Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria.

BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.

Severe malaria is associated with parasite binding to endothelial protein C receptor.

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.

Persistent Plasmodium falciparum Infection in Women With an Intent to Become Pregnant as a Risk Factor for Pregnancy-associated Malaria.

Background: Pregnant women are more susceptible to Plasmodium falciparum than before pregnancy, and infection has consequences for both mother and offspring. The World Health Organization recommends that pregnant woman in areas of transmission receive intermittent preventive treatment (IPTp) starting in the second trimester. Consequently, women are not protected during the first trimester, although P. falciparum infections are both frequent and harmful. Methods: A cohort of nulligravid women was followed up during subsequent pregnancy. Malaria was diagnosed by means of microscopy and polymerase chain reaction. Parasites were genotyped at polymorphic loci. Results: Among 275 nulligravidae enrolled, 68 women became pregnant and were followed up during pregnancy. Before pregnancy, P. falciparum prevalence rates were 15% by microscopy and 66% by polymerase chain reaction. Microscopic infection rates increased to 29% until IPTp administration, and their density increased by 20-fold. Conversely, submicroscopic infection rates decreased. After IPTp administration, all types of infections decreased, but they increased again late in pregnancy. The risk of infection during pregnancy was higher in women with a microscopic (odds ratio, 6.5; P = .047) or submicroscopic (3.06; P = .05) infection before pregnancy and was not related to the season of occurrence. Most infections during pregnancy were persistent infections acquired before pregnancy. Conclusions: Microscopic and submicroscopic malaria infections were frequent in nulligravid women from south Benin. During the first trimester of pregnancy, microscopic infections were more frequent, with a higher parasite density, and mainly derived from parasites infecting the woman before conception. Preventive strategies targeting nonpregnant women with a desire for conception need to be designed.

Plasmodium falciparum Infection Early in Pregnancy has Profound Consequences for Fetal Growth.

Malaria during pregnancy constitutes a large health problem in areas of endemicity. The World Health Organization recommends that interventions are initiated at the first antenatal visit, and these improve pregnancy outcomes. This study evaluated fetal growth by ultrasonography and birth outcomes in women who were infected prior to the first antenatal visit (gestational age, <120 days) and not later in pregnancy. Compared with uninfected controls, women with early Plasmodium falciparum exposure had retarded intrauterine growth between gestational ages of 212 and 253 days (difference between means, 107 g [95% confidence interval {CI}, 26-188]; P = .0099) and a shorter pregnancy duration (difference between means, 6.6 days [95% CI, 1.0-112.5]; P = .0087). The birth weight (difference between means, 221 g [95% CI, 6-436]; P = .044) and the placental weight (difference between means, 84 g [95% CI, 18-150]; P = .013) at term were also reduced. The study suggests that early exposure to P. falciparum, which is not targeted for prevention by current control strategies, has a profound impact on fetal growth, pregnancy duration, and placental weight at term.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced.

Infections with the human malaria parasite Plasmodium falciparum during pregnancy can lead to severe complications for both mother and child, resulting from the cytoadhesion of parasitized erythrocytes in the intervillous space of the placenta. Cytoadherence is conferred by the specific interaction of the parasite-encoded adhesin VAR2CSA with chondroitin-4-sulfate (CSA) present on placental proteoglycans. CSA presented elsewhere in the microvasculature does not afford VAR2CSA-mediated cytoadhesion of parasitized erythrocytes. To address the placenta-specific binding tropism, we investigated the effect of the receptor/ligand arrangement on cytoadhesion, using artificial membranes with different CSA spacing intervals. We found that cytoadhesion is strongly dependent on the CSA distance, with half-maximal adhesion occurring at a CSA distance of 9 ± 1 nm at all hydrodynamic conditions. Moreover, binding to CSA was cooperative and shear stress induced. These findings suggest that the CSA density, together with allosteric effects in VAR2CSA, aid in discriminating between different CSA milieus.

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses.

BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. METHODS: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. RESULTS: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. CONCLUSION: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue: a novel model of placental malaria.

BACKGROUND: Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue. RESULTS: The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. CONCLUSION: The ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.

Clinical development of placental malaria vaccines and immunoassays harmonization: a workshop report.

Placental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays with a goal to define standards that will allow comparative assessment of different placental malaria vaccine candidates. The recommendations of these workshops should guide researchers and clinicians in the further development of placental malaria vaccines.

Bacterial superglue enables easy development of efficient virus-like particle based vaccines.

BACKGROUND: Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components. RESULTS: Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5). CONCLUSIONS: The spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology's ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.

Protective Antibodies against Placental Malaria and Poor Outcomes during Pregnancy, Benin.

Placental malaria is caused by Plasmodium falciparum-infected erythrocytes that bind to placental tissue. Binding is mediated by VAR2CSA, a parasite antigen coded by the var gene, which interacts with chondroitin sulfate A (CSA). Consequences include maternal anemia and fetal growth retardation. Antibody-mediated immunity to placental malaria is acquired during successive pregnancies, but the target of VAR2CSA-specific protective antibodies is unclear. We assessed VAR2CSA-specific antibodies in pregnant women and analyzed their relationships with protection against placental infection, preterm birth, and low birthweight. Antibody responses to the N-terminal region of VAR2CSA during early pregnancy were associated with reduced risks for infections and low birthweight. Among women infected during pregnancy, an increase in CSA binding inhibition was associated with reduced risks for placental infection, preterm birth, and low birthweight. These data suggest that antibodies against VAR2CSA N-terminal region mediate immunity to placental malaria and associated outcomes. Our results validate current vaccine development efforts with VAR2CSA N-terminal constructs.

Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria.

Malaria is a major global health problem. Pregnant women are susceptible to infection regardless of previously acquired immunity. Placental malaria is caused by parasites capable of sequestering in the placenta. This is mediated by VAR2CSA, a parasite antigen that interacts with chondroitin sulfate A (CSA). One vaccine strategy is to block this interaction with VAR2CSA-specific antibodies. It is a priority to define a small VAR2CSA fragment that can be used in an adhesion blocking vaccine. In this, the obvious approach is to define regions of VAR2CSA involved in receptor binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with nanomolar affinity, and that the CSA-binding site lies in the N-terminal part of the protein. In this study we define the minimal binding region by truncating VAR2CSA and analyzing CSA binding using biosensor technology. We show that the core CSA-binding site lies within the DBL2X domain and parts of the flanking interdomain regions. This is in contrast to the idea that single domains do not possess the structural requirements for specific CSA binding. Small-angle x-ray scattering measurements enabled modeling of VAR2CSA and showed that the CSA-binding DBL2X domain is situated in the center of the structure. Mutating classic sulfate-binding sites in VAR2CSA, along with testing dependence of ionic interactions, suggest that the CSA binding is not solely dependent on the sulfated CSA structure. Based on these novel PfEMP1 structure-function studies, we have constructed a small VAR2CSA antigen that has the capacity to induce highly adhesion-blocking antibodies.

Multilaboratory approach to preclinical evaluation of vaccine immunogens for placental malaria.

Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes that adhere to the placental receptor chondroitin sulfate A (CSA) and sequester in the placenta; women become resistant to pregnancy malaria as they acquire antiadhesion antibodies that target surface proteins of placental parasites. VAR2CSA, a member of the P. falciparum EMP1 variant surface antigen family, is the leading candidate for a pregnancy malaria vaccine. Because VAR2CSA is a high-molecular-weight protein, a vaccine based on the full-length protein may not be feasible. An alternative approach has been to develop a vaccine targeting individual Duffy binding-like (DBL) domains. In this study, a consortium of laboratories under the Pregnancy Malaria Initiative compared the functional activity of antiadhesion antibodies elicited by different VAR2CSA domains and variants produced in prokaryotic and eukaryotic expression systems. Antisera were initially tested against laboratory lines of maternal parasites, and the most promising reagents were evaluated in the field against fresh placental parasite samples. Recombinant proteins expressed in Escherichia coli elicited antibody levels similar to those expressed in eukaryotic systems, as did the two allelic forms of the DBL4 and DBL5 domains. The procedures developed for this head-to-head comparison will be useful for future evaluation and down-selection of malaria vaccine immunogens.

