Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering ...multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices.
Despite ongoing efforts, a highly effective vaccine against Plasmodium falciparum remains elusive. Vaccines targeting the pre-erythrocytic stages of the P. falciparum life cycle are the most advanced ...to date, affording moderate levels of efficacy in field trials. However, the discovery that the members of the merozoite PfRH5-PfCyRPA-PfRipr (RCR) complex are capable of inducing strain-transcendent neutralizing antibodies has renewed enthusiasm for the possibility of preventing disease by targeting the parasite during the blood stage of infection. With Phase I/II clinical trials now underway using first-generation vaccines against PfRH5, and more on the horizon for PfCyRPA and PfRipr, this review explores the rationale and future potential of the RCR complex as a P. falciparum vaccine target.
The antigens PfRH5, PfCyRPA, and PfRipr can induce strain-transcendent neutralizing antibodies, and all three targets are essential and highly conserved.PfRH5, PfCyRPA, and PfRipr form a stable complex (RCR) that is involved in the induction of an erythrocytic calcium spike during merozoite invasion.Passive transfer of anti-CyRPA and anti-PfRH5 antibodies can protect against blood-stage P. falciparum in animal models.Structural studies have mapped out the first known critical inhibitory epitopes on PfRH5 and PfCyRPA which can be used for next-generation vaccine design.Early results from the first PfRH5 vaccine clinical trials have been reported with more anticipated soon, which will help guide the development of RCR-based vaccines.
Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in β-thalassemia. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, ...and ERFE suppresses hepatic hepcidin production through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for hepcidin control, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron. In vitro, ERFE decreased SMAD1, SMAD5, and SMAD8 phosphorylation and inhibited expression of BMP target genes. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6, and BMP7 but had little or no effect on hepcidin induction by BMP2, BMP4, BMP9, or activin B. A neutralizing anti-ERFE antibody prevented ERFE from inhibiting hepcidin induction by BMP5, BMP6, and BMP7. Cell-free homogeneous time-resolved fluorescence assays showed that BMP5, BMP6, and BMP7 competed with anti-ERFE for binding to ERFE. We conclude that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially impairing an evolutionarily closely related BMP subgroup of BMP5, BMP6, and BMP7. ERFE can act as a natural ligand trap generated by stimulated erythropoiesis to regulate the availability of iron.
•ERFE suppresses BMP/SMAD signaling in vitro and in vivo.•ERFE inhibits hepcidin induction by BMP5, BMP6, and BMP7.
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Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer ...functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.
In adult mammals, the adipocyte-derived hormone leptin acts on the brain to reduce food intake by regulating the activity of neurons in the arcuate nucleus of the hypothalamus (ARH). Here, we report ...that neural projection pathways from the ARH are permanently disrupted in leptin-deficient ($Lep^{ob}/Lep^{ob}$) mice and leptin treatment in adulthood does not reverse these neuroanatomical defects. However, treatment of$Lep^{ob}/Lep^{ob}$neonates with exogenous leptin rescues the development of ARH projections, and leptin promotes neurite outgrowth from ARH neurons in vitro. These results suggest that leptin plays a neurotrophic role during the development of the hypothalamus and that this activity is restricted to a neonatal critical period that precedes leptin's acute regulation of food intake in adults.
The arcuate nucleus of the hypothalamus (ARH) is a critical component of forebrain pathways that regulate a variety of neuroendocrine functions, including an important role in relaying leptin signals ...to other parts of the hypothalamus. However, neonatal rodents do not lose weight in response to leptin treatment in the same way as do adults, suggesting that certain aspects of leptin signaling pathways in the hypothalamus may not be mature. We tested this possibility by using DiI axonal labeling to examine the development of projections from the ARH to other parts of the hypothalamus in neonatal mice, paying particular attention to the innervation of the paraventricular nucleus (PVH), the dorsomedial nucleus (DMH), and the lateral hypothalamic area (LHA), each of which have been implicated in the regulation of feeding. The results indicate that ARH projections are quite immature at birth and appear to innervate the DMH, PVH, and LHA in succession, within distinct temporal domains. The projections from the ARH to the DMH develop rapidly and are established by the sixth postnatal day (P6), whereas those to the PVH develop significantly later, with the mature pattern of innervation first apparent between postnatal day 8 (P8)-P10. Furthermore, the ability of leptin to activate Fos in the PVH, DMH, and LHA appears to be age-dependent and correlates with the arrival of ARH projections to each nucleus. Taken together, these findings provide new insight into development of hypothalamic circuits and suggest an anatomical basis for the delayed postnatal regulation of food intake and body weight by leptin.
The continued global burden of malaria can in part be attributed to a complex lifecycle, with both human hosts and mosquito vectors serving as transmission reservoirs. In preclinical models of ...vaccine-induced immunity, antibodies to parasite sexual-stage antigens, ingested in the mosquito blood meal, can inhibit parasite survival in the insect midgut as judged by ex vivo functional studies such as the membrane feeding assay. In an era of renewed political momentum for malaria elimination and eradication campaigns, such observations have fueled support for the development and implementation of so-called transmission-blocking vaccines. While leading candidates are being evaluated using a variety of promising vaccine platforms, the field is also beginning to capitalize on global '-omics' data for the rational genome-based selection and unbiased characterization of parasite and mosquito proteins to expand the candidate list. This review covers the progress and prospects of these recent developments.
No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. ...falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.
Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus‐like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid‐19. ...However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre‐existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer‐designed VLP not only to monomers (SARS‐CoV‐2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag‐mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.
Nanoassembly can play a key role in generating effective vaccines for emerging (SARS‐CoV‐2) and established (influenza) pandemic threats. SpyTag‐mediated display was used to adapt the nanocage to the challenge of bacterial or viral antigens with diverse cyclic and dihedral symmetries. Establishing display of both trimeric and tetrameric antigens may lead to an influenza vaccine with broader protection.
Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum ...reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10–15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens.
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