Correction: A conformational epitope in placental malaria vaccine antigen VAR2CSA: What does it teach us?

[This corrects the article DOI: 10.1371/journal.ppat.1011370.].

A conformational epitope in placental malaria vaccine antigen VAR2CSA: What does it teach us?

VAR2CSA is the Plasmodium falciparum variant surface antigen that mediates binding of infected erythrocytes to chondroitin sulfate A (CSA) and their sequestration in intervillous spaces of the placenta, leading to placental malaria (PM). Relatively high polymorphism in VAR2CSA sequences has hindered development of a vaccine that induces broadly neutralizing immunity. Recent research has highlighted that a broadly reactive human monoclonal antibody, called PAM1.4, binds to multiple conserved residues of different subfragments of VAR2CSA, forming a conformational epitope. In this short perspective, we describe evidence that residues located in the interdomain-1 fragment of VAR2CSA within the PAM1.4 binding epitope might be critical to broad reactivity of the antibody. Future investigation into broadly reactive anti-VAR2CSA antibodies may be important for the following: (1) identification of similar conformation epitopes targeted by broadly neutralizing antibodies; and (2) understanding different immune evasion mechanisms used by placenta-binding parasites through VAR2CSA polymorphism in critical epitopes.

Protein-protein conjugation enhances the immunogenicity of SARS-CoV-2 receptor-binding domain (RBD) vaccines.

Several effective SARS-CoV-2 vaccines have been developed using different technologies. Although these vaccines target the isolates collected early in the pandemic, many have protected against serious illness from newer variants. Nevertheless, efficacy has diminished against successive variants and the need for effective and affordable vaccines persists especially in the developing world. Here, we adapted our protein-protein conjugate vaccine technology to generate a vaccine based on receptor-binding domain (RBD) antigen. RBD was conjugated to a carrier protein, EcoCRM®, to generate two types of conjugates: crosslinked and radial conjugates. In the crosslinked conjugate, antigen and carrier are chemically crosslinked; in the radial conjugate, the antigen is conjugated to the carrier by site-specific conjugation. With AS01 adjuvant, both conjugates showed enhanced immunogenicity in mice compared to RBD, with a Th1 bias. In hACE2 binding inhibition and pseudovirus neutralization assays, sera from mice vaccinated with the radial conjugate demonstrated strong functional activity.

A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates.

Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti-adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is known that naturally acquired antibodies target both variant-specific and conserved epitopes. It is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA variants. Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified ~0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. In two independent antibody purification/depletion experiments with permutated order of VAR2CSA variants, IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG. These findings demonstrate that a single VAR2CSA ectodomain variant displays conserved epitopes that are targeted by neutralizing (or binding-inhibitory) antibodies shared by multiple parasite strains, including maternal isolates. This suggests that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

Contracting malaria during pregnancy ­ especially a first pregnancy ­ can lead to a severe, placental form of the disease that is often fatal. Red blood cells infected with the malaria parasite Plasmodium falciparum display a protein, VAR2CSA, which can recognize and bind CSA molecules present on placental cells and in placental blood spaces. This leads to the infected blood cells accumulating in the placenta and inducing harmful inflammation. Having been exposed to the parasite in prior pregnancies generates antibodies that target VAR2CSA, stopping the infected blood cells from latching onto placental CSA or tagging them for immune destruction. Overall, this makes placental malaria less severe in following pregnancies, and suggests that vaccines could be developed based on VAR2CSA. However, this protein has regions that can vary in structure, meaning that P. falciparaum can generate many VAR2CSA variants. Individuals exposed to the parasite naturally generate antibodies that block a wide array of variants from attaching to CSA. In contrast, first-generation vaccines based on VAR2CSA fragments have only induced variant-specific antibodies, therefore offering limited protection against infection. As a response, Doritchamou et al. set out to find VAR2CSA structures that could be recognized by antibodies targeting an array of variants. Blood was obtained from women who had had multiple pregnancies and were immune to malaria. Their plasma was passed over five different large VAR2CSA variants in order to isolate and purify antibodies that attached to these structures. Doritchamou et al. found that antibodies binding to individual VAR2CSA structures could also recognise a wide array of VAR2CSA variants and blocked all tested parasites from sticking to CSA. While further research is needed, these findings highlight antibodies that cross-react to diverse VAR2CSA variants and could be used to design more effective vaccines targeting placental malaria.

