Less severe clinical symptoms of Norwalk virus 8fIIb inoculum compared to its precursor 8fIIa from human challenge studies.

Noroviruses (NoV) are a leading cause of epidemic gastroenteritis. Human challenge studies have been used to examine the infectivity, pathogenicity, and host immune response to NoV as well as vaccine efficacy. The goal of this study was to conduct a meta-analysis of data from five previously completed human challenge trials and compare the response to the secondary NV inoculum (8fIIb) to its precursor (8fIIa). We investigated a total of 158 subjects: 76 subjects were experimentally challenged with NV inoculum 8fIIa, and 82 subjects were challenged with 8fIIb. We compared demographic characteristics, infection, illness, mean severity score, blood types, and duration of viral shedding between the two groups of subjects. There were no statistically significant differences in overall infection and illness rates between subjects inoculated with 8fIIa and 8fIIb. However, individuals challenged with 8fIIa had significantly higher severity scores (5.05 vs. 3.22, p = .008) compared with those challenged with 8fIIb. We also observed that infection with 8fIIb was associated with significantly longer duration of viral shedding compared with 8fIIa (11.0 days vs. 5.0 days, p = .0005). These results have serious implications for the development of new NoV inocula for human challenge studies to test candidate vaccine efficacy-where illness severity and duration of viral shedding are important outcomes.

Immunoinformatics approaches for designing a novel multi epitope peptide vaccine against human norovirus (Norwalk virus).

Norovirus is known as a major cause of several acute gastroenteritis (AGE) outbreaks each year. A study was conducted to develop a unique multi epitope subunit vaccine against human norovirus by adopting reverse vaccinology approach. The entire viral proteome of Norwalk virus was retrieved and allowed for further in silico study to predict highly antigenic epitopes through antigenicity, transmembrane topology screening, allergenicity assessment, toxicity analysis, population coverage analysis and molecular docking approach. Capsid protein VP1 and protein VP2 were identified as most antigenic viral proteins which generated a plethora of antigenic epitopes. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, hydrophilicity, theoretical PI and structural behavior. Molecular docking analysis of the refined vaccine with different MHCs and human immune TLR8 receptor demonstrated higher binding interaction as well. Complexed structure of the modeled vaccine and TLR8 showed minimal deformability at molecular level. The designed construct was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for its heterologous cloning and expression, and sequence of vaccine constructs showed no similarity with human proteins. However, the study could initiate in vitro and in vivo studies regarding effective vaccine development against human norovirus.

Frequent Use of the IgA Isotype in Human B Cells Encoding Potent Norovirus-Specific Monoclonal Antibodies That Block HBGA Binding.

Noroviruses (NoV) are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV). A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors.

Mapping and modeling of a strain-specific epitope in the Norwalk virus capsid inner shell.

Noroviruses are diverse positive-strand RNA viruses associated with acute gastroenteritis. Cross-reactive epitopes have been mapped primarily to conserved sequences in the capsid VP1 Shell (S) domain, and strain-specific epitopes to the highly variable Protruding (P) domain. In this work, we investigated a strain-specific linear epitope defined by MAb NV10 that was raised against prototype (Genogroup I.1) strain Norwalk virus (NV). Using peptide scanning and mutagenesis, the epitope was mapped to amino acids 21-32 (LVPEVNASDPLA) of the NV S domain, and its specificity was verified by epitope transfer and reactivity with a recombinant MAb NV10 single-chain variable fragment (scFv). Comparative structural modeling of the NV10 strain-specific and the broadly cross-reactive TV20 epitopes identified two internal non-overlapping sites in the NV shell, corresponding to variable and conserved amino acid sequences among strains, respectively. The S domain, like the P domain, contains strain-specific epitopes that contribute to the antigenic diversity among the noroviruses.

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans.

Application of salivary antibody immunoassays for the detection of incident infections with Norwalk virus in a group of volunteers.

