Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.
Background Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters. Methodology/Principal Findings To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model. Conclusions Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.
Background The development of an effective malaria vaccine is recognized as one of the most promising approaches that would provide a cost-effective intervention for addition to the currently available malaria control measures. Since the fully annotated P. falciparum genome has become available in 2002, reverse vaccinology represents a new opportunity to identify novel malaria vaccine candidate antigens. Screening of predicted P.falciparum open reading frames for proteins that could elicit parasite-inhibitory antibodies has led to the identification of the Cysteine-Rich Protective Antigen (CyRPA) as promising blood-stage candidate protein for inclusion in a malaria subunit vaccine.
The pathogenesis of malaria is primarily associated with blood-stage infection and there is strong evidence that antibodies specific for parasite blood-stage antigens can control parasitaemia. This provides a strong rationale for incorporation of asexual blood-stage antigen components into an effective multivalent malaria subunit vaccine. On the basis of available genome-wide transcriptomic and proteomic data, previously uncharacterized Plasmodium falciparum open reading frames were screened for new blood stage vaccine candidates. This has led to the identification of the cysteine-rich protective antigen (PfCyRPA), which forms together with PfRH5 and PfRipr a multiprotein complex that is crucial for erythrocyte invasion.Glycosylated and non-glycosylated variants of recombinant PfCyRPA were expressed and produced as secreted protein in mammalian cells. Adjuvanted formulations of purified PfCyRPA were tested to assess whether they can effectively elicit parasite inhibitory antibodies, and to investigate whether or not the glycosylation status affects antibody binding. For this purpose, two sets of PfCyRPA-specific mouse monoclonal antibodies (mAbs) have been raised and evaluated for functional activity.Generated PfCyRPA-specific mAbs, irrespective of the immunogen's glycosylation status, showed substantial parasite in vitro growth-inhibitory activity due to inhibition of erythrocyte invasion by merozoites. Furthermore, passive immunization experiments in P. falciparum infected NOD-scid IL2Rγ (null) mice engrafted with human erythrocytes demonstrated potent in vivo growth-inhibitory activity of generated mAbs.Recombinantly expressed PfCyRPA tested as adjuvanted vaccine formulations in mice elicited antibodies that significantly inhibit P. falciparum asexual blood stage parasite growth both in vitro and in vivo. These findings render PfCyRPA a promising blood-stage candidate antigen for inclusion into a multicomponent malaria subunit vaccine.
Buruli ulcer (BU) caused by Mycobacterium ulcerans is a devastating skin disease, occurring mainly in remote West African communities with poor access to health care. Early case detection and subsequent antibiotic treatment are essential to counteract the progression of the characteristic chronic ulcerative lesions. Since the accuracy of clinical BU diagnosis is limited, laboratory reconfirmation is crucial. However, currently available diagnostic techniques with sufficient sensitivity and specificity require infrastructure and resources only accessible at a few reference centres in the African endemic countries. Hence, the development of a simple, rapid, sensitive and specific point-of-care diagnostic tool is one of the major research priorities for BU. In this study, we have identified a previously unknown M. ulcerans protein, MUL_3720, as a promising target for antigen capture-based detection assays. We show that MUL_3720 is highly expressed by M. ulcerans and has no orthologs in other prevalent pathogenic mycobacteria. We generated a panel of anti-MUL_3720 antibodies and used them to confirm a cell wall location for MUL_3720. These antibodies could also specifically detect M. ulcerans in infected human tissue samples as well as in lysates of infected mouse footpads. A bacterial 2-hybrid screen suggested a potential role for MUL_3720 in cell wall biosynthesis pathways. Finally, we demonstrate that a combination of MUL_3720 specific antibody reagents in a sandwich-ELISA format has sufficient sensitivity to make them suitable for the development of antigen capture-based diagnostic tests for BU.