Identification of a norovirus GII-specific antigenic epitope.

Noroviruses (NoVs) are the chief cause of acute viral gastroenteritis worldwide. By employing the major capsid protein VP1 of a GII.6 NoV strain as an immunogen, we generated two monoclonal antibodies (mAbs) with wide-spectrum binding activities against NoV genogroup II (GII) VP1 proteins. One mAb (10G7) could bind to native and denatured GII-specific VP1 proteins. The other mAb (10F2) could bind to all tested native GII VP1 proteins, but not to denatured GII.3, GII.4, GII.7, or GII.17 VP1 proteins. Using GII.6/GII.4 fusion proteins, the mAb 10F2 binding region was confirmed to be located in the C-terminal P1 domain. An enzyme-linked immunosorbent assay based on peptides covering the P domain did not detect any binding. Using a panel of VP1 proteins with swapped regions, deletions, and mutations, the mAb 10F2 binding region was determined to be located between residues 496 and 513. However, the residue(s) responsible for its varied binding affinity for different denatured GII VP1 proteins remain to be identified. In summary, two NoV GII-specific cross-reactive mAbs were generated, and their binding regions were determined. Our results might facilitate the detection and immunogenic study of NoVs.

Rotavirus vaccines in Africa and Norovirus genetic diversity in children aged 0 to 5 years old: a systematic review and meta-analysis : Rotavirus vaccines in Africa and Norovirus genetic diversity.

Noroviruses are the second leading cause of death in children under the age of 5 years old. They are responsible for 200 million cases of diarrhoea and 50,000 deaths in children through the word, mainly in low-income countries. The objective of this review was to assess how the prevalence and genetic diversity of noroviruses have been affected by the introduction of rotavirus vaccines in Africa. PubMed, Web of Science and Science Direct databases were searched for articles. All included studies were conducted in Africa in children aged 0 to 5 years old with gastroenteritis. STATA version 16.0 software was used to perform the meta-analysis. The method of Dersimonian and Laird, based on the random effects model, was used for the statistical analyses in order to estimate the pooled prevalence's at a 95% confidence interval (CI). Heterogeneity was assessed by Cochran's Q test using the I2 index. The funnel plot was used to assess study publication bias. A total of 521 studies were retrieved from the databases, and 19 were included in the meta-analysis. The pooled norovirus prevalence's for pre- and post-vaccination rotavirus studies were 15% (95 CI, 15-18) and 13% (95 CI, 09-17) respectively. GII was the predominant genogroup, with prevalence of 87.64% and 91.20% respectively for the pre- and post-vaccination studies. GII.4 was the most frequently detected genotype, with rates of 66.84% and 51.24% respectively for the pre- and post-vaccination studies. This meta-analysis indicates that rotavirus vaccination has not resulted in a decrease in norovirus infections in Africa.

Characteristics of Norovirus capsid protein-specific CD8+ T-Cell responses in previously infected individuals.

Norovirus (NV) infection causes acute gastroenteritis in children and adults. Upon infection with NV, specific CD8+ T cells, which play an important role in anti-infective immunity, are activated in the host. Owing to the NV's wide genotypic variability, it is challenging to develop vaccines with cross-protective abilities against infection. To aid effective vaccine development, we examined specific CD8+ T-cell responses towards viral-structural protein (VP) epitopes, which enable binding to host susceptibility receptors. We isolated peripheral blood mononuclear cells from 196 participants to screen and identify predominant core peptides towards NV main and small envelope proteins using ex vivo and in vitro intracellular cytokine staining assays. Human leukocyte antigen (HLA) restriction characteristics were detected using next-generation sequencing. Three conservative immunodominant VP-derived CD8+ T-cell epitopes, VP294-102 (TDAARGAIN), VP2153-161 (RGPSNKSSN), and VP1141-148 (FPHIIVDV), were identified and restrictively presented by HLA-Cw * 0102, HLA-Cw * 0702, and HLA-A *1101 alleles, separately. Our findings provide useful insights into the development of future vaccines and treatments for NV infection.

Self-Powered Immunoassay of Norovirus in Human Stools by π-Electron-Rich Homojunction for Enhanced Charge Transfer.

Norovirus (NoV) stands as a significant causative agent of nonbacterial acute gastroenteritis on a global scale, presenting a substantial threat to public health. Hence, the development of simple and rapid analytical techniques for NoV detection holds great importance in preventing and controlling the outbreak of the epidemic. In this work, a self-powered photoelectrochemical (PEC) immunosensor of NoV capsid protein (VP1) was proposed by the π-electron-rich carbon nitride homojunction (ER-CNH) as the photoanode. C4N2 ring derived from π-rich locust bean gum was introduced into the tri-s-triazine structure, creating a large π-delocalized conjugated carbon nitride homojunction. This strategy enhances the C/N atomic ratio, which widens light utilization, narrows the bandgap, and optimizes the electronic band structure of carbon nitride. By introduction of a π-rich conjugated structure, p-type domains were induced within n-type domains to build the internal electric field at the interface, thus forming a p-n homojunction to boost carrier separation and transfer. The ER-CNH photoanode exhibited excellent photoelectric performance and water oxidation capacity. Since VP1 inhibits the water oxidation of the ER-CNH photoanode, the open-circuit potential of the as-prepared PEC immunosensor system was reduced for detecting NoV VP1. The self-powered PEC immunosensor achieved a remarkably low detection limit (∼5 fg mL-1) and displayed high stability and applicability for actual stool samples. This research serves as a foundation concept for constructing immunosensors to detect other viruses and promotes the application of self-powered systems for life safety.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Effect of Non-Rotavirus Enteric Infections on Vaccine Efficacy in a ROTASIIL Clinical Trial.

This study examined the relative proportion of enteric pathogens associated with severe gastroenteritis (GE) among children younger than 2 years in a phase III efficacy trial of the ROTASIIL® vaccine in India, evaluated the impact of co-infections on vaccine efficacy (VE), and characterized the association between specific pathogens and the clinical profile of severe GE. Stored stool samples collected from cases of severe GE in the phase III trial were tested by quantitative polymerase chain reaction using TaqMan™ Array Cards. Etiology was attributed by calculating the adjusted attributable fraction (AF) for each pathogen. A test-negative design was used to estimate VE. The pathogens with the highest AFs for severe diarrhea were rotavirus (23.5%), adenovirus 40/41 (17.0%), Shigella spp./enteroinvasive Escherichia coli, norovirus GII, enterotoxigenic E. coli, and Cryptosporidium spp. A considerable proportion of the disease in these children could not be explained by the pathogens tested. Severe GE cases associated with rotavirus and Shigella spp. were more likely to have a longer duration of vomiting and diarrhea, respectively. Cases attributed to Cryptosporidium spp. were more severe and required hospitalization. In the intention-to-treat population, VE was estimated to be 43.9% before and 46.5% after adjustment for co-infections; in the per-protocol population, VE was 46.7% before and 49.1% after adjustments. Rotavirus continued to be the leading cause of severe GE in this age group. The adjusted VE estimates obtained did not support co-infections as a major cause of lower vaccine performance in low- and middle-income countries.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Computational Prediction of Potential Vaccine Candidates From tRNA Encoded peptides (tREP) Using a Bioinformatic Workflow and Molecular Dynamics Validations.

Transfer RNAs (tRNA) are non-coding RNAs. Encouraged by biological applications discovered for peptides derived from other non-coding genomic regions, we explore the possibility of deriving epitope-based vaccines from tRNA encoded peptides (tREP) in this study. Epitope-based vaccines have been identified as an effective strategy to mitigate safety and specificity concerns observed in vaccine development. In this study, we explore the potential of tREP as a source for epitope-based vaccines for virus pathogens. We present a computational workflow that uses verified data sources and community-validated predictive tools to produce a ranked list of plausible epitope-based vaccines starting from tRNA sequences. The top epitope, bound to the predicted HLA molecule, for the virus pathogen is computationally validated through 200 ns molecular dynamics (MD) simulations followed by binding free energy calculations. The simulation results indicate that two tRNA encoded epitope-based vaccines, RRHIDIVV and IMVRFSAE for Mamastrovirus 3 and Norovirus GII, respectively, are likely candidates. Peptides originating from tRNAs provide unexplored opportunities for vaccine design. Encouraged by our previous experimental study, which established the inhibitory properties of tREPs against infectious parasites, we have proposed a computationally validated set of peptides derived from tREPs as vaccines for viral pathogens.

Chimeric virus-like particles of human norovirus constructed by structure-guided epitope grafting elicit cross-reactive immunity against both GI.1 and GII.4 genotypes.

IMPORTANCE: Human norovirus (HuNoV) is highly infectious and can result in severe illnesses in the elderly and children. So far, there is no effective antiviral drug to treat HuNoV infection, and thus, the development of HuNoV vaccines is urgent. However, NoV evolves rapidly, and currently, at least 10 genogroups with numerous genotypes have been found. The genetic diversity of NoV and the lack of cross-protection between different genotypes pose challenges to the development of broadly protective vaccines. In this study, guided by structural alignment between GI.1 and GII.4 HuNoV VP1 proteins, several chimeric-type virus-like particles (VLPs) were designed through surface-exposed loop grafting. Mouse immunization studies show that two of the designed chimeric VLPs induced cross-immunity against both GI.1 and GII.4 HuNoVs. To our knowledge, this is the first designed chimeric VLPs that can induce cross-immune activities across different genogroups of HuNoV, which provides valuable strategies for the development of cross-reactive HuNoV vaccines.

High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity.

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

Molecular Epidemiology of Norovirus (NoV) Infection in Mie Prefecture: The Kinetics of Norovirus Antigenemia in Pediatric Patients.

Few studies have shown the presence of norovirus (NoV) RNA in blood circulation but there is no data on norovirus antigenemia. We examined both antigenemia and RNAemia from the sera of children with NoV infections and studied whether norovirus antigenemia is correlated with the levels of norovirus-specific antibodies and clinical severity of gastroenteritis. Both stool and serum samples were collected from 63 children admitted to Mie National Hospital with acute NoV gastroenteritis. Norovirus antigen and RNA were detected in sera by ELISA and real-time RT-PCR, respectively. NoV antigenemia was found in 54.8% (34/62) and RNAemia in 14.3% (9/63) of sera samples. Antigenemia was more common in the younger age group (0-2 years) than in the older age groups, and most patients were male. There was no correlation between stool viral load and norovirus antigen (NoV-Ag) levels (rs = -0.063; Cl -0.3150 to 0.1967; p = 0.6251). Higher levels of acute norovirus-specific IgG serum antibodies resulted in a lower antigenemia OD value (n = 61; r = -0.4258; CI -0.62 to -0.19; p = 0.0006). Norovirus antigenemia occurred more commonly in children under 2 years of age with NoV-associated acute gastroenteritis. The occurrence of antigenemia was not correlated with stool viral load or disease severity.

A split NanoLuc complementation-based human norovirus-like particle entry assay facilitates evaluation of anti-norovirus antibodies in live cells.

Human noroviruses (NoVs) are the most common cause of acute gastroenteritis worldwide. One major obstacle in developing NoV vaccines is the lack of robust cell culture for efficacy evaluation. In this study, we successfully developed a NoV virus-like particle (VLP) entry assay based on split NanoLuc luciferase (LgBiT and HiBiT) complementation. HiBiT-tagged NoV GII.4 VLP (VLP-HiBiT) can be efficiently produced in Pichia pastoris and retain binding activity towards NoV receptor histo-blood group antigens (HBGAs). A 293T-FUT2-LgBiT cell line was established and was shown to stably express cell surface HBGAs and intracellular LgBiT. GII.4 VLP-HiBiT can bind and enter into the 293-FUT2-LgBiT cells, producing strong luminescence signals in live cells. Anti-GII.4 sera can inhibit VLP-HiBiT entry into the 293-FUT2-LgBiT cells in a dose-dependent manner, and neutralizing titers well correlate with their blocking titers measured by HBGAs-binding blockade assay. Moreover, such a surrogate infection/neutralization assay can be applied to other NoV genotypes such as GI.1 and GII.17. Together, the VLP-HiBiT entry assay can mimic both NoV attachment and internalization in live cells and thus facilitate reliable and comprehensive evaluation of NoV vaccine and antibodies.

Antigenicity and immunogenicity of HA2 and M2e influenza virus antigens conjugated to norovirus-like, VP1 capsid-based particles by the SpyTag/SpyCatcher technology.

Virus-like particles (VLPs) modified through different molecular technologies are employed as delivery vehicles or platforms for heterologous antigen display. We have recently created a norovirus (NoV) VLP platform, where two influenza antigens, the extracellular domain of matrix protein M2 (M2e) or the stem domain of the major envelope glycoprotein hemagglutinin (HA2) are displayed on the surface of the NoV VLPs by SpyTag/SpyCatcher conjugation. To demonstrate the feasibility of the platform to deliver foreign antigens, this study examined potential interference of the conjugation with induction of antibodies against conjugated M2e peptide, HA2, and NoV VLP carrier. High antibody response was induced by HA2 but not M2e decorated VLPs. Furthermore, HA2-elicited antibodies did not neutralize the homologous influenza virus in vitro. Conjugated NoV VLPs retained intact receptor binding capacity and self-immunogenicity. The results demonstrate that NoV VLPs could be simultaneously used as a platform to deliver foreign antigens and a NoV vaccine.

GII.P16-GII.2 Recombinant Norovirus VLPs Polarize Macrophages Into the M1 Phenotype for Th1 Immune Responses.

Norovirus (NoV) is a zoonotic virus that causes diarrhea in humans and animals. Outbreaks in nosocomial settings occur annually worldwide, endangering public health and causing serious social and economic burdens. The latter quarter of 2016 witnessed the emergence of the GII.P16-GII.2 recombinant norovirus throughout Asia. This genotype exhibits strong infectivity and replication characteristics, proposing its potential to initiate a pandemic. There is no vaccine against GII.P16-GII.2 recombinant norovirus, so it is necessary to design a preventive vaccine. In this study, GII.P16-GII.2 type norovirus virus-like particles (VLPs) were constructed using the baculovirus expression system and used to conduct immunizations in mice. After immunization of mice, mice were induced to produce memory T cells and specific antibodies, indicating that the VLPs induced specific cellular and humoral immune responses. Further experiments were then initiated to understand the underlying mechanisms involved in antigen presentation. Towards this, we established co-cultures between dendritic cells (DCs) or macrophages (Mø) and naïve CD4+T cells and simulated the antigen presentation process by incubation with VLPs. Thereafter, we detected changes in cell surface molecules, cytokines and related proteins. The results indicated that VLPs effectively promoted the phenotypic maturation of Mø but not DCs, as indicated by significant changes in the expression of MHC-II, costimulatory factors and related cytokines in Mø. Moreover, we found VLPs caused Mø to polarize to the M1 type and release inflammatory cytokines, thereby inducing naïve CD4+ T cells to perform Th1 immune responses. Therefore, this study reveals the mechanism of antigen presentation involving GII.P16-GII.2 recombinant norovirus VLPs, providing a theoretical basis for both understanding responses to norovirus infection as well as opportunities for vaccine development.

Norovirus: Facts and Reflections from Past, Present, and Future.

Human Norovirus is currently the main viral cause of acute gastroenteritis (AGEs) in most countries worldwide. Nearly 50 years after the discovery of the "Norwalk virus" by Kapikian and colleagues, the scientific and medical community continue to generate new knowledge on the full biological and disease spectrum of Norovirus infection. Nevertheless, several areas remain incompletely understood due to the serious constraints to effectively replicate and propagate the virus. Here, we present a narrated historic perspective and summarize our current knowledge, including insights and reflections on current points of interest for a broad medical community, including clinical and molecular epidemiology, viral-host-microbiota interactions, antivirals, and vaccine prototypes. We also include a reflection on the present and future impacts of the COVID-19 pandemic on Norovirus infection and disease.

The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update.

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

Structural Studies on the Shapeshifting Murine Norovirus.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

Identification of Human Norovirus GII.3 Blockade Antibody Epitopes.

Human noroviruses are a common pathogen causing acute gastroenteritis worldwide. Among all norovirus genotypes, GII.3 is particularly prevalent in the pediatric population. Here we report the identification of two distinct blockade antibody epitopes on the GII.3 capsid. We generated a panel of monoclonal antibodies (mAbs) from mice immunized with virus-like particle (VLP) of a GII.3 cluster 3 strain. Two of these mAbs, namely 8C7 and 8D1, specifically bound the parental GII.3 VLP but not VLPs of GII.4, GII.17, or GI.1. In addition, 8C7 and 8D1 efficiently blocked GII.3 VLP binding with its ligand, histo-blood group antigens (HBGA). These data demonstrate that 8C7 and 8D1 are GII.3-specific blockade antibodies. By using a series of chimeric VLPs, we mapped the epitopes of 8C7 and 8D1 to residues 385-400 and 401-420 of the VP1 capsid protein, respectively. These two blockade antibody epitopes are highly conserved among GII.3 cluster 3 strains. Structural modeling shows that the 8C7 epitope partially overlaps with the HBGA binding site (HBS) while the 8D1 epitope is spatially adjacent to HBS. These findings may enhance our understanding of the immunology and evolution of GII.3 noroviruses.

Interferon responses to norovirus infections: current and future perspectives.

Human noroviruses (HuNoVs) are increasingly becoming the main cause of transmissible gastroenteritis worldwide, with hundreds of thousands of deaths recorded annually. Yet, decades after their discovery, there is still no effective treatment or vaccine. Efforts aimed at developing vaccines or treatment will benefit from a greater understanding of norovirus-host interactions, including the host response to infection. In this review, we provide a concise overview of the evidence establishing the significance of type I and type III interferon (IFN) responses in the restriction of noroviruses. We also critically examine our current understanding of the molecular mechanisms of IFN induction in norovirus-infected cells, and outline the diverse strategies deployed by noroviruses to supress and/or avoid host IFN responses. It is our hope that this review will facilitate further discussion and increase interest in this area.

Immunization with Leishmania tarentolae-derived norovirus virus-like particles elicits high humoral response and stimulates the production of neutralizing antibodies.

BACKGROUND: Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. RESULTS: This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 104) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT50) were observed for 1:5-1:320 serum dilutions. CONCLUSIONS: Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.