Norovirus infection is the most common cause of acute gastroenteritis in developed countries. Developing an assay based on a non-invasive biomarker for detecting incident norovirus infections could improve disease surveillance and epidemiological investigations. This project involved analysis of IgA and IgG norovirus-specific antibody responses in saliva samples from a Norwalk virus (Genogroup I, genotype 1 norovirus) challenge study involving infected and symptomatic, and non-infected asymptomatic individuals. Saliva was collected at the challenge, and two weeks and 40 days post-challenge. Samples were analyzed using the Luminex fluorometric and Meso Scale Discovery (MSD) electrochemiluminescence immunoassays. Recombinant P domains of Norwalk virus capsid protein, as well as similar recombinant proteins of two genogroup II noroviruses (VA387 and VA207) were used as antigens. Immunoconversions were defined as >4-fold increase in antibody responses to the norovirus antigens. Various sample pre-treatment options, buffers, saliva dilution ratios, and data adjustment approaches to control for sample-to-sample variability in saliva composition were compared using the Luminex assay. The results suggest that adjusting responses to the norovirus antigens for responses to the protein purification tag, glutathione-S-transferase (GST), significantly improved the odds of producing a correct immunoconversion test result. IgG-based tests were more accurate compared to IgA-based tests. At optimal conditions, both Luminex and MSD assays for Norwalk-specific IgG antibodies correctly identified all infected and non-infected individuals. There was no evidence of cross-reactivity of anti-Norwalk virus antibodies with genogroup II noroviruses. These results suggest that salivary antibody responses can be used for the detection of incident infections with Norwalk virus in prospective surveys.

Mucosal and Cellular Immune Responses to Norwalk Virus.

BACKGROUND: Noroviruses are a leading cause of acute gastroenteritis worldwide. Mucosal and cellular immune responses remain poorly understood, with most studies of noroviruses having focused on serological responses to infection. METHODS: We used saliva, feces, and peripheral blood mononuclear cells collected from persons who were administered Norwalk virus (NV) to characterize mucosal (salivary and fecal immunoglobulin A [IgA]) and cellular (NV-specific IgA and immunoglobulin G [IgG] antibody-secreting cells and total and NV-specific IgA and IgG memory B cells) immune responses following infection. RESULTS: Prechallenge levels of NV-specific salivary IgA and NV-specific memory IgG cells correlated with protection from gastroenteritis, whereas prechallenge levels of NV-specific fecal IgA correlated with a reduced viral load. Antibody-secreting cell responses were biased toward IgA, while memory B-cell responses were biased toward IgG. NV-specific memory B cells but not antibody-secreting cells persisted 180 days after infection. CONCLUSIONS: NV-specific salivary IgA and NV-specific memory IgG cells were identified as new correlates of protection against NV gastroenteritis. Understanding the relative importance of mucosal, cellular, and humoral immunity is important in developing vaccine strategies for norovirus disease prevention.

Experimental human infection with Norwalk virus elicits a surrogate neutralizing antibody response with cross-genogroup activity.

The human noroviruses (NoVs) are genetically diverse, rapidly evolving RNA viruses and are the major cause of epidemic gastroenteritis of humans. Serum antibodies that block the interaction of NoVs and NoV viruslike particles (VLPs) with host attachment factors are considered surrogate neutralizing antibodies in the absence of cell culture and small-animal replication models for the human NoVs. A serological assay for NoV-blocking antibodies was used to assess the breadth of the heterotypic antibody response in the context of an experimental challenge study with a human NoV. Heterotypic histo-blood group antigen (HBGA)-blocking activity against GI.4, GI.7, and GII.4 NoVs increased significantly in the serum of individuals (n = 18) infected with Norwalk virus (GI.1). Although the fold increases and peak titers of heterotypic antibody were more modest than titers of antibody reactive with the challenge antigen, Norwalk virus infection elicited a serological rise even against the novel Sydney variant of GII.4 NoVs. These observations indicate that the development of a broadly cross-protective NoV vaccine containing a limited number of genotypes may be possible.

TLR7 and 9 agonists are highly effective mucosal adjuvants for norovirus virus-like particle vaccines.

Virus-like particles (VLPs) are an active area of vaccine research, development and commercialization. Mucosal administration of VLPs provides an attractive avenue for delivery of vaccines with the potential to produce robust immune responses. Nasal and oral delivery routes are particularly intriguing due to differential activation of mucosa-associated lymphoid tissues. We compared both intranasal and oral administration of VLPs with a panel of toll-like receptor (TLR) agonists (TLR3, 5, 7, 7/8, and 9) to determine the mucosal adjuvant activity of these immunomodulators. We selected Norwalk virus (NV) VLPs because it is an effective model antigen and an active area of research and commercialization. To prioritize these adjuvants, VLP-specific antibody production in serum (IgG, IgG1, IgG2a), vaginal lavages (IgG, IgA), and fecal pellets (IgA) were measured across a longitudinal timeseries in vaccinated mice. Additional distal mucosal sites (nasal, brochoalveolar, salivary, and gastrointestinal) were evaluated for VLP-specific responses (IgA). Intranasal co-delivery of VLPs with TLR7 or TLR9 agonists produced the most robust and broad-spectrum immune responses, systemically and at distal mucosal sites inducing VLP-specific antibodies at all sites evaluated. In addition, these VLP-specific antibodies blocked binding of NV VLPs to histo-blood group antigen (H type 1), supporting their functionality. Oral administration and/or other TLR agonists tested in the panel did not consistently enhance VLP-specific immune responses. This study demonstrates that intranasal co-delivery of VLPs with TLR7 or TLR9 agonists provides dose-sparing advantages for induction of specific and functional antibody responses against VLPs (i.e., non-replicating antigens) in the respiratory, gastrointestinal, and reproductive tract.