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines. https://doi.org/10.7554/eLife.20383.001 eLife digest Malaria is one of the deadliest infectious diseases worldwide, killing over 400,000 people a year. About 200 million people are infected every year, placing a huge social and medical burden especially on developing countries. Microscopic parasites known as Plasmodium are responsible for causing this disease. Plasmodium parasites have a complex life cycle involving both mosquito and mammal hosts. This includes a stage where the parasites infect the mammal's red blood cells, which causes the symptoms of the disease. In 2012, a team of researchers discovered that a protein called CyRPA forms a group (or 'complex') with several other proteins to allow the parasites to enter red blood cells. Developing a vaccine is one of the most promising approaches to prevent malaria. Vaccines help the body to recognise and fight an invading microbe by triggering an immune response that results in the production of proteins called antibodies, which can bind to specific molecules on the surface of the microbe. If the microbe later enters the body, these antibodies can be produced quickly to eliminate the microbe before it causes disease. However, efforts to develop a highly effective vaccine against malaria have so far been unsuccessful. Favuzza et al. – including some of the researchers involved in the 2012 work – used a technique called X-ray crystallography to investigate the three-dimensional structure of the CyRPA protein. The experiments show that an antibody is able to bind to a region of CyRPA – a designated 'protective epitope' – that is similar in the CyRPA proteins of all Plasmodium falciparum strains. These antibodies can prevent the parasite from entering the red blood cells, and vaccines containing CyRPA may therefore be effective at protecting individuals from malaria. The findings of Favuzza et al. also suggest that using CyRPA in combination with another protein in the complex called RH5 could make the vaccine more powerful as it would make it harder for the parasite to become resistant. The next step following on from this work is to design a vaccine containing protective CyRPA epitopes that triggers an immune response in mammals that is strong enough to reduce the numbers of parasites in the blood. A future challenge will be to develop a vaccine that combines several proteins involved in different stages of the parasite's life cycle to provide full protection against malaria. https://doi.org/10.7554/eLife.20383.002 Introduction According to the World Health Organization 2015 Malaria Report (who.int/malaria/publications/world_malaria_report/en), malaria is estimated to have caused 214 million clinical cases and 438,000 deaths in 2015. The disease is transmitted by female Anopheles mosquitoes and caused by parasitic protozoans of the genus Plasmodium, of which P. falciparum and P. vivax are the most prevalent and P. falciparum is causing the most often fatal and medically most severe form of malaria. Debilitating clinical symptoms associated with the infection are caused by the multiplication of the asexual blood-stage parasites in erythrocytes. One of the most promising targets for malaria vaccine development is therefore at the stage where merozoites invade erythrocytes. Invasion of host erythrocytes by merozoites is a complex process, conceptually divisible into four phases: (1) initial recognition of and reversible attachment to the erythrocyte membrane by the merozoite; (2) junction formation leading to irreversible attachment of the merozoite, parasitophorous vacuole formation, and release of the Plasmodium rhoptry-microneme secretory organelles; (3) invagination of the erythrocyte membrane around the merozoite, accompanied by the shedding of the merozoite's surface coat; (4) closing of the parasitophorous vacuole and resealing of the erythrocyte membrane mark the completion of merozoite invasion (Pinder et al., 2000). The initial recognition and the active invasion of erythrocytes depend on specific molecular interactions between parasite