High immunogenicity of virus-like particles (VLPs) decorated with Aeromonas salmonicida VapA antigen in rainbow trout.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Preclinical Efficacy of a Capsid Virus-like Particle-Based Vaccine Targeting IL-1ß for Treatment of Allergic Contact Dermatitis.

Hypersensitivity to a contact allergen is one of the most abundant forms of inflammatory skin disease. Today, more than 20% of the general population are sensitized to one or more contact allergens, making this disease an important healthcare issue, as re-exposure to the allergen can initiate the clinical disease termed allergic contact dermatitis (ACD). The current standard treatment using corticosteroids is effective, but it has side effects when used for longer periods. Therefore, there is a need for new alternative therapies for severe ACD. In this study, we used the versatile Tag/Catcher AP205 capsid virus-like particle (cVLP) vaccine platform to develop an IL-1ß-targeted vaccine and to assess the immunogenicity and in vivo efficacy of the vaccine in a translational mouse model of ACD. We show that vaccination with cVLPs displaying full-length murine IL-1ß elicits high titers of neutralizing antibodies, leading to a significant reduction in local IL-1ß levels as well as clinical symptoms induced by treatment with 1-Fluoro-2,4-dinitrobenzene (DNFB). Moreover, we show that a single amino acid mutation in muIL-1ß reduces the biological activity while maintaining the ability to induce neutralizing antibodies. Collectively, the data suggest that a cVLP-based vaccine displaying full-length IL-1ß represents a promising vaccine candidate for use as an alternative treatment modality against severe ACD.

The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization.

Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson's disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

A cVLP-Based Vaccine Displaying Full-Length PCSK9 Elicits a Higher Reduction in Plasma PCSK9 Than Similar Peptide-Based cVLP Vaccines.

Administration of PCSK9-specific monoclonal antibodies, as well as peptide-based PCSK9 vaccines, can lower plasma LDL cholesterol by blocking PCSK9. However, these treatments also cause an increase in plasma PCSK9 levels, presumably due to the formation of immune complexes. Here, we utilize a versatile capsid virus-like particle (cVLP)-based vaccine platform to deliver both full-length (FL) PCSK9 and PCSK9-derived peptide antigens, to investigate whether induction of a broader polyclonal anti-PCSK9 antibody response would mediate more efficient clearance of plasma PCSK9. This head-to-head immunization study reveals a significantly increased capacity of the FL PCSK9 cVLP vaccine to opsonize and clear plasma PCSK9. These findings may have implications for the design of PCSK9 and other vaccines that should effectively mediate opsonization and immune clearance of target antigens.

Head-to-Head Comparison of Modular Vaccines Developed Using Different Capsid Virus-Like Particle Backbones and Antigen Conjugation Systems.

Capsid virus-like particles (cVLPs) are used as molecular scaffolds to increase the immunogenicity of displayed antigens. Modular platforms have been developed whereby antigens are attached to the surface of pre-assembled cVLPs. However, it remains unknown to what extent the employed cVLP backbone and conjugation system may influence the immune response elicited against the displayed antigen. Here, we performed a head-to-head comparison of antigen-specific IgG responses elicited by modular cVLP-vaccines differing by their employed cVLP backbone or conjugation system, respectively. Covalent antigen conjugation (i.e., employing the SpyTag/SpyCatcher system) resulted in significantly higher antigen-specific IgG titers compared to when using affinity-based conjugation (i.e., using biotin/streptavidin). The cVLP backbone also influenced the antigen-specific IgG response. Specifically, vaccines based on the bacteriophage AP205 cVLP elicited significantly higher antigen-specific IgG compared to corresponding vaccines using the human papillomavirus major capsid protein (HPV L1) cVLP. In addition, the AP205 cVLP platform mediated induction of antigen-specific IgG with a different subclass profile (i.e., higher IgG2a and IgG2b) compared to HPV L1 cVLP. These results demonstrate that the cVLP backbone and conjugation system can individually affect the IgG response elicited against a displayed antigen. These data will aid the understanding and process of tailoring modular cVLP vaccines to achieve improved immune responses.

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens.

Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity.

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.

Advantages and Prospects of Tag/Catcher Mediated Antigen Display on Capsid-Like Particle-Based Vaccines.

Capsid-like particles (CLPs) are multimeric, repetitive assemblies of recombinant viral capsid proteins, which are highly immunogenic due to their structural similarity to wild-type viruses. CLPs can be used as molecular scaffolds to enable the presentation of soluble vaccine antigens in a similar structural format, which can significantly increase the immunogenicity of the antigen. CLP-based antigen display can be obtained by various genetic and modular conjugation methods. However, these vary in their versatility as well as efficiency in achieving an immunogenic antigen display. Here, we make a comparative review of the major CLP-based antigen display technologies. The Tag/Catcher-AP205 platform is highlighted as a particularly versatile and efficient technology that offers new qualitative and practical advantages in designing modular CLP vaccines. Finally, we discuss how split-protein Tag/Catcher conjugation systems can help to further propagate and enhance modular CLP vaccine designs.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.