs230-D2 displayed the next highest similarity when compared to Pfs48/45-D3. Despite only 18.1% sequence identity between the two domains, the TM-score of 0.63 and RMSD of 3.1 Å suggested the possibility that Pfs48/45-D3 may be genetically conjugated to Pfs230-D1 in a manner similar to the native placement of Pfs230-D2. Although this approach would remove Pfs230-D2 from the immunogen, no potent epitopes have been described on this domain.

A theoretical model of Pfs230-D1 tandemly fused to Pfs48/45-D3, herein referred to as Design-1, was constructed by replacing Pfs230-D2 with Pfs48/45-D3 using the domain placement calculated by TMalign on a crystal structure of Pfs230-D1D2. To gauge the theoretical accessibility of potent and non-potent epitopes on Design-1, previously determined crystal structures of antibody-antigen complexes were superimposed on their relevant domains. The epitopes of the potent Pfs230-D1-directed mAbs RUPA-97 and LMIV230-01, as well as the epitopes of the potent Pfs48/45-D3-directed mAbs TB31F and RUPA-44, were found to be fully accessible. While the epitope of the non-functional mAb RUPA-38 on Pfs230-D1 remained accessible, the epitopes on Pfs230-D1 of the non-functional mAbs LMIV230-02 and 15C5 were sterically occluded by the assumed position of Pfs48/45-D3. Unlike RUPA-38, which was identified from an individual naturally exposed to Pf parasites, LMIV230-02 was identified from a study participant immunized with recombinant Pfs230D1M-EPA, and 15C5 was generated from a mouse immunized with recombinant Pfs230-D1, highlighting how immunization with individually excised domains can elicit non-functional antibodies against epitopes that are not accessible on the gametocyte.

Design-1 was expressed and purified to assess the compatibility of the two tandem domains and to validate the predicted epitope masking. Size-exclusion chromatography indicated that Design-1 predominantly existed as a monomer. Binding kinetics experiments confirmed Design-1 maintained epitope fidelity for Pfs230-directed mAbs RUPA-97, LMIV230-01, and RUPA-38, and Pfs48/45-directed TB31F, and RUPA-44. Binding to LMIV230-02 and 15C5 was assessed for Pfs230-D1, Pfs230-D1D2, and Design-1. As would be expected from their three-dimensional structures, the binding observed for both mAbs to Pfs230-D1 was completely undetected for Pfs230-D1D2. For Design-1, binding to LMIV230-02 was partially attenuated by a factor of approximately six-fold, while a greater impact was observed for 15C5 which saw an approximately 60-fold drop in affinity. Taken together, these data indicated that while Pfs230-D1:Pfs48/45-D3 fusions are possible to display potent epitopes, an optimal design that masked non-inhibitory epitopes by recapitulating native 6-Cys domain dispositions required further engineering.

Protein engineering improves the biochemical tractability of stacked antigens
In Design-1, we utilized the inter-domain linker sequence of Pfs230-D1D2 to connect Pfs230-D1 to Pfs48/45-D3; however, partial accessibility of the Pfs230-directed 15C5 and LIMV230-02 epitopes indicated that flexible linkage may have mispositioned the two tandem domains such that the epitopes are not fully occluded. Structural and sequence alignments of Pfs230-D1D2 and Pfs48/45-D2D3 linkers indicated that the backbone positions and amino acid identities are conserved. To better accommodate the conjugation of Pfs48/45-D3, we hypothesized a shortened and hybrid linker sequence may encourage better stacking of the two domains with the effect of blocking the 15C5 and LMIV230-02 epitopes. We designed six shortened and hybrid linkers, which included an N-to-Q mutation to abrogate a putative N-linked glycan sequon. We utilized the potent conformational mAbs RUPA-97 and TB31F that are directed to Pfs230-D1 and Pfs48/45-D3, respectively, to quantify the expression of properly folded, secreted protein designs using BLI. Three of the six designs (Designs-2, -3, and -4) exhibited superior expression relative to Design-1 by BLI quantitation assay. All three new designs were assessed by SDS-PAGE, and Design-4 showed the highest amount of monodisperse species by SEC. For Design-4, binding to LMIV230-02 was reduced by a factor of approximately 26-fold compared to Pfs230-D1, while no binding was observed for 15C5 up to 500 nM, suggesting that Design-4 recapitulated a more desired inter-domain orientation.

To evaluate the structure of Design-4, we determined its co-crystal structure in complex with Fabs RUPA-39 (genetically related and structurally similar to RUPA-97) and RUPA-44 to a resolution of 2.2 Å. Structural alignment with high-resolution crystal structures of Pfs230-D1 and Pfs48/45-D3 demonstrated accurate structural fidelity of each domain with Cα RMSD values of 0.25 Å and 0.30 Å, respectively. Similarly, comparison with crystal structures of RUPA-39 bound to Pfs230-D1 and RUPA-44 bound to Pfs48/45-D3 confirmed identical binding modes in Design-4, with Cα RMSD values for all heavy chain and light chain CDR residues of 0.52 Å and 0.26 Å, respectively. Interestingly, when compared to a crystal structure of Pfs230-D1D2, a dissimilar interdomain orientation was observed for Design-4. When Design-4 was superimposed with the structures of Pfs230-D1 bound by either 15C5 or LMIV230-02, antibody clashing with Pfs48/45-D3 was observed even though binding to LMIV230-02 was partially retained. Therefore, while Design-4 demonstrated an improved expression yield, the sub-optimal occlusion of some non-functional epitopes suggested the inter-domain interface may benefit from further stabilization.

The structure of Design-4 provided an opportunity to apply the fixed-backbone sequence design tool ProteinMPNN to generate a new interdomain interface with improved stability and absent binding to unwanted mAbs. One hundred sequences were generated and filtered using sequence likelihood and recovery metrics, from which 15 diverse sequences were selected for expression testing. Once again, we utilized mAbs RUPA-97 and TB31F, directed to Pfs230-D1 and Pfs48/45-D3, respectively, to quantify the expression of properly folded, secreted protein designs using BLI. Five designs (Designs-8, -14, -16, -17, and -22) exhibited superior expression relative to Design-4 by BLI quantitation assay. All five new designs were assessed by SDS-PAGE, and Design-22 showed the highest expression levels of monodispersed protein by SEC with a yield 12-fold higher than Design-1. Binding kinetics analysis was performed using mAbs RUPA-97, LMIV230-01, RUPA-38, TB31F, and RUPA-44 for Design-4 and Design-22. Binding affinities for all mAbs tested for Design-22 were consistent with those for Pfs230-D1 and Pfs48/45-D3, and within 3-fold of the dissociation constant across all epitopes. Importantly, binding was not detected for either 15C5 or LMIV230-02. Here, we identified Design-22 as a stabilized tandem antigen chimera (abbreviated as STAC) that recapitulated the presentation of native potent epitopes, while fully masking known non-potent epitopes on Pfs230-D1.

Potent epitopes are accurately presented on STAC
To understand the pseudo-native arrangement of Pfs230-D1 and Pfs48/45-D3 in STAC, we performed small-angle X-ray scattering (SAXS) analyses of Designs-1, -4, and STAC, which revealed a reduced radius of gyration (Rg) for both Design-4 and STAC compared to Design-1, supporting that the ProteinMPNN-generated sequence recapitulated the solvated structural profile of the template fold. To further elucidate the inter-domain disposition and antigen presentation of the lead design, we determined the structure of STAC bound by two potent Pfs230-D1-directed mAbs, RUPA-97 and LMIV230-01, and by two potent Pfs48/45-D3-directed mAbs, TB31F and RUPA-44, to a resolution of 3.2 Å by cryogenic electron microscopy (cryo-EM). Alignment of STAC with structures of Pfs230-D1 and Pfs48/45-D3 demonstrated accurate structural fidelity for each domain with Cα RMSD values of 0.26 Å and 0.38 Å, respectively, despite the incorporation of 16 interface mutations in this new design. Similarly, when compared with crystal structures of Pfs230-D1 bound by LMIV230-01 or RUPA-97 and Pfs48/45-D3 with TB31F or RUPA-44, STAC retained identical binding modes in complex with LMIV230-01, RUPA-97, TB31F, and RUPA-44, with combined epitope and paratope Cα RMSD values of 0.35 Å, 0.44 Å, 0.31 Å, and 0.35 Å, respectively. Notably, STAC exhibited distinct interdomain orientations when compared to crystal structures of Pfs230-D1D2 or Pfs4845-D1D2D3, although in both cases, similar interdomain regions were occluded between either Pfs230-D1 or Pfs48/45-D3 and the adjacent domain. Interestingly, STAC did not present an identical interdomain orientation when compared to the structure of Design-4, which was used as the input template for the ProteinMPNN interface re-design. At a resolution of 3.2 Å, we were able to observe sidechain conformers and recognized a general trend in which larger hydrophobic residues packed within the buried interdomain interface, while residues capable of polar interactions were positioned in more solvent accessible regions. Collectively, these mutations contributed to the observed increase in monomeric protein expression yield while also concealing the Pfs230-D1 epitopes of 15C5 and LMIV230-02 in a pseudo-native manner due to steric occlusion by the Pfs48/45-D3 domain.