ligands and receptors on the host erythrocyte membrane. Although several ligand-receptor interactions have already been identified, the entire network of molecular interactions involved in invasion is not yet fully disentangled. In addition, P. falciparum merozoite proteins are antigenically highly diverse and in part functionally redundant, to facilitate parasite escape from host immune surveillance and to ensure erythrocyte invasion via alternative pathways (Cowman et al., 2012). Most efforts in malaria blood stage vaccine research and development have historically concentrated on immuno-dominant, polymorphic antigens that contribute to the invasion of red blood cells by merozoites. Despite major efforts, blood-stage vaccines based on merozoite surface antigens have so far shown limited efficacy in clinical trials (reviewed in Halbroth and Draper, 2015). Extensive antigenic polymorphism represents one major hurdle for the development of an effective blood-stage malaria vaccine (Takala et al., 2009; Dzikowski and Deitsch, 2009). Therefore, the identification of new candidate antigens that are able to induce broad strain-transcending immunity and that are not susceptible to 'vaccine resistance' has become a recent research focus. Availability of pathogen genomes is facilitating the discovery of novel vaccine candidate antigens through 'reverse vaccinology' approaches (Rappuoli, 2001; Donati and Rappuoli, 2013). Sequencing and annotation of the P. falciparum genome (Gardner et al., 2002) has supported the identification of new blood-stage vaccine candidate antigens (Conway, 2015; Proietti and Doolan, 2014), among which the P. falciparum Cysteine-Rich Protective Antigen (PfCyRPA) has a number of noteworthy properties. While PfCyRPA is highly conserved among a plethora of P. falciparum isolates, it also is poorly immunogenic in the context of natural exposure (Dreyer et al., 2012). Moreover, PfCyRPA-specific monoclonal antibodies (mAb) inhibit parasite growth both in vitro and in vivo by blocking merozoite invasion (Dreyer et al., 2012; Favuzza et al., 2016). PfCyRPA is a 42.8 kDa protein of 362 residues with a predicted N-terminal secretion signal. Orthologs of PfCyRPA have been found in the genomes of the human malaria parasite P. vivax and the primate pathogens P. knowlesi, P. cynomolgi, and P. reichenowi (Figure 3—figure supplement 1), but not in the sequenced genomes of other Plasmodium species. P. falciparum PfCyRPA shares on average 42% sequence identity with its orthologs, but within different P. falciparum isolates PfCyRPA is highly conserved: just 13 dimorphic amino acid positions (highlighted in Figure 3—figure supplement 1) were found in 227 P. falciparum field isolates (Manske et al., 2012), and only a single variant (Arg399 instead of Ser399) was found at a frequency of greater than 2%. PfCyRPA is part of a multi-protein complex (Reddy et al., 2015; Volz et al., 2016) including also the PfRH5-interacting protein PfRipr and the reticulocyte binding-like homologous protein PfRH5, which binds to the erythrocyte receptor basigin (Baum et al., 2009; Crosnier et al., 2011; Chen et al., 2011b, 2014). PfRH5, PfCyRPA, and PfRipr colocalize during parasite invasion at the junction between merozoites and erythrocytes. The complex seems to be required both for triggering Ca2+ release and establishment of tight junctions (Volz et al., 2016). While merozoites deficient in PfCyRPA or PfRH5 can still bind to erythrocytes, they do not attach irreversibly and cannot invade the host cells (Volz et al., 2016). Like PfCyRPA (Dreyer et al., 2012), PfRH5 induces invasion-blocking antibodies that are effective across common genetic variants (Douglas et al., 2011; Bustamante et al., 2013; Douglas et al., 2014). 