Increased Galectin-9 Levels Correlate with Disease Activity in Patients with DMARD-Naïve Rheumatoid Arthritis and Modulate the Secretion of MCP-1 and IL-6 from Synovial Fibroblasts.

Background: Fibroblast-like synoviocytes (FLSs) are essential mediators in the expansive growth and invasiveness of rheumatoid synovitis, and patients with a fibroblastic-rich pauci-immune pathotype respond poorly to currently approved antirheumatic drugs. Galectin-9 (Gal-9) has been reported to directly modulate rheumatoid arthritis (RA) FLSs and to hold both pro- and anti-inflammatory properties. The objective of this study was to evaluate clinical and pathogenic aspects of Gal-9 in RA, combining national patient cohorts and cellular models. Methods: Soluble Gal-9 was measured in plasma from patients with newly diagnosed, treatment-naïve RA (n = 98). The disease activity score 28-joint count C-reactive protein (DAS28CRP) and total Sharp score were used to evaluate the disease course serially over a two-year period. Plasma and synovial fluid samples were examined for soluble Gal-9 in patients with established RA (n = 18). A protein array was established to identify Gal-9 binding partners in the extracellular matrix (ECM). Synovial fluid mononuclear cells (SFMCs), harvested from RA patients, were used to obtain synovial-fluid derived FLSs (SF-FLSs) (n = 7). FLSs from patients suffering from knee Osteoarthritis (OA) were collected from patients when undergoing joint replacement surgery (n = 5). Monocultures of SF-FLSs (n = 6) and autologous co-cultures of SF-FLSs and peripheral blood mononuclear cells (PBMCs) were cultured with and without a neutralizing anti-Gal-9 antibody (n = 7). The mono- and co-cultures were subsequently analyzed by flow cytometry, MTT assay, and ELISA. Results: Patients with early and established RA had persistently increased plasma levels of Gal-9 compared with healthy controls (HC). The plasma levels of Gal-9 were associated with disease activity and remained unaffected when adding a TNF-inhibitor to their standard treatment. Gal-9 levels were elevated in the synovial fluid of established RA patients with advanced disease, compared with corresponding plasma samples. Gal-9 adhered to fibronectin, laminin and thrombospondin, while not to interstitial collagens in the ECM protein array. In vitro, a neutralizing Gal-9 antibody decreased MCP-1 and IL-6 production from both RA FLSs and OA FLSs. In co-cultures of autologous RA FLSs and PBMCs, the neutralization of Gal-9 also decreased MCP-1 and IL-6 production, without affecting the proportion of inflammatory FLSs. Conclusions: In RA, pretreatment plasma Gal-9 levels in early RA were increased and correlated with clinical disease activity. Gal-9 levels remained increased despite a significant reduction in the disease activity score in patients with early RA. The in vitro neutralization of Gal-9 decreased both MCP-1 and IL-6 production in an inflammatory subset of RA FLSs. Collectively these findings indicate that the persistent overexpression of Gal-9 in RA may modulate synovial FLS activities and could be involved in the maintenance of subclinical disease activity in RA.

A Virus-like Particle-Based F4 Enterotoxigenic Escherichia coli Vaccine Is Inhibited by Maternally Derived Antibodies in Piglets but Generates Robust Responses in Sows.

F4-positive enterotoxigenic Escherichia coli is associated with diarrhea and poor growth outcomes in neonatal and newly weaned piglets and is thus a major economic and welfare burden in the swine industry. Vaccination of sows with F4 fimbriae protects against the neonatal disease via passive transfer of maternal immunity. However, this strategy does not protect against infection post-weaning. Consequently, prevention and treatment methods in weaner pigs heavily rely on the use of antimicrobials. Therefore, in order to reduce antimicrobial consumption, more effective prophylactic alternatives are needed. In this study, we describe the development of a capsid virus-like particle (cVLP)-based vaccine targeting the major F4 fimbriae subunit and adhesion molecule, FaeG, and evaluate its immunogenicity in mice, piglets, and sows. cVLP-display significantly increased systemic and mucosal antibody responses towards the recombinant FaeG antigen in mice models. However, in piglets, the presence of anti-F4 maternally derived antibodies severely inhibited the induction of active humoral responses towards the FaeG antigen. This inhibition could not be overcome, even with the enhanced immunogenicity achieved via cVLP display. However, in sows, intramuscular vaccination with the FaeG.cVLP vaccine was able to generate robust IgG and IgA responses that were comparable with a commercial fimbriae-based vaccine, and which were effectively transferred to piglets via colostrum intake. These results demonstrate that cVLP display has the potential to improve the systemic humoral responses elicited against low-immunogenic antigens in pigs; however, this effect is dependent on the use of antigens, which are not the targets of pre-existing maternal immunity.

Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice.

BACKGROUND: The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. METHODS: We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically "cold" 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically "hot" CT26 colon carcinoma model. RESULTS: V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. CONCLUSIONS: Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically "cold" tumors "hot" and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.

Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines.

IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

First-in-human use of a modular capsid virus-like vaccine platform: an open-label, non-randomised, phase 1 clinical trial of the SARS-CoV-2 vaccine ABNCoV2.

BACKGROUND: Capsid virus-like particles (cVLP) have proven safe and immunogenic and can be a versatile platform to counter pandemics. We aimed to clinically test a modular cVLP COVID-19 vaccine in individuals who were naive to SARS-CoV-2. METHODS: In this phase 1, single-centre, dose-escalation, adjuvant-selection, open-label clinical trial, we recruited participants at the Radboud University Medical Center in Nijmegen, Netherlands, and sequentially assigned them to seven groups. Eligible participants were healthy, aged 18-55 years, and tested negative for SARS-CoV-2 and anti-SARS-CoV-2 antibodies. Participants were vaccinated intramuscularly on days 0 and 28 with 6 µg, 12 µg, 25 µg, 50 µg, or 70 µg of the cVLP-based COVID-19 vaccine (ABNCoV2). A subgroup received MF59-adjuvanted ABNCoV2. Follow-up was for 24 weeks after second vaccination. The primary objectives were to assess the safety and tolerability of ABNCoV2 and to identify a dose that optimises the tolerability-immunogenicity ratio 14 days after the first vaccination. The primary safety endpoint was the number of related grade 3 adverse events and serious adverse events in the intention-to-treat population. The primary immunogenicity endpoint was the concentration of ABNCoV2-specific antibodies. The trial is registered with ClinicalTrials.gov, NCT04839146. FINDINGS: 45 participants (six to nine per group) were enrolled between March 15 and July 15, 2021. Participants had a total of 249 at least possibly related solicited adverse events (185 grade 1, 63 grade 2, and one grade 3) within a week after vaccination. Two serious adverse events occurred; one was classified as a possible adverse reaction. Antibody titres were dose-dependent with levels plateauing at a vaccination dose of 25-70 µg ABNCoV2. After second vaccination, live virus neutralisation activity against major SARS-CoV-2 variants was high but was lower with an omicron (BA.1) variant. Vaccine-specific IFNγ+ CD4+ T cells were induced. INTERPRETATION: Immunisation with ABNCoV2 was well tolerated, safe, and resulted in a functional immune response. The data support the need for additional clinical development of ABNCoV2 as a second-generation SARS-CoV-2 vaccine. The modular cVLP platform will accelerate vaccine development, beyond SARS-CoV-2. FUNDING: EU, Carlsberg Foundation, and the Novo Nordisk Foundation.