Expression of influenza A virus-derived peptides on a rotavirus VP6-based delivery platform.

Recombinant protein technology enables the engineering of modern vaccines composed of a carrier protein displaying poorly immunogenic heterologous antigens. One promising carrier is based on the rotavirus inner-capsid VP6 protein. We explored different VP6 insertion sites for the presentation of two peptides (23 and 140 amino acids) derived from the M2 and HA genes of influenza A virus. Both termini and three surface loops of VP6 were successfully exploited as genetic fusion sites, as demonstrated by the expression of the fusion proteins. However, further studies are needed to assess the morphology and immunogenicity of these constructs.

Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression.

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.

Experimental VLP vaccine displaying a furin antigen elicits production of autoantibodies and is well tolerated in mice.

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.

Comparison of structure and immunogenicity of CVB1-VLP and inactivated CVB1 vaccine candidates.

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.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion.

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.

Fusion Protein of Rotavirus VP6 and SARS-CoV-2 Receptor Binding Domain Induces T Cell Responses.

Vaccines based on mRNA and viral vectors are currently used in the frontline to combat the ongoing pandemic caused by the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, there is still an urgent need for alternative vaccine technologies inducing/boosting long-lasting and cross-reactive immunity in different populations. As a possible vaccine candidate, we employed the rotavirus VP6-protein platform to construct a fusion protein (FP) displaying receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) at the N-terminus of VP6. The recombinant baculovirus-insect cell produced VP6-RBD FP was proven antigenic in vitro and bound to the human angiotensin-converting enzyme 2 (hACE2) receptor. The FP was used to immunize BALB/c mice, and humoral- and T cell-mediated immune responses were investigated. SARS-CoV-2 RBD-specific T cells were induced at a high quantity; however, no RBD or S-specific antibodies were detected. The results suggest that conformational B cell epitopes might be buried inside the VP6, while RBD-specific T cell epitopes are available for T cell recognition after the processing and presentation of FP by the antigen-presenting cells. Further immunogenicity studies are needed to confirm these findings and to assess whether, under different experimental conditions, the VP6 platform may present SARS-CoV-2 antigens to B cells as well.

Internalization and antigen presentation by mouse dendritic cells of rotavirus VP6 preparations differing in nanostructure.

Nanoparticles are highly immunogenic due to the multivalent, repetitive antigen expression and direct activation of antigen presenting cells (APCs), key players of adaptive immune responses. Different virus-like particles (VLPs) have been used as display platforms to amplify immune responses to biologically relevant, but poorly immunogenic foreign antigens. A candidate platform based on rotavirus (RV) inner-capsid protein VP6 oligomers, such as nanotubes (T-VP6) and nanospheres (S-VP6), is also considered. Different VP6 nanostructures were compared for internalization and antigen presentation by the APCs. We found, that a lack of a high-order structures, T-VP6 and S-VP6, did not negatively affect VP6 uptake and presentation by murine bone-marrow derived dendritic cells (BMDCs) in vitro. Furthermore, T-VP6 was preferable to norovirus (NoV) VLPs for BMDC internalization resulting in significantly higher VP6-specific immune responses when T-VP6 and NoV VLP pulsed BMDCs were transferred to syngeneic mice. These results support the use of different VP6 nanostructures as foreign antigen delivery platforms.