'Structural vaccinology', a combination of immunological, structural, and bioinformatics approaches, is increasingly used for the design of improved vaccine antigens (Dormitzer et al., 2008; Cozzi et al., 2013; Malito et al., 2015). To this end, the crystal structures of PfRH5 in complex with basigin and neutralizing inhibitory mAb have been determined (Chen et al., 2014; Wright et al., 2014). Here, we describe the crystal structure of the promising vaccine candidate PfCyRPA alone and in complex with the antigen-binding fragment (Fab) of the parasite growth inhibitory mAb c12 (Dreyer et al., 2012). The structure of PfCyRPA represents a step toward elucidating its biological function. Furthermore, definition of the specific epitope–paratope interactions from the crystal structure of the PfCyRPA/c12 complex will support rational design of an epitope-focused PfCyRPA-based candidate vaccine. Results Fine specificities of anti-PfCyRPA antibodies The finding that PfCyRPA and PfRH5 form a complex essential for parasite invasion prompted us to investigate the fine specificities of previously generated parasite inhibitory and non-inhibitory anti-PfCyRPA mAbs. The 16 anti-PfCyRPA mAbs available for analysis (Dreyer et al., 2012; Favuzza et al., 2016), showed six distinctive reactivity patterns with seven overlapping recombinant protein fragments of PfCyRPA, assigning them to the epitope groups A – F, with groups A, B, C, and F comprising the parasite inhibitory and groups D and E the non-inhibitory mAbs (Figure 1). All mAbs bound to the full-length PfCyRPA (without the signal sequence; fragment 1). mAbs belonging to epitope group A exclusively bound this fragment, indicating that they recognize conformational epitopes not present in any of the shorter PfCyRPA sequence stretches. Lack of binding to fragments 2 and 7 indicates that the epitope may comprise sequences from both ends of the polypeptide chains. Epitope group B antibodies, including mAb c12, bound only to fragments 1, 2, and 3. Epitope group F mAbs bound to fragments 1, 2, and 5, but not to fragment 3, indicating that in contrast to group B, residues located in the sequence stretch between aa 181–251 are required for their binding. The single mAb c04 constitutes the epitope group C showing binding to fragment 7 (only in IFA, not confirmed by Western blotting analysis). The non-inhibitory mAbs clustered into the distinct epitope groups D and E. Figure 1 with 1 supplement see all Download asset Open asset Binding of anti-PfCyRPA mAbs to fragments of PfCyRPA. Binding of 16 mAbs to PfCyRPA fragments (black bars) expressed on the cell surface of HEK cells as assessed by Western blotting analysis and live-cell immunofluorescence staining. (x) indicates staining and (–) no staining; (a) indicates no reactivity in Western blot analysis of HEK cell lysates. Expression on the surface of the HEK cells has been demonstrated for all PfCyRPA fragments by immunofluorescence analysis using anti-Histidine tag HIS-6/9 mAb (Figure 1—figure supplement 1). For reference, the 17 residues constituting the epitope on PfCyRPA identified from the complex crystal structure with the Fab of mAb c12 is shown in all constructs as red bars. According to their reactivity pattern, anti-PfCyRPA mAbs were assigned to different epitope groups: A: c10, SB2.5; B: c02, c06, c08, c09, c12, SB3.7; C: c04; D: c05; E: c13, SB3.9; F: SB1.6, SB2.1, SB2.3, SB3.3. https://doi.org/10.7554/eLife.20383.003 The parasite growth inhibitory activity of anti-PfCyRPA antibodies is enhanced by anti- PfRH5 antibodies Since it may prove useful to incorporate a combination of PfCyRPA and PfRH5 in a multivalent malaria vaccine, we investigated whether mAbs against these two vaccine candidate antigens have additive or synergistic effects. In a first step, we tested a combination of inhibitory mAbs against the two antigens in an in vitro parasite growth inhibition assay. Parasites were cultured for one cycle of merozoite invasion in the presence of the anti-PfCyRPA c12 mAb with or without the anti-PfRH5 BS1.2 mAb at concentrations of 500, 250, and 125 μg/mL. Either mAbs showed potent inhibitory activity, consistently reducing parasite growth of all four tested P. falciparum strains in a concentration-dependent manner and to the same extent as the well characterized inhibitory anti-MSP-1 mAb 12.10 (Blackman et al., 1990) (Figure 2A and Figure 2—figure supplement 1). When combining the anti-PfCyRPA c12 mAb with the anti-RH5 BS1.2 mAb, we found a significantly enhanced inhibitory activity: while mAbs c12 and BS1.2 at a concentration of 250 µg/mL inhibited growth by 21% ± 2.2% and 31% ± 4.6%, respectively, the combination of both mAbs (250 µg/mL each) inhibited growth by 59% ± 1.4%; (Figure 2A). The functional activity of both mAbs was not enhanced by the addition of a malaria-unrelated control mAb. Figure 2 with 1 supplement see all Download asset