Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations.
ABSTRACT: Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 "Consensus" VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)(3)) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.
Project description:Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.
Project description:Norovirus (NoV) genogroup I (GI) and GII are responsible for most human infections with NoV. Because of the high genetic variability of NoV, natural infection does not induce sufficient protective immunity to different genotypes or to variants of the same genotype and there is little or no cross-protection against different genogroups. NoV-derived virus-like particles (VLPs) are promising vaccine candidates that induce high levels of NoV-specific humoral and cellular immune responses. It is believed that a bivalent NoV vaccine consisting of a representative VLP from GI and GII is a minimum requirement for an effective vaccine. Here, we compared the abilities of monovalent immunizations with NoV GI.1-2001, GI.3-2002, GII.4-1999, and GII.4-2010 New Orleans VLPs to induce NoV type-specific and cross-reactive immune responses and protective blocking antibody responses in BALB/c mice. All of the VLPs induced comparable levels of type-specific serum IgG antibodies, as well as blocking antibodies to the VLPs used for immunization. However, the abilities of different VLP genotypes to induce cross-reactive IgG and cross-blocking antibodies varied remarkably. Our results confirm previous findings of a lack of cross-protective immune responses between GI and GII NoVs. These data support the rationale for including NoV GI.3 and GII.4-1999 VLPs in the bivalent vaccine formulation, which could be sufficient to induce protective immune responses across NoV genotypes in the two common genogroups in humans.
Project description:Norovirus is the primary cause of viral gastroenteritis in humans with multiple genotypes currently circulating worldwide. The development of a successful norovirus vaccine is contingent on its ability to induce both systemic and mucosal antibody responses against a wide range of norovirus genotypes. Norovirus virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered intranasally. Incorporation of these VLPs into an intranasal powder vaccine offers the advantage of simplicity and induction of neutralizing systemic and mucosal antibodies. Nasal immunization, which provides the advantage of ease of administration and a mucosal delivery mechanism, faces the real issue of limited nasal residence time due to mucociliary clearance. Herein, we describe a novel dry powder (GelVac™) formulation of GI or GII.4 norovirus VLPs, two dominant circulating genotypes, to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs. Systemic and mucosal immunogenicity of each of the VLPs was observed in a dose-dependent manner. In addition, a boosting effect was observed after the second dosing of each VLP antigen. With the GelVac™ formulation, a total antigen dose of ? 15 ?g was determined to be the maximally immunogenic dose for both GI and GII.4 norovirus VLPs based on evaluation for 56 days. Taken together, these results indicate that norovirus VLPs could be used as potential vaccine candidates without using an immunostimulatory adjuvant and provide a basis for the development of a GelVac™ bivalent GI/GII.4 norovirus VLP vaccine.
Project description:The global health community is beginning to understand the burden of norovirus-associated disease, which has a significant impact in both developed and developing countries. Norovirus virus like particle (VLP)-based vaccines are currently under development and have been shown to elicit systemic and mucosal immune responses when delivered intranasally. In the present study, we describe the use of a dry powder formulation (GelVac™) with an in situ gelling polysaccharide (GelSite™) extracted from Aloe vera for nasal delivery of a bivalent vaccine formulation containing both GI and GII.4 norovirus VLPs. Dose-ranging studies were performed to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs and determine any antigenic interference. A dose-dependent increase in systemic and mucosal immunogenicity against each of the VLPs were observed as well as a boosting effect for each VLP after the second dosing. A total antigen dose of ?50 ?g of each GI and GII.4 VLPs was determined to be the maximally immunogenic dose in guinea pigs. The immunogenicity results of this bivalent formulation, taken together with previous work on monovalent GelVac™ norovirus vaccine formulation, provides a basis for future development of this norovirus VLP vaccine.
Project description:INTRODUCTION:Noroviruses are the leading cause of foodborne illness worldwide, account for approximately one-fifth of acute gastroenteritis (AGE) cases globally, and cause a substantial economic burden. Candidate norovirus vaccines are in development, but there is currently no licensed vaccine. AREAS COVERED:Noroviruses cause approximately 684 million cases and 212,000 deaths per year across all age groups, though burden estimates vary by study and region. Challenges to vaccine research include substantial and rapidly evolving genetic diversity, short-term and homotypic immunity to infection, and the absence of a single, well-established correlate of protection. Nonetheless, several norovirus vaccine candidates are currently in development, utilizing virus-like particles (VLPs), P particles, and recombinant adenoviruses. Of these, a bivalent GI.1/GII.4 VLP-based intramuscular vaccine (Phase IIb) and GI.1 oral vaccine (Phase I) are in clinical trials. EXPERT COMMENTARY:A norovirus vaccine should target high-risk populations, including the young and the elderly, and protect them against the most common circulating norovirus strains. A norovirus vaccine would be a powerful tool in the prevention and control of norovirus while lessening the burden of AGE worldwide. However, more robust burden and cost estimates are needed to justify investments in and guide norovirus vaccine development.
Project description:Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ?10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ?15 Å off the shell domain and rotated ?40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.
Project description:Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.
Project description:Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. A virus-like particle (VLP) candidate vaccine induces the production of serum histo-blood group antigen (HBGA)-blocking antibodies, the first identified correlate of protection from HuNoV gastroenteritis. Recently, virus-specific IgG memory B cells were identified to be another potential correlate of protection against HuNoV gastroenteritis. We assessed B-cell responses following intramuscular administration of a bivalent (genogroup I, genotype 1 [GI.1]/genogroup II, genotype 4 [GII.4]) VLP vaccine using protocols identical to those used to evaluate cellular immunity following experimental GI.1 HuNoV infection. The kinetics and magnitude of cellular immunity to G1.1 infection were compared to those after VLP vaccination. Intramuscular immunization with the bivalent VLP vaccine induced the production of antibody-secreting cells (ASCs) and memory B cells. ASC responses peaked at day 7 after the first dose of vaccine and returned to nearly baseline levels by day 28. Minimal increases in ASCs were seen after a second vaccine dose at day 28. Antigen-specific IgG memory B cells persisted at day 180 postvaccination for both GI.1 and GII.4 VLPs. The overall trends in B-cell responses to vaccination were similar to the trends in the responses to infection, where there was a greater bias of an ASC response toward IgA and a memory B-cell response to IgG. The magnitude of the ASC and memory B-cell responses to the GI.1 VLP component of the vaccine was also comparable to that of the responses following GI.1 infection. The production of IgG memory B cells and persistence at day 180 is a key finding and underscores the need for future studies to determine if IgG memory B cells are a correlate of protection following vaccination. (This study has been registered at ClinicalTrials.gov under registration no. NCT01168401.).
Project description:There is currently no licensed vaccine for noroviruses, and development is hindered, in part, by an incomplete understanding of the host adaptive immune response to these highly heterogeneous viruses and rapid GII.4 norovirus molecular evolution. Emergence of a new predominant GII.4 norovirus strain occurs every 2 to 4 years. To address the problem of GII.4 antigenic variation, we tested the hypothesis that chimeric virus-like particle (VLP)-based vaccine platforms, which incorporate antigenic determinants from multiple strains into a single genetic background, will elicit a broader immune response against contemporary and emergent strains. Here, we compare the immune response generated by chimeric VLPs to that of parental strains and a multivalent VLP cocktail. Results demonstrate that chimeric VLPs induce a more broadly cross-blocking immune response than single parental VLPs and a similar response to a multivalent GII.4 VLP cocktail. Furthermore, we show that incorporating epitope site A alone from one strain into the background of another is sufficient to induce a blockade response against the strain donating epitope site A. This suggests a mechanism by which population-wide surveillance of mutations in a single epitope could be used to evaluate antigenic changes in order to identify potential emergent strains and quickly reformulate vaccines against future epidemic strains as they emerge in human populations.Noroviruses are gastrointestinal pathogens that infect an estimated 21 million people per year in the United States alone. GII.4 noroviruses account for >70% of all outbreaks, making them the most clinically important genotype. GII.4 noroviruses undergo a pattern of epochal evolution, resulting in the emergence of new strains with altered antigenicity over time, complicating vaccine design. This work is relevant to norovirus vaccine design as it demonstrates the potential for development of a chimeric VLP-based vaccine platform that may broaden the protective response against multiple GII.4 strains and proposes a potential reformulation strategy to control newly emergent strains in the human population.
Project description:Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987-1997), contemporary (2004-2009), and broad (1987-2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294-298 and 368-372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393-395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution.