Rotavirus VP6 Adjuvant Effect on Norovirus GII.4 Virus-Like Particle Uptake and Presentation by Bone Marrow-Derived Dendritic Cells in Vitro and in Vivo

We have previously shown that rotavirus (RV) inner capsid protein VP6 has an adjuvant effect on norovirus (NoV) virus-like particle- (VLP-) induced immune responses and studied the adjuvant mechanism in immortalized cell lines used as antigen-presenting cells (APCs). Here, we investigated the uptake and presentation of RV VP6 and NoV GII.4 VLPs by primary bone marrow-derived dendritic cells (BMDCs). The adjuvant effect of VP6 on GII.4 VLP presentation and NoV-specific immune response induction by BMDC in vivo was also studied. Intracellular staining demonstrated that BMDCs internalized both antigens, but VP6 more efficiently than NoV VLPs. Both antigens were processed and presented to antigen-primed T cells, which responded by robust interferon γ secretion. When GII.4 VLPs and VP6 were mixed in the same pulsing reaction, a subpopulation of the cells had uptaken both antigens. Furthermore, VP6 copulsing increased GII.4 VLP uptake by 37% and activated BMDCs to secrete 2-5-fold increased levels of interleukin 6 and tumor necrosis factor α compared to VLP pulsing alone. When in vitro-pulsed BMDCs were transferred to syngeneic BALB/c mice, VP6 improved NoV-specific antibody responses. The results of this study support the earlier findings of VP6 adjuvant effect in vitro and in vivo

Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)3) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses

Modular vaccine platform based on the norovirus-like particle

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.publishedVersionPeer reviewe

Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)3) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses

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.publishedVersionPeer reviewe

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.publishedVersionPeer reviewe