Open asset Anti-PfCyRPA and anti-PfRH5 mAbs have both in vitro and in vivo an additive inhibitory effect on parasite growth. (A) Growth inhibition in vitro. Synchronized P. falciparum 3D7 blood-stage parasites were cultured for one cycle of merozoite invasion (48 hr) in the presence of anti-PfCyRPA c12 mAb, anti-PfRH5 BS1.2 mAb, and their combinations. An isotype-matched, malaria-unrelated mAb (NR4.2) (Rose et al., 2016) was used as negative control. Inhibitory and non-inhibitory anti-MSP-1 mAbs (12.10 and 2F10, respectively) were also included as reference (Blackman et al., 1990, 1994). Percent parasite growth inhibition was calculated against the parasitemia of PBS control wells. Each bar represents the mean of a triplicate experiment, and error bars indicate the standard deviation (SD). Differences in parasite growth inhibition between mAbs c12 and BS1.2 alone and their combinations are statistically significant (unpaired t test with Welch's correction, 95% confidence interval, two-tailed p value). (B) Growth inhibition in vivo. NODscidIL2Rγnull mice received purified anti-PfCyRPA c12 mAb and/or anti-PfRH5 BS1.2 mAb by i.v. injections. Mice were then infected with P. falciparum 3D7 and parasitemia was monitored over 6 days. Values represent the mean parasitemia in human erythrocytes in peripheral blood of three mice per group. Error bars indicate the SD. PBS and an unrelated control mAb were used as negative control. https://doi.org/10.7554/eLife.20383.005 In a second step, the in vivo parasite inhibitory activity of the mAbs was evaluated in the P. falciparum SCID murine model that employs non-myelodepleted NODscidIL2Rγnull mice engrafted with human erythrocytes (Dreyer et al., 2012; Jiménez-Díaz et al., 2009). Groups of three mice received 2.5 or 0.5 mg of mAbs c12 or BS1.2 or a combination of both mAbs by i.v. injection. The control groups received either 2.5 mg of an isotype-matched malaria-unrelated mAb or the same volume of PBS without Ab. Mice were infected with parasitized erythrocytes 1 day after the antibody transfer and parasitemia was subsequently monitored (Figure 2B). In the control groups, parasitemia reached 19.6% ± 0.8% on day 9 after mAb injection. Parasitemia in mice having received 2.5 mg c12 or BS1.2 mAb increased only marginally, reaching 2.2 ± 0.5 and 2% ± 0.3% on day nine after mAb injection, respectively. At the lower dose of 0.5 mg c12 and BS1.2 inhibited parasite growth to 10.1 ± 2.3 and 10.8% ± 6.9% parasitemia, respectively (Figure 2B). In accord with the in vitro data, parasitemia decreased significantly (p=0.0356; unpaired t test, 95% confidence interval, two-tailed) and reached only 4.8% ± 1.9% on day 9 if mice received 0.5 mg of the anti-PfRH5 BS1.2 mAb in addition to 0.5 mg of the anti-PfCyRPA c12 mAb. These results demonstrated that anti-PfCyRPA and anti-PfRH5 antibodies have an additive parasite growth inhibitory effect, justifying the combination of both antigens in a subunit vaccine. While the structure of PfRH5 in complex with an inhibitory antibody has been determined, structural information for PfCyRPA is lacking. We therefore determined the crystal structures of PfCyRPA and its complex with the Fab of the growth inhibitory mAb c12. Biophysical analysis and crystal structure of PfCyRPA The far-UV CD spectrum of PfCyRPA is consistent with an all-β structure connected by loop regions and the absence of α-helices (Figure 3—figure supplement 2A). Mass spectrometric analysis of proteolytic fragments of PfCyRPA revealed at least four disulfide bonds that are sequential along the sequence (Figure 3—figure supplement 2B). Intrinsic fluorescence showed that the two Trp residues present in PfCyRPA are buried in the native state. Also, the disulfide bonds seem to be buried, because addition of 50 mM reducing agent had no significant effect on the fluorescence of PfCyRPA (Figure 3—figure supplement 2C). Taken together, these data are consistent with PfCyRPA forming a compact, disulfide-stabilized molecule of predominantly β-sheet structure. In order to crystallize PfCyRPA, we needed to pre-treat the protein with Actinase E (Figure 4—figure supplement 1A). The crystal structure of PfCyRPA, determined to a resolution of 2.5 Å (detailed in Supplementary file 1), confirmed our biophysical analyses (Figure 3). PfCyRPA adopts a six-bladed