Allelic variants of full-length VAR2CSA, the placental malaria vaccine candidate, differ in antigenicity and receptor binding affinity.

Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta via surface protein VAR2CSA, which binds chondroitin sulfate A (CSA) expressed on the syncytiotrophoblast surface, causing placental malaria (PM) and severe adverse outcomes in mothers and their offspring. VAR2CSA belongs to the PfEMP1 variant surface antigen family; PfEMP1 proteins mediate IE adhesion and facilitate parasite immunoevasion through antigenic variation. Here we produced deglycosylated (native-like) and glycosylated versions of seven recombinant full-length VAR2CSA ectodomains and compared them for antigenicity and adhesiveness. All VAR2CSA recombinants bound CSA with nanomolar affinity, and plasma from Malian pregnant women demonstrated antigen-specific reactivity that increased with gravidity and trimester. However, allelic and glycosylation variants differed in their affinity to CSA and their serum reactivities. Deglycosylated proteins (native-like) showed higher CSA affinity than glycosylated proteins for all variants except NF54. Further, the gravidity-related increase in serum VAR2CSA reactivity (correlates with acquisition of protective immunity) was absent with the deglycosylated form of atypical M200101 VAR2CSA with an extended C-terminal region. Our findings indicate significant inter-allelic differences in adhesion and seroreactivity that may contribute to the heterogeneity of clinical presentations, which could have implications for vaccine design.

Plasmodium Preerythrocytic Vaccine Antigens Enhance Sterile Protection in Mice Induced by Circumsporozoite Protein.

Preerythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading preerythrocytic vaccine, RTS,S/AS01E (Mosquirix), entered implementation programs in 2019 and targets the major sporozoite surface antigen, circumsporozoite protein (CSP). However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver-stage proteins that could potentially be used in combination with CSP; they are referred to as preerythrocytic vaccine antigens (PEVAs). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (protein prime-adenovirus 5 [Ad5] boost) and Plasmodium berghei (DNA prime-Ad5 boost) in mice. When combined as individual antigens with P. yoelii CSP (PyCSP), three of eight P. yoelii PEVAs significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when three P. berghei PEVAs and P. berghei CSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP plus PEVA vaccinations. Both P. yoelii and P. berghei CSP plus PEVA combination vaccines induced robust CD8+ T cell responses, including signature gamma interferon (IFN-γ) increases. In the P. berghei model system, IFN-γ responses were significantly higher in hepatic versus splenic CD8+ T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.

A human monoclonal antibody blocks malaria transmission and defines a highly conserved neutralizing epitope on gametes.

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites. Among nine Pfs230 human monoclonal antibodies (mAbs) that we generated, one potently blocks transmission to mosquitoes in a complement-dependent manner and reacts to the gamete surface; the other eight show only low or no blocking activity. The structure of the transmission-blocking mAb in complex with vaccine antigen reveals a large discontinuous conformational epitope, specific to domain 1 of Pfs230 and comprising six structural elements in the protein. The epitope is conserved, suggesting the transmission-blocking mAb is broadly functional. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination.

Pfs230 yields higher malaria transmission-blocking vaccine activity than Pfs25 in humans but not mice.

BACKGROUNDVaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication, and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced serum functional activity in both US and Malian adults. However, antibody levels declined rapidly, and transmission-reducing activity required 4 vaccine doses. Functional immunogenicity and durability must be improved before advancing transmission-blocking vaccines further in clinical development. We hypothesized that the prefertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity.METHODSTransmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody levels were measured by ELISA and transmission-reducing activity was assessed by the standard membrane feeding assay.RESULTSPfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In US adults, 2 vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in 5 Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone.CONCLUSIONThe complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model.TRIAL REGISTRATIONClinicalTrials.gov NCT02334462.FUNDINGIntramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Structural basis for placental malaria mediated by Plasmodium falciparum VAR2CSA.

Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment. In the present study, we determined the cryo-electron microscopy structures of the full-length ectodomain of VAR2CSA from P. falciparum strain NF54 in complex with CSA, and VAR2CSA from a second P. falciparum strain FCR3. The architecture of VAR2CSA is composed of a stable core flanked by a flexible arm. CSA traverses the core domain by binding within two channels and CSA binding does not induce major conformational changes in VAR2CSA. The CSA-binding elements are conserved across VAR2CSA variants and are flanked by polymorphic segments, suggesting immune selection outside the CSA-binding sites. This work provides paths for developing interventions against placental malaria and cancer.

Placental malaria vaccine candidate antigen VAR2CSA displays atypical domain architecture in some Plasmodium falciparum strains.

Two vaccines based on Plasmodium falciparum protein VAR2CSA are currently in clinical evaluation to prevent placental malaria (PM), but a deeper understanding of var2csa variability could impact vaccine design. Here we identified atypical extended or truncated VAR2CSA extracellular structures and confirmed one extended structure in a Malian maternal isolate, using a novel protein fragment assembly method for RNA-seq and DNA-seq data. Extended structures included one or two additional DBL domains downstream of the conventional NTS-DBL1X-6ɛ domain structure, with closest similarity to DBLɛ in var2csa and non-var2csa genes. Overall, 4/82 isolates displayed atypical VAR2CSA structures. The maternal isolate expressing an extended VAR2CSA bound to CSA, but its recombinant VAR2CSA bound less well to CSA than VAR2CSANF54 and showed lower reactivity to naturally acquired parity-dependent antibody. Our protein fragment sequence assembly approach has revealed atypical VAR2CSA domain architectures that impact antigen reactivity and function, and should inform the design of VAR2CSA-based vaccines.

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.

ATP-independent control of autotransporter virulence protein transport via the folding properties of the secreted protein.

Autotransporter (AT) proteins are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. The mechanism by which AT proteins cross the bacterial outer membrane (OM), in the absence of ATP or another external energy source, is unknown. Here we demonstrate a linear correlation between localized regions of stability (ΔG(folding)) in the mature virulence protein (the AT "passenger") and OM secretion efficiency. Destabilizing the C-terminal ß-helical domain of a passenger reduced secretion efficiency. In contrast, destabilizing the globular N-terminal domain of a passenger produced a linearly correlated increase in secretion efficiency. Thus, C-terminal passenger stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion demonstrate a crucial role for the passenger itself in directing its secretion, suggesting a novel type of ATP-independent, folding-driven transporter.

Disulfide bond-mediated passenger domain stalling as a structural probe of autotransporter outer membrane secretion in vivo.

Autotransporters (ATs) are the largest class of extracellular virulence proteins secreted by Gram-negative pathogenic bacteria, but the details of their outer membrane (OM) secretion mechanism remain unclear. Recently, a novel strategy has been developed to study OM secretion of AT proteins by introducing pairs of cysteine (Cys) residues into the central passenger domain sequence. Upon oxidation in the periplasm, these Cys residues form a long loop that stalls AT OM secretion. This Cys-loop stalling technique has been used to investigate such questions as the directionality of AT OM secretion and the extent of AT passenger domain folding during secretion. Here, we will describe how to use the Cys-loop approach to produce disulfide-bonded, stalled AT OM secretion intermediates, and how these stalled "snapshots" can be used to investigate structural aspects of the AT OM secretion mechanism.

A conserved stable core structure in the passenger domain beta-helix of autotransporter virulence proteins.

In Gram-negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right-handed parallel beta-helical structure, and the three crystal structures available for AT passenger domains each contain a long right-handed parallel beta-helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three-state folding and has a C-terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three-state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N-terminal sequencing demonstrate that the Pet stable core is also located near the C-terminus of the passenger domain. Moreover, sequence analysis suggests that three-state folding and a C-terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo.