Enteroviruses, including coxsackievirus B1 (CVB1), cause severe diseases such as myocarditis and meningitis, but vaccines are lacking for most enteroviruses. Conserved and immunodominant epitopes, such as VP4 region and VP1 N-terminus may limit vaccine efficacy by inducing non-neutralizing antibody responses. Virus-like particles (VLPs) mimic native viruses without genetic material and can be engineered to exclude epitopes. To address these challenges, we developed a CVB1-VLP lacking VP4. Sequence conservation of CVB VP4 protein and the VP1 N-terminal PALXA region was assessed, and BALB/c mice were sequentially immunized with different formalin inactivated CVB vaccines. VLPΔVP4 was produced using baculovirus-insect cell expression system, was purified, and characterized by SDS-PAGE, transmission electron microscopy, dynamic light scattering, cryogenic electron microscopy, three-dimensional image reconstruction and atomic modelling. VLPΔVP4 stability was monitored over five years at 8 °C. Comprehensive preclinical experiments were conducted in mice with VLPΔVP4, VLPΔpalxa and inactivated CVB1. Vaccine immunogenicity was evaluated by neutralization assay, ELISA, ELISpot, and in vitro infection assays. VP4- and PALXA-regions were conserved among CVB serotypes and sequential mouse vaccinations confirmed the induction of antibodies against these regions, that should be avoided in vaccination. VLPΔVP4 exhibited > 95

Current COVID-19 vaccines are effective at preventing severe disease but provide limited protection against infection and transmission, particularly as new variants emerge. Vaccines capable of inducing both systemic and mucosal immunity and robust T cell responses, may offer broader and more long-lasting protection. This study aimed to evaluate protein-based vaccine candidates incorporating SARS-CoV-2 spike (S) and its receptor-binding domain (RBD), as well as nucleocapsid protein (N) antigens administered through different immunization schemes. Mice were immunized three times at four-week intervals with vaccine formulations containing Fc-fused RBD proteins, S, and/or N proteins. Vaccines were administered intranasally, subcutaneously, or with subcutaneous or intramuscular priming followed by intranasal boosting. Branched polyethylenimine (BPEI) was used as a mucosal adjuvant, and Adjuvant system 04 (AS04) for intramuscular administration. Depending on the experiment, BPEI or AS04 was used for subcutaneous immunizations. Systemic antibody responses were assessed from serum samples and mucosal antibody responses from bronchoalveolar lavage samples by ELISA. Cellular responses were measured from splenocytes after antigen stimulation by FluoroSpot analysis of cytokine secretion. Fc-fused RBD antigens elicited higher antibody responses than whole S protein. Inclusion of low amount of N protein enhanced RBD- and S-specific systemic and mucosal IgG and IgA responses, and significantly increased splenocyte IL-2 and IFN-γ secretion. Intranasal vaccination alone induced variable mucosal antibody responses, whereas intramuscular priming followed by intranasal boosting consistently produced higher systemic IgG levels, robust mucosal responses, and T cell activity. Neutralizing antibodies were negligible in intranasally primed groups but were detectable in most animals receiving intramuscular priming. Among all regimens, the combination of intramuscular priming with N-containing formulations generated the highest magnitude and breadth of humoral and cellular responses. All vaccine formulations were well tolerated with no adverse effects observed. Protein-based vaccines incorporating N together with Fc-fused RBD antigens significantly broaden and enhance immune responses in mice. Intramuscular priming followed by intranasal boosting proved superior to other regimens, inducing strong systemic, mucosal, and cellular immunity. These findings suggest that inclusion of conserved internal virus antigens and heterologous prime-boost strategies may improve durability and breadth of protection, supporting their development as next-generation COVID-19 vaccines.

Enteroviruses greatly affect public health worldwide. Therefore, methods to reliably and quickly identify virus infections and screen treatments or preventatives against them are needed. Here, we describe protocols for analyzing the concentrations of infective enteroviruses and neutralizing enterovirus antibodies and the efficacy of antiviral compounds against enteroviruses and compound cytotoxicity with a cell-based method in a microtiter plate format accompanied with python scripts for data and statistical analyses. Detailed instructions for performing the methods and running the scripts are provided, and scripts are available in a public repository. The assays described here are faster and less labor intensive than state-of-the-art methods, producing results in less than 72 h. Developed scripts can reduce the amount of manual handling of the data and calculations, enabling analysis of large datasets without the use of commercial software. The developed methods can be utilized by researchers and healthcare providers for diagnostic and screening purposes for viruses growing in cell cultures.

Coxsackievirus B1 (CVB1) is a common cause of acute and chronic myocarditis, cardiomyopathy, and meningitis. CVBs replicate in mucosal membranes. Therefore, vaccines inducing robust mucosal immune responses are needed. We investigated the immunogenicity of virus-like particles (VLP) and inactivated virus vaccines for CVB1, administered to mice either subcutaneously or intranasally, formulated with and without commercial and an experimental adjuvant. In this study, epigallocatechin-3-gallate (EGCG) was used both as a potential adjuvant and as an inactivating agent. EGCG adjuvanted CVB1-VLP enhanced immunogenicity via the parenteral route, but not intranasally. EGCG-adjuvanted and non-adjuvanted CVB1-VLPs triggered an immune response after intranasal administration, although the response remained weak. Intranasal administration of formalin-inactivated virus elicited robust CVB1-specific humoral, cellular, and mucosal immune responses, but after EGCG-inactivation, the mucosal antibody response was lower than after formalin-inactivation. To identify the link between structure and mucosal immunogenicity, we solved the structures of CVB1-VLP and formalin-inactivated CVB1 virus at resolutions ranging from 2.15 to 4.1 Å. The structural difference between VLP and formalin-inactivated CVB1 was the presence of the genome and cross-linked amino acid residues in the formalin-inactivated virus. Formalin-inactivated CVB1 vaccine shows promise for mucosal immunizations and the structural data supports the development of next-generation VLP-vaccines in the future.

Enteroviruses, including Coxsackie B (CVB) viruses, can cause severe diseases such as myocarditis, pancreatitis, and meningitis. Vaccines can prevent these complications, but conserved non-neutralizing epitopes in the viral capsid may limit their effectiveness. The immunodominant PALXAXETG motif, located in the VP1 N-terminus, is a highly conserved region in enteroviruses that elicits non-neutralizing antibody responses. Virus-like particles (VLPs) offer a safe and effective vaccine platform because of their structural similarity to native viruses but lack viral genetic material. Importantly, VLPs can be structurally modified to exclude specific epitopes. Here, we produced a modified CVB1 virus-like particle (VLP) vaccine lacking 15 amino acids from the PALXAXETG motif (designated VLPΔpalxa) using the baculovirus-insect cell expression system. To confirm the structural integrity, we determined the crystal structure of the modified VLP with 3.2 Å resolution. We then conducted comprehensive immunogenicity studies in mice, including dose titration, comparison of two versus three immunizations, and post-vaccination viral challenge. In addition, we evaluated the impact of the AS04 adjuvant on the immunogenicity of unmodified and modified CVB1-VLP vaccines and the formalin-inactivated CVB1 vaccine. The yield of CVB1-VLPΔpalxa was 29.5 mg/L, and the particles were shown to assemble similarly to unmodified CVB1-VLP. CVB1-VLPΔpalxa induced robust antibody responses, with neutralizing antibody titres comparable to or exceeding those elicited by unmodified VLP or inactivated virus vaccines. A 2 µg dose was identified as optimal, providing the highest neutralizing antibody titres. A third immunization significantly increased antibody levels, and all non-adjuvanted vaccines protected the mice from CVB1 challenge after the third dose. The addition of AS04 significantly enhanced the antibody response, particularly in both VLP groups. We demonstrated that with targeted structural modification of the CVB1-VLP capsid, immunodominant antibody responses against the conserved PALXAXETG motif can be avoided. We demonstrate that structural modification of CVB1-VLP is a viable strategy. Since the deleted epitope is known to be non-neutralizing, its deletion may help focus the immune response on more protective targets and thereby improve vaccine efficacy. The modified VLPs, particularly when adjuvanted, offer a promising approach for developing safe and effective enterovirus vaccines.

Proprotein convertase (PCSK) enzymes serve a wide range of regulatory roles in mammals, for example in metabolism and immunity, and altered activity of PCSKs is associated with disorders, such as cardiovascular disease and cancer. Inhibition of PCSK9 activity with therapeutic antibodies or small interfering RNAs is used in the clinic to lower blood cholesterol, and RNA interference -based silencing of FURIN (PCSK3) is being evaluated in clinical trials as a cancer treatment. Inhibiting these proteins through vaccine-induced autoantibodies could be a patient-friendly way to reduce the frequency of intervention and the overall price of treatment. Here, we show that a self-directed immune response against PCSK9 and furin can be generated in mice by presenting fragments of the proteins on norovirus-like particles (noro-VLPs). We genetically fused three PCSK peptides and the P domain of furin to the SpyCatcher linker protein and covalently conjugated them on noro-VLPs via SpyCatcher/SpyTag linkage. Both PCSK9 peptides and the furin P domain generated antigen specific IgGs even without conventional adjuvants. Importantly, vaccinating against furin did not cause adverse events or immune-mediated inflammatory disease. This study adds further support for the feasibility of VLP-based anti-PCSK9 vaccines and shows that the same principles can be applied to make novel vaccine candidates against other endogenous proteins such as furin. We also demonstrate that the noro-VLP can be used as a vaccine platform for presenting self-antigens.

Enteroviruses (EVs) are the most prevalent viruses in humans. EVs can cause a range of acute symptoms, from mild common colds to severe systemic infections such as meningitis, myocarditis, and flaccid paralysis. They can also lead to chronic diseases such as cardiomyopathy. Although more than 280 human EV serotypes exist, only four serotypes have licenced vaccines. No antiviral drugs are available to treat EV infections, and global surveillance of EVs has not been effectively coordinated. Therefore, poliovirus still circulates, and there have been alarming epidemics of non-polio enteroviruses. Thus, there is a pressing need for coordinated preparedness efforts against EVs.This review provides a perspective on recent enterovirus outbreaks and global poliovirus eradication efforts with continuous vaccine development initiatives. It also provides insights into the challenges and opportunities in EV vaccine development. Given that traditional whole-virus vaccine technologies are not suitable for many clinically relevant EVs and considering the ongoing risk of enterovirus outbreaks and the potential for new emerging pathogenic strains, the need for new effective and adaptable enterovirus vaccines is emphasized.This review also explores the difficulties in translating promising vaccine candidates for clinical use and summarizes information from published literature and clinical trial databases focusing on existing enterovirus vaccines, ongoing clinical trials, the obstacles faced in vaccine development as well as the emergence of new vaccine technologies. Overall, this review contributes to the understanding of enterovirus vaccines, their role in public health, and their significance as a tool for future preparedness.

Coxsackievirus B1 (CVB1) is a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. In this study, we investigated the immunogenicity of virus-like particle (VLP) and inactivated whole-virus vaccines for CVB1 when administrated to mice via either subcutaneous or intranasal routes formulated with and without commercial and experimental adjuvants. Here, the potential of utilizing epigallocatechin-3-gallate (EGCG) as a mucosal adjuvant synergistically with its ability to inactivate the virus were investigated. EGCG had promising adjuvant properties for CVB1-VLP when administered via the parenteral route but limited efficacy via intranasal administration. However, intranasal administration of the formalin-inactivated virus induced high CVB1-specific humoral, cellular, and mucosal immune responses. Also, based on CVB1-specific IgG-antibody responses, we conclude that CVB1-VLP can be taken up by immune cells when administrated intranasally and further structural engineering for the VLP may increase the mucosal immunogenicity. The preparations contained mixtures of compact and expanded A particles with 85% expanded in the formalin-inactivated virus, but only 52% in the VLP observed by cryogenic electron microscopy. To correlate the structure to immunogenicity, we solved the structures of the CVB1-VLP and the formalin-inactivated CVB1 virus at resolutions ranging from 2.15 A to 4.1 A for the expanded and compact VLP and virus particles by image reconstruction. These structures can be used in designing mutations increasing the stability and immunogenicity of CVB1-VLP in the future. Overall, our results highlight the potential of using formalin inactivated CVB1 vaccine in mucosal immunization programs and provide important information for future development of VLP-based vaccines against all enteroviruses.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

Coxsackievirus Bs (CVBs) are common human RNA viruses that belong to the enterovirus genus of Picornaviridae. Most commonly, infections with these viruses manifest with mild flu-like symptoms. However, CVBs are also known to cause severe diseases, including aseptic meningitis, myocarditis, and chronic dilated cardiomyopathy (DCM), encephalitis, pancreatitis, and hand-foot-and-mouth disease. In this study, we aimed for a versatile characterization of a modified CVB virus-like particle vaccine in a murine model. The modified CVB-VLP was produced using baculovirus-insect cell production platform. Purified and characterized vaccine was administered to mice via s.c. or i.m. routes, either with or without an adjuvant. Immunological characterization of vaccine response included both humoral and cellular characterization. Antigen-specific IgG levels and neutralizing antibodies in the sera were measured to determine the humoral immune response. To characterize the cellular immunity, activation markers of the lymphocytes were identified using flow cytometry. Additionally, the cytokine secretion of the stimulated splenocytes was determined with FluoroSpot assay. The modified CVB-VLP induced high levels of IgG and neutralizing antibodies in the sera of vaccinated mice. Further, the in vitro stimulation of splenocytes induced activation of mainly CD4+ T-cells, and adjuvanted vaccine induced a significant production level of IFN-γ and IL-2. These results indicate that the virus-like particle combined with an adjuvant is a promising vaccine candidate. In addition, our current challenge study with coxsackie B1 virus will shed us more insight on the protective role of this vaccine. Supported by grants from Finnish Foundations (Instrumentarium Science Foundation, Paulo Foundation and Ida Montini Foundation)

Coronaviruses have caused three deadly human outbreaks over the last 20 years, the latest causing the COVID-19 pandemic which has now lasted over three years. The animal reservoir of coronaviruses is extensive, and history has shown that coronaviruses are able to crossover from animals to humans. Thus, there is a clear need for a universal coronavirus vaccine. In this study, we produced S1 subunits of spike proteins of six different human coronaviruses fused to the antibody Fc fragment in HEK293 cells. In addition, we produced another coronavirus protein (CP) in High Five™ insect cells. The vaccine compositions included either the six S1-Fc or the six S1-Fc together with CP, and mice were immunized either intranasally or with a combination method utilizing both subcutaneous and intranasal injections. Our results showed that both vaccine compositions elicited both humoral and cellular immune responses in mice. Antigen-specific IgG levels were higher in mice immunized solely intranasally but using a combination of different immunization routes seemed to boost cellular immune responses. As a conclusion, all the vaccine compositions were highly immunogenic and generated both antibody and T cell responses in mice. Further studies are needed to optimize the immunization scheme. Additionally in the future, in order to better characterize the T cell responses generated by intranasal immunization, the nasal associated lymphoid tissue and cervical lymph nodes from the mice will be collected and analyzed. Supported by a grant from The Research Foundation of the Pulmonary Diseases (Finland)

Introduction Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher. Methods To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice. Results and discussion We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.

Viral respiratory tract infections exacerbate airway disease and facilitate life-threatening bacterial colonization in cystic fibrosis (CF). Annual influenza vaccination is recommended and vaccines against other common respiratory viruses may further reduce pulmonary morbidity risk. Enteroviruses have been found in nasopharyngeal samples from CF patients experiencing pulmonary exacerbations. Using serology tests, we found that infections by a group of enteroviruses, Coxsackievirus Bs (CVBs), are prevalent in CF. We next showed that a CVB vaccine, currently undergoing clinical development, prevents infection and CVB-instigated lung damage in a murine model of CF. Finally, we demonstrate that individuals with CF have normal vaccine responses to a similar, commonly used enterovirus vaccine (inactivated poliovirus vaccine). Our study demonstrates that CVB infections are common in CF and provides experimental evidence indicating that CVB vaccines could be efficacious in the CF population. The role of CVB infections in contributing to pulmonary exacerbations in CF should be further studied.

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.

Viruses play a major role in modern society and create risks from global pandemics and bioterrorism to challenges in agriculture. Virus infectivity assays and genome copy number determination methods are often used to obtain information on virus preparations used in diagnostics and vaccine development. However, these methods do not provide information on virus particle count. Current methods to measure the number of viral particles are often cumbersome and require highly purified virus preparations and expensive instrumentation. To tackle these problems, we developed a simple and cost-effective time-resolved luminescence-based method for virus particle quantification. This mix-and-measure technique is based on the recognition of the virus particles by an external Eu3+-peptide probe, providing results on virus count in minutes. The method enables the detection of non-enveloped and enveloped viruses, having over tenfold higher detectability for enveloped, dynamic range from 5E6 to 3E10 vp/mL, than non-enveloped viruses. Multiple non-enveloped and enveloped viruses were used to demonstrate the functionality and robustness of the Protein-Probe method.

Enteroviruses, including the Coxsackievirus Bs (CVB), have been implicated as causal agents in human type 1 diabetes. Immunization of at-risk individuals with a CVB vaccine provides an attractive strategy for elucidating the role of CVBs in the disease etiology. Previously we have shown that an inactivated whole-virus vaccine covering all CVB serotypes (CVB1-6) is safe to administer and highly immunogenic in preclinical models, including non-human primates. Before initiating clinical trials with this type of vaccine it was also important to address whether a) the vaccine itself induces adverse immune reactions including accelerating diabetes onset in a diabetes prone host and b) the vaccine can prevent CVB induced diabetes in a well-established disease model. Here we present results from studies in which female NOD mice were left untreated, mock-vaccinated or vaccinated with CVB1-6 vaccine and monitored for insulitis occurrence or diabetes development. We demonstrate that vaccination induces virus neutralizing antibodies without altering insulitis scores or the onset of diabetes. We also show that NOD mice vaccinated with a CVB1 vaccine are protected from CVB-induced accelerated disease onset. Taken together, these studies show that CVB vaccines do not alter islet inflammation or accelerate disease progression in an animal model that spontaneously develops autoimmune type 1 diabetes. However, they can prevent CVB-mediated disease progression in the same model.

Enteroviruses, including the Coxsackievirus Bs (CVB), have been implicated as causal agents in human type 1 diabetes. Immunization of at-risk individuals with a CVB vaccine provides an attractive strategy for elucidating the role of CVBs in the disease etiology. Previously we have shown that an inactivated whole-virus vaccine covering all CVB serotypes (CVB1-6) is safe to administer and highly immunogenic in preclinical models, including non-human primates. Before initiating clinical trials with this type of vaccine it was also important to address whether a) the vaccine itself induces adverse immune reactions including accelerating diabetes onset in a diabetes prone host and b) the vaccine can prevent CVB induced diabetes in a well-established disease model. Here we present results from studies in which female NOD mice were left untreated, mock-vaccinated or vaccinated with CVB1-6 vaccine and monitored for insulitis occurrence or diabetes development. We demonstrate that vaccination induces virus neutralizing antibodies without altering insulitis scores or the onset of diabetes. We also show that NOD mice vaccinated with a CVB1 vaccine are protected from CVB-induced accelerated disease onset. Taken together, these studies show that CVB vaccines do not alter islet inflammation or accelerate disease progression in an animal model that spontaneously develops autoimmune type 1 diabetes. However, they can prevent CVB-mediated disease progression in the same model.

BackgroundVirus-like particle (VLP) vaccines have recently emerged as a safe and effective alternative to conventional vaccine technologies. The strong immunogenic effects of VLPs can be harnessed for making vaccines against any pathogen by decorating VLPs with antigens from the pathogen. Producing the antigenic pathogen fragments and the VLP platform separately makes vaccine development rapid and convenient. Here we decorated the norovirus-like particle with two conserved influenza antigens and tested for the immunogenicity of the vaccine candidates in BALB/c mice.ResultsSpyTagged noro-VLP was expressed with high efficiency in insect cells and purified using industrially scalable methods. Like the native noro-VLP, SpyTagged noro-VLP is stable for months when refrigerated in a physiological buffer. The conserved influenza antigens were produced separately as SpyCatcher fusions in E. coli before covalent conjugation on the surface of noro-VLP. The noro-VLP had a high adjuvant effect, inducing high titers of antibody production against the antigens presented on its surface.ConclusionsThe modular noro-VLP vaccine platform presented here offers a rapid, convenient and safe method to present various soluble protein antigens to the immune system for vaccination and antibody production purposes.

Coxsackievirus B (CVB) enteroviruses are common human pathogens known to cause severe diseases including myocarditis, chronic dilated cardiomyopathy, and aseptic meningitis. CVBs are also hypothesized to be a causal factor in type 1 diabetes. Vaccines against CVBs are not currently available, and here we describe the generation and preclinical testing of a novel hexavalent vaccine targeting the six known CVB serotypes. We show that the vaccine has an excellent safety profile in murine models and nonhuman primates and that it induces strong neutralizing antibody responses to the six serotypes in both species without an adjuvant. We also demonstrate that the vaccine provides immunity against acute CVB infections in mice, including CVB infections known to cause virus-induced myocarditis. In addition, it blocks CVB-induced diabetes in a genetically permissive mouse model. Our preclinical proof-of-concept studies demonstrate the successful generation of a promising hexavalent CVB vaccine with high immunogenicity capable of preventing CVB-induced diseases.