β-propeller structure that buries the disulfide bonds and the Trp residues. Each blade of the propeller is constructed by a four-stranded anti-parallel β-sheet (Figure 3B). The five disulfide bonds in PfCyRPA are located within blades 2–6 (Figure 3A), stabilizing each individual blade. The first blade has no disulfide bond; it is formed by β-strands from the N- and C-terminal regions of PfCyRPA, potentially enabling conformational changes in PfCyRPA by opening and closing. A domain alignment search (DALI; [Holm and Rosenström, 2010]) for related structures revealed that PfCyRPA adopts a heavily modified sialidase/neuraminidase fold. The closest structural relative is the catalytic domain of Vibrio cholerae sialidase (Moustafa et al., 2004) (Figure 3C). The two proteins have only 9% sequence identity and the structures have a large root mean square distance (rmsd) of 3.7 Å over 285 residues, clearly showing that while the overall fold is similar, the structures are very different with respect to the inclination of the blades (Figure 3C) and the length and conformations of the surface loops connecting the β-strands (Figure 3D). PfCyRPA also contains a signature sequence motif for sialidases known as an Asp-box (201-SHDKGETW-208; conserved residues are underlined) (Roggentin et al., 1989), which serve structural roles in the β-propellers of sialidases. However, while bacterial sialidases contain between three to five Asp-boxes, PfCyRPA contains only a single one. Figure 3 with 3 supplements see all Download asset Open asset PfCyRPA adopts the neuraminidase fold. (A) Structure-sequence relationship of PfCyRPA. Indicated are an Actinase E cleavage site at Asp189 (red), a sialidase-typical Asp-box (dotted underlined), the two Trp residues (dotted underlined), and the sequential disulfide bonds (connected by lines and in same color). β-strands are shown as arrows colored according to the blade they form. The epitope recognized by mAb c12 is underlined in bold. (B) Cross-eyed stereo view of the ribbon representation of a superposition of the two PfCyRPA molecules in the asymmetric unit with the blades numbered 1–6 from the N-terminus and colored individually. Blade one is made up of an N-terminal (black) and three C-terminal β-strands (red). One protomer is shown with white, the other with black loop regions, which may differ substantially (arrows in blade 5). The Trp and Cys residues are drawn as stick models. (C) The same orientation of the catalytic domain of Vibrio cholerae sialidase (PDB-ID 1w0o), the next structural homolog of PfCyRPA with a DALI score of 18 (Z < 5 is structurally dissimilar). Sialic acid and residues in the Vibrio enzyme are displayed as balls and sticks. Structural Ca2+ ions are marked as magenta spheres. None of the residues necessary for metal ion binding, substrate binding, or catalysis is present in PfCyRPA. (D) Superposition of PfCyRPA with the V. cholerae sialidase. While both proteins are 6-bladed β-propellers, the blades have very different angles, extents, and loop lengths and conformations connecting the β-strands. The four Asp-boxes in the bacterial sialidase (grey) are colored black. PfCyRPA (orange) has only a single Asp-box connecting the third and fourth β-strands in blade 3 (colored blue). Other β-strand connections are made by sequences unrelated to the Asp-box motif, in agreement with poor conservation of the Asp-box in other, e.g. viral, sialidases. The view in (D) is rotated by 180° about the horizontal axis compared to (B) and (C). https://doi.org/10.7554/eLife.20383.007 Two molecules of PfCyRPA are present in the asymmetric unit and have conformational differences in several surface loops, suggesting possible flexibility of these loops in solution (Figure 3B). From the point of peptidomimetics that could be derived from PfCyRPA for vaccine development, these surface loops connecting the blades on the back and front of the β-propeller are natural candidates. Although some of the loops, e.g. in blade 5 (arrows in Figure 3B), have significant structural plasticity, it should be possible to stabilize them in a conformation suitable to raise an immune response. PfCyRPA has no sialidase activity The sialidase fold of PfCyRPA and the presence of an Asp-box motif raised the question whether PfCyRPA exhibits sialidase activity. In view of the