The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the ...need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 3
turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.
•30 recombinant proteins, which covered the entire Pfs230 molecule, were expressed.•Antibodies against each construct were tested by Western blot, IFA and SMFA.•All constructs containing CM domain 1 ...induced SMFA-positive antibodies.•All constructs without CM domain 1 elicited SMFA-negative antibodies.
A transmission-blocking vaccine (TBV) against Plasmodium falciparum is likely to be a valuable tool in a malaria eradication program. Pfs230 is one of the major TBV candidates, and multiple Pfs230-based vaccines induced antibodies, which prevented oocyst formation in mosquitoes as determined by a standard membrane-feeding assay (SMFA). Pfs230 is a >300 kDa protein consisting of 14 cysteine motif (CM) domains, and the size and cysteine-rich nature of the molecule have hampered its production as an intact protein. Except for one early study with maltose-binding protein fusion Pfs230 constructs expressed in Esherichia coli, all other studies have focused on only the first four CM domains in the Pfs230 molecule. To identify all possible TBV candidate domains, we systematically produced either single-CM-domain (a total of 14), 2-CM-domain (7), or 4-CM-domain (6) recombinant protein fragments using a eukaryotic wheat germ cell-free expression system (WGCFS). In addition, two more constructs which covered previously published regions, and an N-terminal prodomain construct spanning the natural cleavage site of Pfs230 were produced. Antisera against each fragment were generated in mice and we evaluated the reactivity to native Pfs230 protein by Western blots and immunofluorescence assay (IFA), and functionality by SMFA. All 30 WGCFS-produced Pfs230 constructs were immunogenic in mice. Approximately half of the mouse antibodies specifically recognized native Pfs230 by Western blots with variable band intensities. Among them, seven antibodies showed higher reactivities against native Pfs230 determined by IFA. Interestingly, antibodies against all protein fragments containing CM domain 1 displayed strong inhibitions in SMFA, while antibodies generated using constructs without CM domain 1 showed no inhibition. The results strongly support the concept that future Pfs230-based vaccine development should focus on the Pfs230 CM domain 1.
The circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on ...CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species.
Using a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge.
Bioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody.
The PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.
Interventions that can block the transmission of malaria-causing Plasmodium falciparum (Pf) between the human host and Anopheles vector have the potential to reduce the incidence of malaria. Pfs48/45 ...is a gametocyte surface protein critical for parasite development and transmission, and its targeting by monoclonal antibody (mAb) 85RF45.1 leads to the potent reduction of parasite transmission. Here, we reveal how the Pfs48/45 6C domain adopts a (SAG1)-related-sequence (SRS) fold. We structurally delineate potent epitope I and show how mAb 85RF45.1 recognizes an electronegative surface with nanomolar affinity. Analysis of Pfs48/45 sequences reveals that polymorphisms are rare for residues involved at the binding interface. Humanization of rat-derived mAb 85RF45.1 conserved the mode of recognition and activity of the parental antibody, while also improving its thermostability. Our work has implications for the development of transmission-blocking interventions, both through improving vaccine designs and the testing of passive delivery of mAbs in humans.
Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein ...(CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)₃ peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine.
A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the ...problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.
Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles ...mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines.
The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ...ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.
Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for ...vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.
The development of highly effective and durable vaccines against the human malaria parasites Plasmodium falciparum and P. vivax remains a key priority. Decades of endeavor have taught that achieving ...this goal will be challenging; however, recent innovation in malaria vaccine research and a diverse pipeline of novel vaccine candidates for clinical assessment provides optimism. With first-generation pre-erythrocytic vaccines aiming for licensure in the coming years, it is important to reflect on how next-generation approaches can improve on their success. Here we review the latest vaccine approaches that seek to prevent malaria infection, disease, and transmission and highlight some of the major underlying immunological and molecular mechanisms of protection. The synthesis of rational antigen selection, immunogen design, and immunization strategies to induce quantitatively and qualitatively improved immune effector mechanisms offers promise for achieving sustained high-level protection.
Development of highly effective vaccines against human malaria remains a public health priority. In this Review, Draper et al. describe the latest vaccine approaches to prevent malaria alongside immunological and molecular mechanisms of protection. Rational antigen selection, immunogen design, and novel immunization strategies offer promise for achieving high-level protection.
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