Improved stacked antigen stability does not compromise functional immunogenicity
To assess the immunogenicity of STAC relative to Pfs230-D1, Pfs48/45-D3, and their combination at low dose, we immunized mice with each antigen group loaded onto CPQ nanoparticles (1 µg each) or in combination (0.5 µg + 0.5 µg). While anti-Pfs48/45 ELISA titers were slightly lower in the STAC group compared to Pfs48/45-D3 or the combination groups, anti-Pfs230 ELISA titers were similar among STAC, Pfs230-D1 and the combination groups. On CPQ liposomes, STAC is linked to CoPoP by a C terminal 6x His tag which may bias the response to the apical Pfs230-D1 domain over the basal Pfs48/45-D3 domain. To determine whether antigen orientation on CoPoP influenced immunodominance, we immunized mice with N-tagged STAC (N-STAC). Antibody titers and functional assays confirmed that N-STAC elicited responses comparable to the C-tagged STAC (C-STAC), suggesting a minimal effect of domain orientation on CPQ liposomes for immunodominance in mice.

We previously identified stabilizing mutations in Pfs48/45-D3, incorporated into 6C.mAgE1, enhanced recombinant protein yield and thermostability, both of which were associated with increased potency. In contrast, the development of Pfs230-D1-based immunogens, such as D1M, has not been as adversely affected by poor thermostability. We utilized differential scanning calorimetry (DSC) to assess the thermostability of several tandem antigen chimeras comprising Pfs230-D1 and Pfs48/45-D3, representing the design series starting from Design-1 and leading to STAC. Melting temperatures (Tm) and their onset values T onset were also measured for the individual domains of Pfs230-D1 (D1+) and Pfs48/45-D3 (6C.mAgE1). We observed Tm and T onset values of 60.6 °C and 54.2 °C for Pfs230-D1 and values of 69.2 °C and 63.1 °C for Pfs48/45-D3, which closely matched the previously reported thermostability data for these antigens. We found Design-1 possessed the lowest measured Tm and T onset values of 52.9 °C and 45.8 °C within the design series. The linker optimization in Design-4 resulted in a minor increase in Tm and T onset values to 54.3 °C and 46.2 °C, while interface re-design further enhanced thermostability with STAC showing the greatest improvement with Tm and T onset values of 59.1 °C and 52.3 °C, which most closely approached the parameters observed for Pfs230-D1.

While STAC possessed multiple stabilizing mutations that improved the expression yield and biochemical tractability over both Design-1 and Design-4, whether these alterations would adversely impact functional immunogenicity was undetermined. To address this question, we immunized mice using the same prime-boost and 5 µg dosing regimens as described in Fig. 1 with CPQ liposomes loaded with either Design-1, -4, or STAC antigens. Mouse immunizations with each of these three antigens elicited equivalent high antibody titers against both domains and all resulted in similarly potent antibody responses as determined by SMFA from three antibody dilutions, thereby confirming the fidelity of the potent epitopes in the structurally engineered STAC antigen.

STAC on protein nanoparticles elicits robust transmission-reducing activity
Protein nanoparticles are an established strategy for enhancing the potency of subunit-based vaccines by promoting B cell receptor clustering and processing by antigen-presenting dendritic cells. Two clinically validated platforms, H. pylori ferritin and I53-50, display 24 and 60 antigen copies, respectively. To assess the functional activity elicited by STAC relative to Pfs230-D1 and Pfs48/45-D3 using a multivalent display platform, we conducted an immunization study similar to the previous CPQ liposome experiments but using protein nanoparticles with the adjuvant AddaVax delivered via subcutaneous injections in a prime-boost regimen. To ensure comparability across antigen groups and nanoparticle platforms of different valencies, we maintained a consistent molar stoichiometry for each antigen across all immunization groups.

For I53-50-based immunogens, STAC, Pfs48/45-D3 (6C.mAgE1), and Pfs230-D1 (D1+) were genetically conjugated to the I53-50A component homotrimer which assembles into nanoparticles in the presence of I53-50B component. Self-assembling H. pylori ferritin-based immunogens were similarly constructed for each antigen but with two additional nanocage enhancements: engineered N-linked high mannose glycans and the PADRE pan-DR T cell epitope, which have been previously employed for an next-generation PfCSP H. pylori ferritin immunogen. Cocktails were generated by mixing independently assembled immunogens of Pfs230 and Pfs48/45 on either I53-50 or H. pylori ferritin, while mosaics were formed by co-assembling mixtures of Pfs230 and Pfs48/45 on I53-50A with I53-50B. Importantly, the molar stoichiometries of all cocktail and mosaic groups were twice that of the individual antigen groups to account for the two antigenic domains present in the STAC design. Immunizations elicited robust and specific antibody responses, as detected by ELISA. When Pfs230-D1 and Pfs48/45-D3 were combined as cocktails or mosaics on ferritin or I53-50, ELISA signals remained consistent with those of the respective single-antigen immunogens. Notably, for STAC presented on H. pylori ferritin or I53-50, Pfs230-D1 titers remained comparable to those of single-antigen immunogens, whereas sera reactivity to Pfs48/45-D3 was lower. To further characterize the immunodominance hierarchies of epitopes on STAC, we depleted mouse sera after immunization with STAC on H. pylori ferritin using monomeric STAC, Pfs230-D1, Pfs48/45-D3, or a combination of Pfs230-D1 and Pfs48/45-D3, followed by ELISA to detect remaining STAC-specific antibodies. Depletions with STAC, Pfs230-D1, or the combination of Pfs230-D1 and Pfs48/45-D3 removed >99%, 97%, and 99% of STAC-specific antibodies, respectively, while depletion with Pfs48/45-D3 alone only depleted 44% of the STAC-reactive antibodies, similar to the 35% depleted after the negative-depletion control. TRA was evaluated using the depleted sera from each depletion group by SMFA. Total sera, Pfs48/45-depleted, or negative-depleted control exhibited TRA values of 98.3%, 94.2%, and 93.2%, respectively. In contrast, no detectable TRA was measured for sera depleted for STAC-, Pfs230-D1-, or both Pfs230-D1 and Pfs48/45-D3-specifc antibodies. Together, these data indicate that the mouse antibody response and corresponding inhibitory activity are predominantly directed to the Pfs230-D1 domain of STAC, consistent with results from STAC immunization on CPQ nanoparticles.

Functional immunogenicity elicited by each immunogen group was evaluated by SMFA with serial dilutions of pooled sera. SMFAs performed with sera diluted 27-fold demonstrated >80% TRA across all antigen-containing I53-50 immunogen groups, whereas sera from negative controls exhibited no TRA. At 81-fold dilution, sera from the I53-50 immunogen group retained strong TRA, while the Pfs230-D1 and Pfs48/45-D3 groups both dropped below 80% TRA, and the mosaic and cocktail groups also saw modest decreases in activity. Notably, at 243-fold dilution, only the STAC group retained more than 80% TRA, and had significantly higher TRA than other groups. In contrast to the I53-50 immunogens, sera diluted 81-fold from all antigen-containing H. pylori ferritin immunogen groups demonstrated >80% TRA, potentially due to the engineering of N-linked glycans and helper T cell antigen in the H. pylori ferritin component. As seen with the I53-50 immunogens, at 243-fold dilutions of sera elicited by H. pylori ferritin immunogens, only the STAC group demonstrated >80% TRA, and this group had significantly higher TRA than the other groups. Taken together, these data indicate that when formulated as protein nanoparticle immunogens, STAC can elicit a more potent functional response at higher sera dilutions when compared to Pfs230-D1 and Pfs48/45-D3, both as individual antigens and in combination as cocktails or mosaics.

Methods
Recombinant protein expression and purification
Cell lines. For expression of recombinant proteins, female human cell lines (HEK293F, FreeStyle™ 293-F cells, Thermo Fisher Scientific; HEK 293S, GnT I−/− cells, ATCC) were cultured in suspension in GIBCO™ FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) for 6–7 days at 37°C, with 70% humidity and 8% CO2 and rotating at 150 rpm. These cell lines were not authenticated beyond the manufacturer.

Antigen monomers. All Pf antigens were based on the consensus 3D7 sequence, gene synthesized and cloned into the pcDNA3.4 expression vector (GeneArt) and expressed and purified as previously described. Briefly, antigens were transiently expressed in HEK293F or HEK293S cells (Thermo Fisher Scientific) and purified using a 5mL HisTrap FF column (GE Healthcare), followed by size exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare). Sequences of designed antigens are available in Supplementary Fig. 6.

Antibodies. Antibodies were expressed and purified as explained previously. Briefly, variable light (VL) and heavy (VH) chains of Fabs used in these studies were gene-synthesized and cloned (GeneArt) into custom pcDNA3.4 expression vectors immediately upstream of human Igκ and Igγ1-CH1 domains. Fab heavy chain and Fab light chain plasmids were co-transfected at a 2:1 ratio into FreeStyle 293-F cells (Thermo Fisher Scientific) at a cell density of 0.8×106 cells/ml for transient expression using PEI MAX transfection reagent (Polysciences). Cells were cultured in GIBCO FreeStyle 293 Expression Medium for 6–7 days, and supernatants were isolated by centrifugation and filtered through a 0.22-μm membrane. Fabs were purified following a scheme of HiTrap KappaSelect affinity chromatography (Cytiva) and cation exchange chromatography (MonoS, Cytiva).

H. pylori ferritin protein nanoparticles. Antigen-conjugated nanoparticles containing the mutations from native Helicobacter pylori ferritin sequence (K75N, E77T, E99N, and I101T) to add two N-linked glycan sequons, as well as addition of a PADRE sequence at the C-terminus (AKFVAAWTLKAAA) were transiently expressed in HEK293F cells (Thermo Fisher Scientific) in the presence of 10µM Kifunensine (Cayman Chem) to obtain high-mannose glycans. All purification instruments were washed with 500 mM sodium hydroxide and purified water prior to protein isolation to avoid endotoxin contamination. Purification was completed using a 5 mL HisTrap FF column (GE Healthcare), followed by size exclusion chromatography (Superose 6 10/300 GL, GE Healthcare). Endotoxin was assessed using the EndoSafe Nexgen-PTS System (Charles River). The threshold for samples suitable for immunization was <5 EU/ml. If required, endotoxin removal using a ToxinEraser endotoxin removal kit (GenScript) was performed, and endotoxin levels below threshold were reconfirmed. Antigen-conjugated nanoparticle structural integrity was confirmed by negative stain electron microscopy. Proteins were diluted to 20–50 μg/ml and adsorbed onto homemade carbon film-coated grids, which were stained with 2% uranyl formate. Specimens were imaged with a Hitachi HT7800 electron microscope operating at 120 kV with a Xarosa CMOS camera.

I53-50 protein nanoparticles. Plasmids encoding antigen-conjugated I53-50A and I53-50B.4PT1 were synthesized by GenScript and cloned into pET29b between the NdeI and XhoI restriction sites. I53-50B.4PT1 incorporated a double stop codon directly before the C-terminal polyhistidine tag to make a tagless construct. Antigen-conjugated I53-50A proteins were produced in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33°C, 70% humidity, 8% CO2, rotating at 150 rpm. Cultures were transfected using PEI-MAX (Polyscience) with cells grown to a density of 3.0 million cells per mL and cultivated for 3 days. Supernatants were clarified by centrifugation (5 min at 4000 xg), the addition of PDADMAC solution to a final concentration of 0.0375% (Sigma Aldrich, #409014), and a second spin (5 min at 4000 xg). Proteins containing His tags were purified from clarified supernatants via an AKTA AVANT FPLC using prepacked 5 mL Ni Sepharose Excel resin columns (Cytiva), with each clarified supernatants supplemented with 1 M Tris-HCl, pH 8.0, to a final concentration of 45 mM and 5 M NaCl to a final concentration of ∼310 mM, prior to column application. The resin was washed with 5 column volumes (CV) of 20 mM Tris pH 7.0, 150 mM NaCl, and the protein was eluted over 10 CV up to 100% B buffer, which was composed of 20 mM Tris pH 7.0, 150 mM NaCl, 500 mM imidazole. The pentamer was expressed in Lemo21 (DE3) cells (NEB) in LB medium (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) in a 10 L BioFlo 320 Fermenter (Eppendorf). At inoculation, culture conditions were set to 37°C, 225 RPM impeller speed, and 5 SLPM (standard liter per minute) gas flow, with O2 supplementation active as part of the dissolved oxygen cascade. At the onset of a dissolved oxygen spike (OD~12), the culture was supplemented with 100 mL of 100% glycerol and induced with 1 mM IPTG. Simultaneously, the temperature was lowered to 18°C, and O2 supplementation was stopped. Protein expression continued overnight until reaching an OD~20. Cells were harvested by centrifugation, and pellets were resuspended in dPBS (Cat# 14190144), homogenized, and lysed using a Microfluidics M110P microfluidizer at 18,000 psi for three passes. Lysates were clarified by centrifugation (24,000 xg, 30 min, 4°C), and the supernatant was discarded to isolate inclusion bodies. Inclusion bodies were first washed with dPBS, 0.1% Triton X-100 (pH 8.0), followed by centrifugation for sample clarification. The resulting pellet was then washed with dPBS supplemented with 1 M NaCl (pH 8.0). After the second wash, pentamer protein was extracted from the pellet using dPBS, 2 M urea, 0.75% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), pH 8.0. Extracted protein was loaded onto a DEAE Sepharose FF column (Cytiva) using an AKTA Avant 150 FPLC system (Cytiva). After binding, the column was washed with 5 CV of dPBS + 0.1% Triton X-100 (pH 8.0), followed by 5 CV of dPBS + 0.75% CHAPS (pH 8.0). Elution was performed using 3 CV of dPBS + 500 mM NaCl (pH 8.0). Eluted fractions for both antigen-conjugated I53-50A and I53-50B.4PT1 were confirmed by SDS-PAGE, pooled, concentrated using 10K MWCO centrifugal filters (Millipore), sterile-filtered (0.22 μm), and applied to a HiLoad S200 pg GL SEC column (Cytiva) using either dPBS or 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer, respectively. After sizing, peak fractions for each protein were confirmed by SDS-PAGE, pooled, concentrated using 10K MWCO centrifugal filters (Millipore) to 50 μM, sterile-filtered (0.22 μm), and assessed for low endotoxin content before freezing in single-use aliquots stored at −80°C. Endotoxin levels in protein samples were measured using the EndoSafe Nexgen-MCS System (Charles River). Samples were diluted 1:50 in Endotoxin-free LAL reagent water and applied to wells of an EndoSafe LAL reagent cartridge. Charles River EndoScan-V software was used to analyze endotoxin content, automatically back-calculating for the dilution factor. Endotoxin values were reported as EU/mL, which were then converted to EU/mg based on UV/vis measurements. The threshold for samples suitable for immunization was <10 EU/ml.

Liposome nanoparticles. CPQ liposomes were prepared as previously described with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC; Corden, catalog number LP-R4-070), cholesterol (CHOL; PhytoChol, Evonik), Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (PHAD-3D6A; Avanti catalog number 699855), and QS-21 (Desert King). Liposomes had a [DOPC: CHOL: CoPoP: PHAD-3D6A: QS-21] mass ratio of [20: 5: 1: 0.4: 0.4] and were prepared using ethanol injection and nitrogen pressurized extrusion through stacked 200 nm, 100 nm, and 80 nm polycarbonate membranes (Whatman) in PBS, carried out at 50°C. Ethanol was then removed by dialysis against PBS twice at 4°C. QS-21 (1 mg/mL) was added to the liposomes after formation. Liposomes were sterile filtered and stored at 4°C. Liposome sizes were determined by dynamic light scattering (DLS) with a NanoBrook 90 plus PALS instrument after 200-fold dilution in PBS. To assess the stability of constructs of interest bound in particle form, a Ni-NTA competition assay was carried out as previously described. Ni-NTA magnetic beads (Thermo Fisher Scientific, catalog no. 88831) were added to the CPQ liposome-incubated antigen constructs (1:4 mass ratio of total protein: CoPoP) and incubated with the beads for 30 min at RT, then the supernatant and beads were separated using a magnetic separator (Thermo Fisher Scientific, catalog no. 12321D). Denaturing reducing loading dye was then added to the samples (supernatant or beads) and heated at 95°C for 10 min. The samples were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using Novex 4 to 12% bis-tris acrylamide gels (Invitrogen, catalog no. NP0321BOX) and MES running buffer (Invitrogen, catalog no. NP0002).

Animal immunizations
Protein nanoparticles. Female Crl:CD1 (ICR) outbred mice were purchased from Charles River (order code 022) at 7 weeks of age and were housed in a specific pathogen-free facility within the Department of Comparative Medicine at the University of Washington, Seattle. Animals had access to standard rodent chow and water ad libitum and were maintained in an ABSL1 housing room with environmental parameters set to 68–79°F, 30–70% humidity, and a dark/light cycle of 10 h of dark and 14 h of light. For each immunization, low-endotoxin immunogens were mixed with 1:1 vol/vol AddaVax (InvivoGen vac-adx-10) to reach the specified final dose for each immunogen per 0.1 mL injection. At 9 weeks of age, 5 mice per group were injected subcutaneously in the inguinal region at week 0 for the prime immunization and at 13 weeks of age for the boost immunization at week 4. Mice were bled via the submental route at weeks 0 and 2 and terminally at week 6. Blood was collected in serum separator tubes (BD #365967) and rested for 30 min at room temperature for coagulation. Serum tubes were then centrifuged for 10 min at 2000 xg, and serum was collected and stored at −80°C until use.

Lipid nanoparticles. The immunogenicity of Pfs230-D1, Pfs48/45-D3, and various designed antigen constructs was assessed using CPQ liposomes. To prepare the final vaccine, CPQ liposomes were incubated with the constructs for 3 h at RT. For each group, six 6–8 week old female CD-1 mice (Envigo) were immunized intramuscularly with indicated doses of protein bound to liposomes in a prime/boost dose regimen at day 0 and 21; sera were collected at day 42.

Ethical Statement. Animal experiments involving protein nanoparticles were conducted in accordance with the University of Washington’s Institutional Animal Care and Use Committee (IACUC protocol 4470-01). The University of Washington’s animal use program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Reference Assurance: #000523). Immunization studies involving lipid nanoparticles were carried out in compliance with the University at Buffalo IACUC protocols (protocol BME05044Y).

ELISA, antibody depletion and SMFA
Lipid nanoparticles. All ELISA were conducted using Pfs230-D1 or Pfs48/45-D3 as coating antigens using the method described previously. For the ELISA data, log-transformed values were compared by a Student's t-test (two groups), or one-way ANOVA followed by Tukey’s multiple comparison tests (more than two groups). The ability of antibodies to reduce the development of P. falciparum NF54 strain oocysts in the mosquito midgut was evaluated by SMFA as described elsewhere. In brief, IgG was purified from whole sera using Protein G affinity chromatography, and then indicated concentrations of IgG were mixed with 0.15%–0.2% stage V gametocytemia and then fed to 3-6-day-old female Anopheles stephensi. All experiments were done in the presence of human complement. The mosquitoes were maintained for 8 days and then dissected to count the number of oocysts per midgut in 20 mosquitoes. For the statistical analysis of SMFA results, a zero-inflated negative binomial model was used.

Protein nanoparticles. To measure antibody titers in total sera, 50 μL of 2 μg/mL of Pfs230-D1 or Pfs48/45-D3 was plated onto 96-well Nunc Maxisorp (Thermo Fisher) plates in TBS. Plates were incubated at 25°C for 1 h, then blocked with 200 μL of 2% BSA in TBST for an additional 1 h at 25°C. Plates were washed 3× in TBST using a plate washer (BioTek), 1:5 serial dilutions of mouse sera were made in 50 μL TBST starting at 1:100 and incubated at 25°C for 1 h. Plates were washed 3× in TBST, then anti-mouse horseradish peroxidase-conjugated horse IgG (Cell Signaling Technology, #7076S) was diluted 1:2,000 in 2% BSA in TBST, and 50 μL was added to each well and incubated at 25°C for 30 min. Plates were washed 3× in TBST, and 100 μL of TMB (SeraCare) was added to every well for 2 min at room temperature. The reaction was quenched with the addition of 100 μL of 1N HCl. Plates were immediately read at 450 nm on a SpectraMax M5 plate reader (Molecular Devices), and data were plotted and fit in Prism (GraphPad) using nonlinear four-parameter logistic sigmoidal regression, where X is log(concentration) to determine EC50 values from curve fits. For depletion experiments, NHS-activated magnetic beads (Pierce) were coated with Pfs230-D1, Pfs48/45-D3, STAC or PBS, following the manufacturer’s instructions. After coupling, the beads were washed and stored in PBS. Sera were incubated with beads for 1.5 h at room temperature while gently mixing the suspension. After incubation, beads were separated on a magnetic stand, and serum was collected for analysis by ELISA and SMFA. One batch of serum was depleted with beads coated with Pfs48/45-D3, followed by another round of depletion with beads coated with Pfs230-D1. Depletion of antigen-specific antibodies was tested by ELISA. In short, 96-well Nunc Maxisorp (Thermo Fisher) plates were coated with 1 μg/mL STAC in PBS overnight at 4°C. Plates were washed with PBS and blocked with 5% milk in PBS for 1 h at 25°C. After washing three times with PBS, serial dilutions of (depleted) sera in 1% milk in PBST were added in duplicate and incubated for three hours at 25°C. Plates were washed with PBS, and 1:3000 diluted polyclonal rabbit anti-mouse HRP (P0260, Dako) in PBST was added, and plates were then incubated for 1 h at 25°C. After washing, the plates were incubated with TMB, and reactions were stopped with H2SO4. Plates were read at 450 nm. EC50 values were calculated using nonlinear four-parameter logistic sigmoidal regression in Prism (GraphPad). SMFA experiments were conducted as previously described. Blood meals containing cultured P. falciparum NF54 gametocytes mixed with mouse sera were fed to Anopheles stephensi mosquitoes (Nijmegen colony). Serum dilution factors indicate the proportion of serum to the total blood meal volume. All SMFA experiments were conducted in the presence of active human complement. For each condition, 20 fully-fed mosquitoes were dissected, and oocysts were counted 6-8 days after feeding. Transmission-reducing activity was estimated using a mix-effects negative binomial regression model as previously described.

Biolayer interferometry
Kinetics. Biolayer interferometry (Octet RED96, Sartorius) experiments at 25°C were conducted to determine the binding kinetics of antibody-antigen interactions using recombinant His-tagged antigen proteins and Fabs. Antigen protein was diluted in kinetics buffer (PBS, pH 7.4, 0.01% (w/v) BSA, 0.002% (v/v) Tween-20) at 20 μg/ml and immobilized onto Ni-NTA (NTA) biosensors (FortéBio) to a threshold binding signal of 1.0 nm. Following a 60 s baseline measurement, biosensors were dipped into wells containing a two fold dilution series of Fab, from 500 nM to 15.6 nM, to monitor the association rate and then dipped back into kinetics buffer to monitor the dissociation rate. Kinetics data were analyzed using FortéBio’s Data Analysis software 9.0, and kinetic curves were fitted to a 1:1 binding model using at least four concentrations.

Quantitation. For quantification experiments, RUPA-97 or TB31F Fabs were diluted in kinetics buffer to a concentration of 20 μg/ml and immobilized on an anti-human Fab CH1 biosensor (Sartorius). Pfs230-D1 (D1+) and Pfs48/45-D3 (6C.mAgE1) were serially diluted in clarified and filtered HEK293F supernatant to a concentration range of 0–50 μg/mL, which was used to generate a standard curve from the association rate. For supernatants of all antigen designs quantitated, the initial association slopes were used for calculations, and all analyses were done using Sartorius’ Data Analysis software 9.0. To contrast the relative signals for each antigenic domain, relative binding signals were additionally calculated by normalizing each binding signal relative to the maximum observed binding signal for each specific antigen.

X-ray crystallography
For the ternary complex of RUPA-39 and RUPA-44 Fabs bound to Design-4, a 1.5:1.5:1 molar ratio of Fab:Fab:antigen was used. RUPA-44 and Design-4 were both expressed from HEK293S cells and previously treated with EndoH to truncate N-linked glycans present on both molecules. For the complex of RUPA-39 Fab bound to Pfs230-D1+, a 1:1.5 molar ratio of Fab:antigen was used. Both Fab-antigen complexes were incubated for 1 h at 4°C followed by size exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva) in 20 mM Tris and 150 mM sodium chloride at pH 8.0. Purified RUPA-39:Pfs230-D1+ complex was concentrated to 9.2 mg/ml and mixed in a 1:1 ratio with crystallization buffer (0.2 M calcium acetate and 20% (w/v) PEG 3350). Crystals grew from a sitting-drop by vapor diffusion at 20°C and were cryoprotected in crystallization buffer supplemented with 15% ethylene glycol, before being flash-frozen in liquid nitrogen. Diffraction data were collected at the CMCF-ID beamline at the Canadian Light Source and then processed and scaled using XDS and Aimless. Purified RUPA-39:RUPA-44:Design-4 complex was concentrated to 13.0 mg/ml and mixed in a 1:1 ratio with crystallization buffer (0.1 M sodium cacodylate, pH 6.5, 40% PEG 300, 0.2 M calcium acetate). Crystals grew from a sitting-drop by vapor diffusion at 20 oC and were cryoprotected in crystallization buffer supplemented with 10% ethylene glycol, before being flash-frozen in liquid nitrogen. Diffraction data were collected at the 23-ID-D beamline at the Argonne National Laboratory Advanced Photon Source. The diffraction data was processed with XDS and scaled with correction for anisotropy using the Staraniso server to a diffraction-limit cutoff (I/σ(I)) of 1.2. Both structures were determined by molecular replacement using Phaser. Structural refinements were performed using PHENIX, and models were manually checked and improved with Coot. Images were generated using PyMOL (The PyMOL Molecular Graphics System, v2.3.4, Schrödinger, LLC.). Access to all software was supported through SBGrid.

Computational design
Mutable residue selection for the Design-4 interdomain interface was defined as residues containing sidechain atoms within 7 Å of the neighboring domain as observed from the crystal structure of Design-4 bound by RUPA-39 and RUPA-44 Fabs. For fixed-backbone sequence redesign, the crystal structure was used as input to ProteinMPNN (v1.0.1) and the 34 positions located around the interdomain interface were selected for redesign. The input model backbone coordinates were either noised by 0.02 Å or left unaltered, and three temperatures (0.1, 0.2, and 0.3) were sampled using the default weights for all twenty amino acids, with 20 sequences generated per temperature for a total of 120 sequences generated using the v_48_020 model of ProteinMPNN. Generated sequences were ranked by the ProteinMPNN sequence likelihood score with preference for higher sequence recovery to ensure proper domain folding and down-selected to 15 sequences, which were predicted with AlphaFold2 using 6 recycling steps without structural templating. All 15 designs adopted domain folds that matched Pfs230-D1 and Pfs48/45-D3 crystal structures with Cα RMSD < 1.0 Å for beta-sheet residues and possess interface residues with pLDDT > 80, although interdomain orientations were not identical with the input structure for all designs. All 15 designed sequences were selected for expression testing.

Cryo-EM
Data collection. The complex of the stabilized tandem antigen chimera (STAC) of Pfs230 and Pfs48/45 bound to RUPA-97, LMIV230-01, TB31F, and RUPA-44 was concentrated to 5 mg/ml with 0.13 CMC of DDM in 20 mM Tris and 150 mM sodium chloride at pH 8.0. 3.2 µl of the sample was deposited on glow-discharged homemade holey gold grids. A Leica EM GP2 Automatic Plunge freezer operated at 4°C and 90% humidity was used to blot away excess sample (2.7 s), and grids were subsequently plunge-frozen in liquid ethane. Data were collected on a Thermo Fisher Scientific Titan Krios G3 operated at 300 kV and equipped with a Selectris X energy filter with a slit width of 10 eV and a Falcon 4i camera. Data acquisition was automated with EPU software. A nominal magnification of 130,000× with a calibrated pixel size of 0.93 Å was used for data acquisition, and exposures were collected as 6.4 or 6.5 s movies with a total exposure of ~50 or ~53.7 electrons/Å2. Movies were recorded in Electron Event Representation mode and fractionated into 40 frames prior to data analysis. 4676 and 5995 raw movies were obtained at 0° and 35° tilt, respectively, across two data collections.

Image processing. Cryo-EM data were processed using CryoSPARC v4.5.2. The movies from the un-tilted and 35° tilted datasets were corrected using patch motion correction, and contrast transfer function (CTF) parameters were determined using patch CTF estimation. A subset of un-tilted micrographs was used to pick and extract particles for 2D classification. Selected 2D classes were used as templates for the template picker on a set of 4085 un-tilted micrographs (CTF < 5 Å). 2X Fourier cropped particles were extracted, underwent 2D classification and selection, and were used for ab initio reconstruction. After heterogeneous refinement, particles from the selected class were re-extracted from micrographs without Fourier cropping (1,094,268 particles) and used for ab initio reconstruction with two classes, followed by heterogeneous refinement and non-uniform refinement to generate a 3.2 Å map (399,207 particles). This volume was used to generate templates for template particle picking on a curated dataset including both tilted and un-tilted data using thresholds for average defocus values (1000-30000 Å), CTF fit resolution (<3.5 Å for un-tilted micrographs, <4.2 Å for tilted micrographs), and relative ice thickness (0.95-1.13). A total of 5287 micrographs were accepted, including 3122 untilted and 2165 35° tilted micrographs. Template picker resulted in 2,674,017 extracted particles (360×360 px box size). 2D classification and selection were used to remove poor-quality particles, and the resulting particles were used for ab initio 3D reconstruction (done twice with two different sets of 2D classes selected). Generated maps were then used in several rounds of heterogeneous refinement to further classify particles. 2D classes corresponding to rare views of the complex were selected for Topaz particle picking, training and extraction. After subsequent extraction and 2D classification, selected particles were combined with a previous set of 814,237 particles, followed by duplicate particle removal and heterogeneous refinement. Topaz training, particle picking, and heterogeneous refinement were done twice in parallel. Particles from selected heterogeneous refinement classes from each job were combined, and duplicates were removed, resulting in 2,216,915 particles. This set of particles was used for heterogeneous refinement with 5 volumes, and particles from one class (645,647 particles) were subsequently used for 3D classification with 5 classes. The selected class underwent one round of heterogeneous refinement, and the class corresponding to the complex was put through duplicate removal (147,546 particles), followed by non-uniform refinement. Local refinement with a mask containing the stabilized tandem antigen chimera and the variable regions of RUPA-97, LMIV230-01, TB31F, and RUPA-44 was done to achieve a 3.22 Å map (147,546 particles). A detailed workflow for cryo-EM data processing is depicted in Supplementary Fig. 8.

Modeling building. Starting structural models were obtained by fitting previously determined structural models (PDB: 6E63, Pfs48/45-D3:TB31F; 7UXL, Pfs48/45-D3:RUPA-44; 7UVQ, Pfs230-D1:RUPA-97; and 7UFW, Pfs230-D1D2:LMIV230-01) into the experimentally determined map. Model building and refinements were done using PHENIX and Coot. Images were generated using ChimeraX, and access to all software was supported through SBGrid.

Small-angle X-ray scattering
An Anton Paar SAXSpace instrument (Anton-Paar GmbH, Austria) with SAXSDrive software was used to collect SAXS scattering curves for monomeric antigen proteins at concentrations between 3–4 mg/ml in a 1x PBS buffer. SAXS data were collected in 6×10 min exposure frames at 20°C under vacuum. The zero position for each scattering curve was calibrated using SAXSTreat before buffer subtraction using SAXSquant. SAXStreat software (Anton-Paar GmbH, Austria) was used to define the zero position of the scattering curve and convert the data to 1D scattering profiles. SAXSQuant (Anton-Paar GmbH, Austria) was then used to desmear the data and perform buffer subtraction. Analyses of the processed SAXS data were done using the ATSAS package (version 3.2.1; EMBL, Hamburg, Germany). PRIMUS was used to perform Guinier analysis, and GNOM was used to calculate the P(r) distance distribution. Additional SAXS data collection parameters are available in Supplementary table 4.