involvement of viral and microbial sialidases in the unmasking of cryptic host ligands, host cell adhesion, and invasion (Chen et al., 2011a; Lewis and Lewis, 2012; Matrosovich et al., 2015), sialidase activity could make sense for PfCyRPA-mediated invasion of erythrocytes. We detected no sialidase activity, however, when we tested recombinant PfCyRPA for neuraminidase activity using a colorimetric assay (Figure 3—figure supplement 3). The active site of sialidases usually contains a triad of Arg residues that bind to the substrate, a Glu/Tyr pair where the acid acts as a general base to activate the Tyr nucleophile, and a hydrophobic pocket with a conserved Trp that accommodates the acetyl group of sialic acid (Buschiazzo and Alzari, 2008). In addition, many sialidases bind to Ca2+ ions, of which one is close to the active site and is bound by three main-chain carbonyl groups and oxygen atoms in the side-chains of Asn, Thr, and Asp. PfCyRPA contains none of the residues necessary for catalysis, nor does it harbor a Ca2+ binding site, providing structural correlates of the absence of sialidase activity in PfCyRPA. PfCyRPA and sialidases may have evolved from a common ancestor, or PfCyRPA could have evolved from a genuine sialidase to adopt other functionalities. Characterization of the epitope recognized by the parasite growth inhibitory anti-PfCyRPA mAb c12 The parasite inhibitory anti-PfCyRPA c12 mAb binds tightly to PfCyRPA with a Kd of ca. 1 nM as determined by surface plasmon resonance analysis (Dreyer et al., 2012). This mAb recognizes PfCyRPA independent of its glycosylation, as revealed by Western blot analyses (Figure 4A). Because of these favorable properties, we chose c12 for epitope mapping by determining the structure of a PfCyRPA/c12 complex. In a first step, we determined the crystal structure of c12 in isolation (Figure 4B and C). We obtained three different crystal forms containing a total of four crystallographically independent Fab molecules. Superposition of the structures showed very little structural plasticity of the CDR loops (Figure 4C), suggesting that they retain their structures upon epitope binding. Figure 4 with 1 supplement see all Download asset Open asset Recognition of PfCyRPA by the mAb c12 and structure of c12. (A) Reducing SDS-PAGE of glycosylated (left) and non-glycosylated (right) PfCyRPA detected by Coomassie-staining (blue) and Western blotting with mAb c12 (black). Recognition by c12 is independent of the glycosylation. (B) Overview of the c12 structure with a glycan located at heavy-chain Asn37. mFo-DFc electron density for the glycan is shown as a red mesh drawn at the three rmsd level. The light and heavy chains are colored light pink and light blue, respectively. Heavy chain CDR1-3 are colored cyan, magenta, and yellow, and light chain CDR1-3 are drawn in dark blue, pink, and green. (C) Comparison of the four c12 structures shows little conformational variability of the CDR loops. The four c12 molecules superimpose onto their variable VHVL di-domains with an average rmsd of 0.35 Å, which reveals a minor spread of the elbow angles between 133.1° and 135.8°. The high structural congruence indicates that the CDR conformations are genuine and not dominated by crystal contacts. The view is from above on top of the CDR loops. https://doi.org/10.7554/eLife.20383.011 We then purified the PfCyRPA/c12 complex, analyzed it by limited proteolysis, and crystallized it. Actinase E treatment of the PfCyRPA/c12 complex resulted in the same proteolysis pattern as observed for PfCyRPA alone (Figure 4—figure supplement 1B), suggesting that the epitope for mAb c12 is distant from the Actinase E recognition site at Asp189. Actinase E treatment was not necessary to crystallize the PfCyRPA/c12 complex, however. We determined the crystal structure of the PfCyRPA/c12 complex at a resolution of 4.0 Å by molecular replacement using the individual high-resolution structures of c12 and PfCyRPA as search models (Figure 5A). Despite the limited resolution, novel molecular features were visible in the electron density maps of the complex, providing confidence in the relative orientation of PfCyRPA and c12 (Figure 5—figure supplement 1). First, the electron density visible for PfCyRPA after molecular replacement with c12 was used as a search model for structure determination of PfCyRPA alone (see Materials and methods). This strategy would have been impossible had the placement of c12 been wrong. Second, a loop region that is absent in the PfCyRPA search model due to Actinase E proteolysis (see above) exhibits omit electron density in the PfCyRPA/c12 complex (Figure 4—figure supplement 1). Third, consistent with the similar conformations of the CDR loops in the individual c12 Fab structures (Figure 4) there are negligible conformational changes in the c12 CDR loops when bound to PfCyRPA. Similarly, the epitope conformation in PfCyRPA is very similar in the unbound and complexed form. Minor adjustments of side-chains were required during rebuilding of the complex structure. While the resolution of the PfCyRPA/c12 complex is limited and many side-chains lack clear electron density, as is typical for this resolution, knowledge of the relative orientation of PfCyRPA with respect to c12 in the complex is sufficient for designing peptidomimetics to target the immune response to the protective epitope. Figure 5 with 2 supplements see all Download asset Open asset Structure of the PfCyRPA/c12 complex. (A) Overview showing that the majority of the interface is made by interactions between the light chain of c12 and blade 2 of PfCyRPA. (B) Details of the interface viewed from top onto the CDR loops. The light and heavy chain surfaces buried by PfCyRPA are colored pink and blue, respectively. Possible hydrogen bonds and van der Waals interactions are indicated by dashed green and black lines. The CDR loops are color-coded as in Figure 4. The Asp66-Arg50 salt bridge is circled. (C) Close-up of (B). https://doi.org/10.7554/eLife.20383.013 The epitope recognized by mAb c12 is a surface composed of blade two and part of blade 3 of PfCyRPA (Figure 5A). The most frequent amino acid dimorphism of PfCyRPA at position 339 is thus located outside the epitope recognized by mAb c12, consistent with the observation that mAb c12 binds to P. falciparum independent of the PfCyRPA variant they express (Dreyer et al., 2012). mAb c12 buries a total surface area of 950 Å2 on PfCyRPA with the major contributor being the light chain, which buries 520 Å2, while the heavy chain buries only 430 Å2. The surface complementarity coefficient Sc of the complex is 0.67, a typical value for antibody-antigen interactions (Lawrence and Colman, 1993); a value of one would denote perfect complement
Abstract Background The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context. Results We describe a strategy to generate monoclonal antibodies against membrane or membrane-associated proteins that completely bypasses any need for purified recombinant antigen. This approach utilises stably transfected mammalian cells expressing recombinant antigens on their cell surface for immunisation of mice. The transfected cells are also used for measuring seroconversion, hybridoma selection and antibody characterisation. By presenting the antigen in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of antibodies capable of binding to the endogenous protein. In the present study, we applied this approach successfully for three predicted GPI-anchored proteins of the malaria parasite Plasmodium falciparum . Conclusions The described entirely cell-based technology is a fast and efficient approach for obtaining antibodies reactive with endogenous cell-surface proteins in their native conformation.
Diarrheal diseases are the third leading cause of death globally, disproportionally affecting low- to middle-income countries like Kenya, with Shigella species being the leading cause of bacterial diarrhea, especially in children. The low infectious dose and high antibiotic resistance levels complicate treatment, leading to long-term sequelae that necessitate control measures such as vaccines to reduce morbidity and mortality rates, especially among children under 5 years of age.
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.
BackgroundShigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis.MethodsIn this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed.FindingsSixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002).InterpretationThe Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.)FundingFunding for this study was through a grant from the Wellcome Trust.