Differential scanning calorimetry
Antigen samples at 1 mg/ml were buffer exchanged into 1X PBS, and all measurements were conducted on a Nano DSC (TA Instruments) in duplicate, unless otherwise stated. The sample was heated from 20 to 95°C at a constant heating rate of 1.0°C/min. Runs with only 1x PBS buffer were performed and subtracted from the melting curves, and the data were fit to a single-stage unfolding model. The melting temperature (Tm) for each protein was determined by the NanoAnalyze software (TA Instruments) by fitting the data to the Gaussian curve with a melting temperature onset (T onset) model.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Ivanochko et al presents an interesting protein design story. Two related proteins, Pfs230 and Pfs48/45 each contain domains which elicit antibodies which can block transmission of malaria parasites and a structural comparison suggested to the authors that two of these domains can be combined into a novel chimeric antigen, while still presenting the key epitopes for transmission-blocking antibodies.\nThey conducted a stepwise design of this chimera, using a fusion, a linker design and finally a Protein MPNN-based design of the interface, validating at each step using mAb binding. They also used structural biology during the design process and at the end to validate the final design. This is a high-quality piece of work, with the design and validation well-presented and the data of high quality too. It resulted in a successful design.\nThe authors then tested their design (“STAC”), either attached to liposomes or fused to I53-50 or ferritin nanoparticles. On liposomes, STAC performed similarly to Pfs230D1. On ferritin it also behaved similarly, albeit with some statistically significant improvement at one concentration point. On I53-50, STAC seemed substantially improved over the individual components, generating transmission blocking activity which survived greater dilution.\nThis is well conducted study, with an interesting design and STAC shows potential for use in future transmission blocking vaccine strategies. I have one experiment which I would strongly recommend. The sera generated and tested in Figure 5 could be used to assess whether the transmission blocking activity induced by STAC is primarily due to antibodies targeting 230 or 48/45 or equally due to both. To do this, the authors could simply deplete antibodies from these sera against either 230D1 of 48/45D3 and assess the TRA of what is left. This would give an interesting answer about the relative importance of the two fusion partners in STAC. In my view, this would be a very interesting experiment which would complete this high-quality study.\nSome questions:\nIn the first experiment, liposomes contained either the 230 antigen or the 48/45 antigen, or both mixed. Why did the authors not mix liposomes containing just 230 with liposomes containing just 48/45? As a B cell makes one type of antibody would it not be interesting to see if mixing two populations of liposomes, each liposome containing one antigen had a different effect to having all liposomes containing both antigens? Could the authors comment on why they didn’t do this? Also the lower efficacy of the dual-conjugated over 230 alone seems to not justify the subsequent approach?\nIn the design the authors sensibly present that the key TB antibodies should still be able to bind. However, there might be antibodies not structurally characterised which could also be useful for transmission block. Are they likely to bind? In the case of 48/45 could the authors estimate what fraction of the surface area of 48/45 D3 exposed in 48/45 is also exposed in their tandom design?\nDesign-1 bound to the desired neutralising antibodies but also bound to the non-inhibitory antibodies, whose epitopes should have been blocked in the new tandom construct. The authors make it clear that this indicates that further design is needed (lines 246-8). It would be good to be more explicit here. I think that these means that the two domains are not associated in the correct relative orientation but instead are flexibly linked so that the epitopes are not occluded? Also 278.\nLine 408-9 is too strongly worded. STAC only strongly outperforms on I53-50, with perhaps improvement (at one dilution point) for ferritin and none for liposomes. I think that the final conclusion should therefore be toned down a little.\nMinor comments:\nFigure 1c – could do with x axis labels for the black, yellow and blue circles.\nFigure 1 – why not show binding data for the transmission blocking antibodies as well as the non-blockers which shouldn’t bind?\nThroughout, it would help the reader to have a clearer explanation of what the different mAbs do/bind. More “Pfs230-binding mAb XXXX” etc…\nFigure 4b, should that be STAC not design-22?\nFigure 4f is missing x axis labels for the proteins.", "reviewer_2": "This paper generates a chimeric molecule combining two leading transmission-blocking vaccine candidates, Pfs230D1 and Pfs Pfs48/45-D3. These subdomains have been previously described to contain critical protective epitopes and have been used to elicit antibodies that block gametocyte ferritization in mosquitoes in complement-dependent (Pfs230D1) and complement-independent fashion, Pfs48/45. These candidate antigens are expressed on late-stage gametocytes, are immunogenic, and can boost antibody responses to vaccines based on these domains. This study uses a detailed structure study to generate a chimeric molecule that optimizes functional epitopes while masking epitopes that elicit non-functional Abs. They have successfully and convincingly accomplished this goal. The structure study and analysis well be done and clever. Overall comments on the structure studies initially described and followed by comments on the immunological and functional aspects.\nComments related to the structural design\nThe authors of this study report a stabilized tandem antigen chimera of Pfs230-D1 (179 residues) and Pfs48/45-D3 (137 residues), representing the most potent epitopes of each antigen, thereby removing distracting epitopes from both. The individual constructs were combined with liposomes and used to immunize mice. They then examined the potential of linking both antigens in a single construct and evaluated, using BLI and known mAbs, the binding to the construct to confirm correctly folded domains and the masking of unwanted epitopes.\nDesign4 resulted in a crystal structure that is indistinguishable by rmsd from the native individual structures of Pfs230 and Pfs48/45, indicating correctly folded proteins despite linking them together.\nIn a next step, the authors applied AI-supported methods ProteinMPNN to improve the interface design and reduce unwanted binding, resulting in additional suitable designs. The improved designs increased expression yields and binding affinities to the tested mAbs. It would be interesting to note if the authors used the default weights for amino acids for their interface design or skewed towards residues typically found in interfaces.\nParagraph 304-307\nHow many residues are involved in the interface in total? Are the 16 introduced mutations 20% of the interface, or is it more like 80%? This shows how dynamic an interface could be, redesigned while maintaining or improving the interface interaction so that others can derive their own interface designs for other proteins of interest.\nAlso there is a discrepancy in Figure 3c showing 18 mutated residues instead of 16?\n9N8I\nChain A and E might be worth revisiting Mopprobity to examine those atom clashes and potentially choose a different rotamer to improve the structure if the electron density supports that conformation.\nThis seems to be supported by section 6.1 with the rotamer outliers as well for Chain E\nAlso Rfree/Rwork seem to suggest a slight overfit, might be better to cut the resolution limit to 1.9 Å instead of 1.85 Å\n9N8N\nRotamers could be optimized.\nWhy did the authors model certain residues differently than the sequence construct suggests? I’m referring to the Table on Page 5, particularly residues 186, 195, 284, and 289.\nAgain it would be worth running Molprobity to optimize the rotamer outliers of Chain I\nMinor\nLine 175 define TRA\nComments related to immunology and the functional characteristics of the vaccine design\nThe standard membrane feeding assay (SMFA) is used to evaluate IgG isotypes enriched from mice after vaccination with various constructs to measure the reduction in oocyte formation. (Do you think other isotypes might be participate for protection?) These assays are well executed and are the typical functional tests to assess transmission-blocking activity. There was one biological replicate. The best chimeric molecule, STAC, shows comparable transmission-reducing activity (TRA) to the individual component antigens. In fact, its TRA is slightly lower than Pfs48/45 as a single molecule, suggesting that. There is only a slight improvement in TRA with STAC linked to nanoparticle immunization compared to the individual immunogens based on the SMFA model. Still, as the authors point out, the molecule is easy to produce and stable, which is important for vaccine candidate developability.\nThe other aspect of the study was a vaccine molecule designed to mask the stimulation of non-functional antibodies. Theoretically, this is important, as a non-functional antibody could cause a steric hindrance to functional antibodies. Is there data to support this concerning Pfs230 or Pvs48/45? If so, this should be discussed or highlighted as a limitation.\nFor example if you add non-blocking mAbs to antibodies elicited by the different constructs do you observed alterations in TRA activity?\nMinor points\n1. In Fig. 2, the relative binding shown in c and h can be better described. Is this relative binding of the new construct to the Pfs230 or Pfs48/45 individually? Please make this clearer.\n2. In some figures they used Design-22 and STAC interchangeably – this can be confusing. Please stick to one or the other designation as I presume these are the same constructs.\n3. Figure 5 is critical. The different color codes are difficult to follow, particularly with small figures with poor resolution. Suggest using different shapes and colors for the symbols denoting the different constructs.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the Reviewer for their positive assessment of our study where we present a stepwise design strategy to fuse distinct malarial transmission-blocking antigens together, as a proof-of-principle approach to occlude a non-functional epitope on Pfs230-D1. Indeed, while STAC performed similarly to Pfs230-D1 when presented on lipid nanoparticles, we found that STAC elicited significantly more potent functional activity at a high serum dilution when presented on either Hp ferritin or I53-50 nanoparticles, when compared to Pfs230-D1 or Pfs48/45-D3 on the same platforms. We agree with the Reviewer on the importance of understanding the relative contribution of each component of STAC to the overall antibody response and transmission-reducing activity. In the Result section related to the protein nanoparticle immunizations (lines 400–403), we note the following characterization of the Pfs230- and Pfs48/45-specific immune response:\nNotably, for STAC presented on H. pylori ferritin or I53-50, Pfs230-D1 titers remained comparable to those of single-antigen immunogens, whereas sera reactivity to Pfs48/45-D3 was lower. This suggests the immune response in mice predominantly targeted the Pfs230-D1 domain of STAC (Fig 5c), consistent with results from STAC immunization on CPQ nanoparticles (Fig 4).\nWe would like to thank the Reviewer for their suggestion of depletion assays to further interrogate the functional antibody responses presented in Figure 5. In fact, this is an experiment that has already been performed in the context of a follow-on study currently under way. Indeed, while the current manuscript aims to present the design of STAC and validate a non-inferior/superior immunogenicity and functional activity in WT mice, we had generated this depletion data as a component of a subsequent study dissecting the immunogenicity and individual contribution of each domain in mouse, rat, aotus and human Ig repertoire mouse at both the polyclonal and monoclonal levels. Given the ongoing nature of these experiments, and their extensive scope to properly characterize the immunodominance hierarchies of epitopes on STAC across preclinical species, we believe that this content is beyond the scope of the present article. Nonetheless, in transparency to the Reviewer, we are sharing the results of a depletion experiment from WT mouse sera after immunization with STAC on H. pylori ferritin, which further corroborates the reactogenicity data already present in the manuscript that the transmission-reducing activity elicited by STAC in WT mice is principally orchestrated by the Pfs230-D1-directed antibodies.\nFigure showing reactogenicity (left) and functional activity (right) of WT mouse sera from immunization with STAC on H. pylori ferritin depleted with specific antigens. (Left) ELISAs demonstrating residual STAC-reactive sera after depletion with indicated antigens. Percentages indicate reduction in titers compared to those in total serum. (Right) TRA of the same depleted sera at an 81-fold dilution. TRA values are estimates from two independent experiments.\nIn the revised manuscript, we have added to the discussion of immunodominance in the Discussion section, as well as provided further clarity on the importance for future work in this area to better understand its implication (lines 480–490):\nImportantly, the genetic conjugation of distinct epitopes may confer immunodominance hierarchies between epitopes, as we have observed here in WT mice immunizations with STAC that elicited a Pfs230-D1-dominant response. Several factors may contribute to this phenomenon, including germline-encoded bias of specific B cell lineages for residue signatures at specific epitopes70, proteolysis sensitivity in B cell follicles for specific epitopes71 or epitope prominence by antigen orientations on nanoparticles72. Investigating the rules of immunodominance for different epitopes, and their contributions to function across preclinical species and in the context of the human Ig repertoire will be important next steps to further understand responses to STAC-based immunogens, and whether further engineering is warranted to obtain a more balanced response to the potent Pfs230 and Pfs48/45 epitopes in a human Ig repertoire context.\nSome questions:\nIn the first experiment, liposomes contained either the 230 antigen or the 48/45 antigen, or both mixed. Why did the authors not mix liposomes containing just 230 with liposomes containing just 48/45? As a B cell makes one type of antibody would it not be interesting to see if mixing two populations of liposomes, each liposome containing one antigen had a different effect to having all liposomes containing both antigens? Could the authors comment on why they didn’t do this? Also the lower efficacy of the dual-conjugated over 230 alone seems to not justify the subsequent approach?\nTo provide additional clarity as to the overall rationale of the study, i.e. including two antigens in one construct, we added to the Introduction (lines 131–135):\nIn addition, seroprevalence of antibodies to Pfs230 and Pfs48/45 indicate that some individuals in endemic regions elicit detectable antibody titers exclusively to one individual antigen but not to the other29,30, so it is conceivable that an immunogen presenting both Pfs230 and Pfs48/45 could have the advantage of boosting the naturally acquired immunity for a broader diversity of individuals.\nAnd to the Discussion (lines 457–462 and 470–472) with the following sentence:\nMoreover, given the limited clinical data available, it remains unclear whether Pfs230-D1 or Pfs48/45-D3 elicits the stronger functional immune response in malaria-endemic populations, where seroprevalence of antibodies to either Pfs230, Pfs48/45, or both antigens has been reported29,30. This underscores the challenge of selecting an optimal TBV candidate, and highlight the advantage of an approach that instead focuses on combining potent epitope bins present across both antigens. […] In addition, a single chimeric protein, instead of two separate immunogens, is likely to reduce the burden of production, which may lead to a lower the cost of goods.\nBy carrying out the first experiment with equivalent mass amounts of Pfs230-D1 and Pfs48/45-D3 on the same lipid nanoparticles, we did observe lower overall efficacy, which we rationalized was due to a sub-stoichiometric ratio of 0.74-to-1 of the larger antigen, Pfs230-D1. The experiment suggested by the Reviewer to compare a cocktail of two separate nanoparticle immunogens vs one nanoparticle containing two immunogens wasn’t performed on the CoPoP platform, but was carried out in the context of the I53-50 nanocage (Fig. 5). As suggested by the Reviewer, these differential display approaches could lead to differences in the B cell response elicited, and we have expanded on this topic in the Discussion (lines 481–486):\nImportantly, the genetic conjugation of distinct epitopes may confer immunodominance hierarchies between epitopes, as we have observed in rodent immunizations with STAC which elicits a Pfs230-D1-dominant response. Several factors may contribute to this phenomenon, including germline-encoded bias of specific B cell lineages for residue signatures at specific epitopes70, proteolysis sensitivity in B cell follicles for specific epitopes71 or epitope prominence by antigen orientations on nanoparticles72.\nIn the design the authors sensibly present that the key TB antibodies should still be able to bind. However, there might be antibodies not structurally characterised which could also be useful for transmission block. Are they likely to bind? In the case of 48/45 could the authors estimate what fraction of the surface area of 48/45 D3 exposed in 48/45 is also exposed in their tandom design?\nIt is certainly possible that yet-undiscovered epitopes exist on Pfs48/45-D3, however functional antibodies are unlikely to bind to Pfs48/45-D3 at the interface with Pfs48/45-D2 due to steric occlusion – analogous to the non-functional antibodies which bind to the Pfs230-D1-D2 interface (e.g 15C5 and LMIV230-02). This is because the same surface on Pfs48/45-D3 that is occluded by Pfs230-D1 on STAC is also occluded by Pfs48/45-D2 in the native protein, thereby indicating a similar relative surface area exposure of Pfs48/45-D3 in both the native Pfs48/45 and STAC proteins. To address this important question raised by the Reviewer, we have incorporated a structural alignment of STAC with a crystal structure of full-length Pfs48/45 (PDB 7ZXF) at Supplementary figure 6f and updated the accompanying Results text (lines 318–321) with the following panel and sentence:\nNotably, STAC exhibited distinct interdomain orientations when compared to crystal structures of Pfs230-D1D2 or Pfs4845-D1D2D3, although in both cases similar interdomain regions were occluded between either Pfs230-D1 or Pfs48/45-D3 and the adjacent domain (Sup fig 6e-f).\nDesign-1 bound to the desired neutralising antibodies but also bound to the non-inhibitory antibodies, whose epitopes should have been blocked in the new tandom construct. The authors make it clear that this indicates that further design is needed (lines 246-8). It would be good to be more explicit here. I think that these means that the two domains are not associated in the correct relative orientation but instead are flexibly linked so that the epitopes are not occluded? Also 278.\nWe fully agree with the Reviewer’s description, and have integrated additional language in this revised Results section (lines 254–257):\nIn Design-1, we utilized the inter-domain linker sequence of Pfs230-D1D2 to connect Pfs230-D1 to Pfs48/45-D3, however partial accessibility of the Pfs230-directed 15C5 and LIMV230-02 epitopes indicated that flexible linkage may have mispositioned the two tandem domains such that the epitopes are not fully occluded.\nWe have also corrected phrasing at the end of this paragraph (line 270) to state that “Design-4 recapitulated a more desired inter-domain orientation.”\nLine 408-9 is too strongly worded. STAC only strongly outperforms on I53-50, with perhaps improvement (at one dilution point) for ferritin and none for liposomes. I think that the final conclusion should therefore be toned down a little.\nThe conclusion sentence has been updated to accurately summarize the results presented in this section and based on the statistical analyses performed. Specifically, we now state (lines 416–419):\nTaken together, these data indicate that when formulated as protein nanoparticle immunogens, STAC can elicit a more potent functional response at higher sera dilutions when compared to Pfs230-D1 and Pfs48/45-D3, both as individual antigens and in combination as cocktails or mosaics.\nMinor comments:\nFigure 1c – could do with x axis labels for the black, yellow and blue circles.\nThis information has been newly incorporated in the figure legend.\nFigure 1 – why not show binding data for the transmission blocking antibodies as well as the non-blockers which shouldn’t bind?\nFigure 1f-h demonstrates that certain Pfs230-D1 epitopes are masked in the context of a D2-containing Pfs230 construct and shows that while the Design-1 construct is partially able to mask these epitopes, it is not sufficient. Here, we have presented the binding that is most relevant to the design in Figure 1. The remaining data for representative antibodies with functional and non-functional TRA, which serve as controls that are not expect to change in affinity, are available to the reader in Supplementary figure 2 for the purpose of demonstrating the availability of other important epitopes for Design-1 compared to Pfs230-D1 and Pfs48/45-D3.\nThroughout, it would help the reader to have a clearer explanation of what the different mAbs do/bind. More “Pfs230-binding mAb XXXX” etc…\nWe thank the Reviewer for this suggestion. To improve clarity, we have added Pfs230 and Pfs48/45 antibody descriptors to the text, particularly in sections related to Figures 1, 2, and 3.\nFigure 4b, should that be STAC not design-22?\nThis has been corrected.\nFigure 4f is missing x axis labels for the proteins.\nThis information has been newly incorporated in the figure legend.\nWe thank the Reviewer for their positive assessment of our work.\nComments related to the structural design\nThe authors of this study report a stabilized tandem antigen chimera of Pfs230-D1 (179 residues) and Pfs48/45-D3 (137 residues), representing the most potent epitopes of each antigen, thereby removing distracting epitopes from both. The individual constructs were combined with liposomes and used to immunize mice. They then examined the potential of linking both antigens in a single construct and evaluated, using BLI and known mAbs, the binding to the construct to confirm correctly folded domains and the masking of unwanted epitopes.\nDesign4 resulted in a crystal structure that is indistinguishable by rmsd from the native individual structures of Pfs230 and Pfs48/45, indicating correctly folded proteins despite linking them together.\nIn a next step, the authors applied AI-supported methods ProteinMPNN to improve the interface design and reduce unwanted binding, resulting in additional suitable designs. The improved designs increased expression yields and binding affinities to the tested mAbs. It would be interesting to note if the authors used the default weights for amino acids for their interface design or skewed towards residues typically found in interfaces.\nWe thank the Reviewer for their interest in the ProteinMPNN parameters used for this study. We have updated the Methods for the Computational design section (line 713) to indicate that we ran ProteinMPNN “using the default weights for all twenty amino acids”.\nParagraph 304-307\nHow many residues are involved in the interface in total? Are the 16 introduced mutations 20% of the interface, or is it more like 80%? This shows how dynamic an interface could be, redesigned while maintaining or improving the interface interaction so that others can derive their own interface designs for other proteins of interest.\nWe identified 34 residues at the interface which had sidechains which did or could project towards the linked domain. These residues were redesigned using ProteinMPNN. However, we pursued a somewhat conservative approach by selecting outputs with higher sequence recoveries to ensure proper domain folding. We have updated the Methods for the Computational design section to make this detail clear (lines 715–717):\nGenerated sequences were ranked by the ProteinMPNN sequence likelihood score with preference for higher sequence recovery to ensure proper domain folding and down selected to 15 sequences which were predicted with AlphaFold2.\nAlso there is a discrepancy in Figure 3c showing 18 mutated residues instead of 16?\nWe thank the Reviewer for catching this discrepancy. One stabilizing mutation on the Pfs48/45-D3 domain, N299Q, was missing and has been added to Figure 3c. The other mutation in Pfs230-D1, N585Q, is to remove a potential N-glycosylation site and is not found at the interface. N585Q is present in the clinical-stage Pfs230D1M-EPA candidate, as well as all the Pfs230-D1 constructs in this study. We have updated the Figure 3c caption to explain that N585Q is omitted for clarity.\n9N8I\nChain A and E might be worth revisiting Mopprobity to examine those atom clashes and potentially choose a different rotamer to improve the structure if the electron density supports that conformation.\nThis seems to be supported by section 6.1 with the rotamer outliers as well for Chain E\nAlso Rfree/Rwork seem to suggest a slight overfit, might be better to cut the resolution limit to 1.9 Å instead of 1.85 Å\nWe thank the Reviewer for taking the time to inspect the validation reports of the structures presented in our manuscript. We had initially deposited a model with the anti-Kappa VHH sequence identity partially masked (using UNK pseudo-atoms in the model). We now incorporate the complete sequence and we have updated the model and validation report. With this change, the reported Rfree value has improved from 0.259 to 0.234 and the RSRZ outliers have improved from 5.8% to 2.7%, while maintaining no Ramachandran or Sidechain outliers and a relatively low Clashscore. An update validation report is provided.\n9N8N\nRotamers could be optimized.\nWhy did the authors model certain residues differently than the sequence construct suggests? I’m referring to the Table on Page 5, particularly residues 186, 195, 284, and 289.\nAgain it would be worth running Molprobity to optimize the rotamer outliers of Chain I\nWe have re-refined the molecular model of Design-4 bound to 2 potent Fabs. This allowed elimination of the two rotamer outliers and improved the RSRZ outliers from 6.8% to 5.8%, while maintaining no Ramachandran outliers and a relatively low Clashscore and Rfree value. Regarding the sequences reported in the validation report, these are actually the differences between our engineered structure and the reference sequences of the native proteins displayed by Uniprot. We thank the Reviewer for flagging this potential issue. To be certain of the accuracy of our deposited models, we have double checked and confirmed that no sequence errors exist in any of the structures. For reference to the readers, we have included the amino acid sequences of the NF54 reference sequences for Pfs230 D1 and Pfs48/45 D3, as well as all mutations found in our relevant designed constructs in Supplementary figure 6d.\nIn addition to improvements in the molecular models determined by X-ray crystallography, we have also taken this opportunity to make minor improvements to the map and model from the cryo-EM dataset. All changes have been indicated in the Methods section Cryo-EM – Image processing. To this effect, the cFAR score has improved from 0.10 to 0.31, which has improved the backbone inclusion from 0.773 to 0.783. These changes also maintain no Ramachandran or Sidechain outliers, a low relative Clashscore and a favourable relative Q-score. All updated validation reports have been included and any relevant changes have been introduced in Supplementary tables 2 and 3, and Supplementary figures 7 and 8.\nMinor\nLine 175 define TRA\nWe have altered this section title at lines 177–178 to state “Mixing Pfs230-D1 and Pfs48/45-D3 on adjuvanted liposomes elicits strong transmission-reducing activity”. For reference, TRA remains defined in the Introduction at line 141.\nComments related to immunology and the functional characteristics of the vaccine design\nThe standard membrane feeding assay (SMFA) is used to evaluate IgG isotypes enriched from mice after vaccination with various constructs to measure the reduction in oocyte formation. (Do you think other isotypes might be participate for protection?) These assays are well executed and are the typical functional tests to assess transmission-blocking activity. There was one biological replicate. The best chimeric molecule, STAC, shows comparable transmission-reducing activity (TRA) to the individual component antigens. In fact, its TRA is slightly lower than Pfs48/45 as a single molecule, suggesting that. There is only a slight improvement in TRA with STAC linked to nanoparticle immunization compared to the individual immunogens based on the SMFA model. Still, as the authors point out, the molecule is easy to produce and stable, which is important for vaccine candidate developability.\nWe thank the Reviewer for this positive assessment of our characterization of functional antibody responses to our nanoparticle immunogens. The SMFAs following immunizations with lipid nanoparticles and protein nanoparticles were performed by separate laboratories at the National Instituites of Health and Radboud University Medical Center, respectively, using distinct protocols (described in Methods). It has been previously shown that purified mouse polyclonal IgGs from Pfs230-D1 immunizations exhibited significantly greater SMFA activity in the presence of human complement (Miura, et al., Vaccine. 2019). Therefore, for lipid and protein nanoparticle immunizations, mouse polyclonal antibodies were evaluated by SMFA in the presence of human complement proteins. For SMFAs following immunizations with lipid nanoparticles, IgG was purified from whole sera using Protein G affinity chromatography, so the effects of complement-fixing isotypes, like IgM, were not measured. In contrast, for SMFAs following immunization with protein nanoparticles, mouse sera were directly assessed by SMFA, again in the presence of human complement and this detail has been clarified for the reader in the Method section (lines 658–659) with the following sentence:\nAll SMFA experiments were conducted in the presence of active human complement.\nThe other aspect of the study was a vaccine molecule designed to mask the stimulation of non-functional antibodies. Theoretically, this is important, as a non-functional antibody could cause a steric hindrance to functional antibodies. Is there data to support this concerning Pfs230 or Pvs48/45? If so, this should be discussed or highlighted as a limitation.\nFor example if you add non-blocking mAbs to antibodies elicited by the different constructs do you observed alterations in TRA activity?\nThis is a very interesting point regarding both the quality of antibody responses, as well as the interplay of various monoclonals within a polyclonal mixture. As we describe in detail throughout the manuscript, our previous studies elucidating the epitopes on Pfs230-D1 and Pfs48/45-D3 targeted by antibodies from naturally infected individuals (Fabra-García, et al., 2023 and Ivanochko, et al., 2023) provided the rationale for the STAC concept. Importantly, non-functional antibodies directed to the buried D1-D2 interface on Pfs230 are only known to arise from immunizations with Pfs230-D1 subunit-based immunogens (also referenced in publications from the groups of P. Duffy and N. Tolia). We are not aware of studies that looked at mixing potent Pfs230 mAbs with these non-potent ones in SMFA, although such data is emerging for other malarial antigens such as RH5 where functional interplay has been demonstrated (Barrett, at al. Cell, 2025). To date, we are also not aware of any structural epitope mapping published for non-functional Pfs48/45-D3-directed mAbs, but fully agree that ongoing efforts to this effect will be valuable to address in the future. We have thus added this content to the Discussion (lines 451–461):\nAt present, a comprehensive epitope map of Pfs48/45-D3 is not available, with multiple surfaces unmapped for any known antibody epitope bins. Structural elucidation of full-length Pfs48/45 in complex with Pfs230 suggests certain epitopes on Pfs48/45-D3 may be occluded by Pfs230 in the heterodimer complex19,56. These would become exposed in a Pfs48/45 domain-based subunit vaccine format, conceivably leading to the elicitation of non-functional antibodies directed to natively buried epitopes, as was the case for the Pfs230-D1 subunit vaccine. Moreover, given the limited clinical data available, it remains unclear whether Pfs230-D1 or Pfs48/45-D3 elicits the stronger functional immune response in malaria-endemic populations, where seroprevalence of antibodies to either Pfs230, Pfs48/45, or both antigens has been reported29,30. This underscores the challenge of selecting an optimal TBV candidate, and highlight the advantage of an approach that instead focuses on combining potent epitope bins present across both antigens.\nMinor points\n1. In Fig. 2, the relative binding shown in c and h can be better described. Is this relative binding of the new construct to the Pfs230 or Pfs48/45 individually? Please make this clearer.\nHere, we present the binding signal relative to the maximum binding signal observed in the assays (for each specific antigen), which normalizes signal to 1 and enables the reader to see a relative rank ordering across both the Pfs230 and Pfs48/45 antigens of each designed molecule compared on an equivalent scale. We have also provided the absolute binding data for the reader in Supplementary figure 3a and 3c. The Methods section has been updated accordingly under the Biolayer interferometry – Quantitation section (lines 679–681):\nTo contrast the relative signals for each antigenic domain, relative binding signals were additionally calculated by normalizing each binding signal relative to the maximum observed binding signal for each specific antigen.\n2. In some figures they used Design-22 and STAC interchangeably – this can be confusing. Please stick to one or the other designation as I presume these are the same constructs.\nWe have corrected the Figures and Figures legends for Figures 3 and 4, and Supplementary figures 6 and 7 to exclusively use ‘STAC’ after its definition on line 294.\n3. Figure 5 is critical. The different color codes are difficult to follow, particularly with small figures with poor resolution. Suggest using different shapes and colors for the symbols denoting the different constructs.\nWe thank the Reviewer for recognizing the importance of Figure 5 and appreciate that the low resolution in the initial submission and similar symbols made it difficult to interpret this data. To mitigate this effect, this figure and all other figures have been provided at higher resolution and we have changed the symbols used for the I53-50-based immunogens (now depicted as triangles) to differentiate them from the ferritin-based immunogens (depicted as closed circles.)\nWe appreciate this initiative from Nature Communications for recognizing the contributions from early career researchers and trainees during the peer review process and would also like to thank Reviewer 3 for taking time to review our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have largely responded positively to my review and have made changes to the manuscript.\nThe one area in which I don't agree with their response relates to the depletion experiments. The aim of their overall study is to generate an immunogen which combines the best parts of Pfs48/45 and Pfs230 into a single immunogen which elicits inhibitory antibodies against both components. I therefore suggested a depletion experiment in which they deplete sera for Pfs230D1 and Pfs48.45 antibodies and see the contribution to SMFA, assessing whether they have achieved their objectives of raising both sets of antibodies. They have done this experiment and present it in the response to reviewers, but prefer to save it for a future paper.\nThe data is nice and makes it clear that TRA is almost entirely due to Pfs230D1 antibodies. This shows that the strategy to design an immunogen which elicits transmission blocking antibodies against both Pfs230 and Pfs48/45 has not been entirely successful as transmission blocking antibodies have not been elicited against Pfs48/45.\nIn the end, this is an editorial decision. However, my view is that this data should be included and discussed in the paper as it directly relates to whether the aims of this study have been achieved.", "reviewer_2": "The authors have adequately address our concerns", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank Reviewer 1 for their appreciation of the additional data shared during revisions. Given the Editor also viewed its inclusion favorably, the data has now been incorporated in the Results section and in Supplementary figure 10c-d. As requested, the implications of our findings are also further discussed in the Discussion section: “By mitigating non-functional antibody interference, STAC may overcome limitations of leading TBV candidates and represent an important epitope-masking strategy with broader applications in vaccine development. Importantly, the genetic conjugation of distinct epitopes may confer immunodominance hierarchies between epitopes, as we have observed in rodent immunizations with STAC which elicits a Pfs230-D1-dominant response. Several factors may contribute to this phenomenon, including germline-encoded bias of specific B cell lineages for residue signatures at specific epitopes70, proteolysis sensitivity in B cell follicles for specific epitopes71 or epitope prominence by antigen orientations on nanoparticles72. Investigating the rules of immunodominance for different epitopes, and their contributions to function across preclinical species and in the context of the human Ig repertoire will be important next steps to further understand responses to STAC-based immunogens, and whether further engineering is warranted to obtain a more balanced response to the potent Pfs230 and Pfs48/45 epitopes in a human Ig repertoire context.”\nWe thank Reviewer 2 for taking time to review our manuscript and for their favorable assessment of our work.\nWe appreciate this initiative from Nature Communications for recognizing the contributions from early career researchers and trainees during the peer review process and would also like to thank Reviewer 3 for taking time to review our manuscript."}]

Decision letter