Development of Norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of Norwalk virus gastroenteritis.

Passive immunoprophylaxis or immunotherapy with norovirus-neutralizing monoclonal antibodies (MAbs) could be a useful treatment for high-risk populations, including infants and young children, the elderly, and certain patients who are debilitated or immunocompromised. In order to obtain antinorovirus MAbs with therapeutic potential, we stimulated a strong adaptive immune response in chimpanzees to the prototype norovirus strain Norwalk virus (NV) (genogroup I.1). A combinatorial phage Fab display library derived from mRNA of the chimpanzees' bone marrow was prepared, and four distinct Fabs reactive with Norwalk recombinant virus-like particles (rVLPs) were recovered, with estimated binding affinities in the subnanomolar range. Mapping studies showed that the four Fabs recognized three different conformational epitopes in the protruding (P) domain of NV VP1, the major capsid protein. The epitope of one of the Fabs, G4, was further mapped to a specific site involving a key amino acid residue, Gly365. One additional specific Fab (F11) was recovered months later from immortalized memory B cells and partially characterized. The anti-NV Fabs were converted into full-length IgG (MAbs) with human γ1 heavy chain constant regions. The anti-NV MAbs were tested in the two available surrogate assays for Norwalk virus neutralization, which showed that the MAbs could block carbohydrate binding and inhibit hemagglutination by NV rVLP. By mixing a single MAb with live Norwalk virus prior to challenge, MAbs D8 and B7 neutralized the virus and prevented infection in a chimpanzee. Because chimpanzee immunoglobulins are virtually identical to human immunoglobulins, these chimpanzee anticapsid MAbs may have a clinical application.

Helium beam shadowing for high spatial resolution patterning of antibodies on microstructured diagnostic surfaces.

We have developed a technique for the high-resolution, self-aligning, and high-throughput patterning of antibody binding functionality on surfaces by selectively changing the reactivity of protein-coated surfaces in specific regions of a workpiece with a beam of energetic helium particles. The exposed areas are passivated with bovine serum albumin (BSA) and no longer bind the antigen. We demonstrate that patterns can be formed (1) by using a stencil mask with etched openings that forms a patterned exposure, or (2) by using angled exposure to cast shadows of existing raised microstructures on the surface to form self-aligned patterns. We demonstrate the efficacy of this process through the patterning of anti-lysozyme, anti-Norwalk virus, and anti-Escherichia coli antibodies and the subsequent detection of each of their targets by the enzyme-mediated formation of colored or silver deposits, and also by binding of gold nanoparticles. The process allows for the patterning of three-dimensional structures by inclining the sample relative to the beam so that the shadowed regions remain unaltered. We demonstrate that the resolution of the patterning process is of the order of hundreds of nanometers, and that the approach is well-suited for high throughput patterning.

Antibody responses to norovirus genogroup GI.1 and GII.4 proteases in volunteers administered Norwalk virus.

An assay was developed to detect antibodies against two norovirus proteases among participants in a Norwalk virus (GI.1) challenge study. Prechallenge seroprevalence was lower against the protease from the homologous GI.1 virus than against protease from a heterologous GII.4 strain. Seroresponses were detected for 14 of 19 (74%) infected persons.

Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers.

Noroviruses are the most frequent cause of acute gastroenteritis in humans of all ages. No vaccines are currently available. An intranasally delivered Norwalk (NV) virus-like particle (VLP) vaccine was recently shown to be well tolerated, immunogenic and to protect against infection in Phase 1 studies. Here, we examined B memory (B(M)) responses in volunteers who received the highest dosage levels of the NV-VLP vaccine (50 µg and 100 µg). We measured the frequency of NV-specific IgG and IgA-secreting B(M) cells in peripheral blood and the level of antibodies produced by these cells in culture. All subjects immunized with 100 µg of the NV-VLP vaccine and 90% of those who received 50 µg had significant IgA or IgG B(M) responses. The B(M) cell frequencies correlated with serum antibody levels and mucosally-primed antibody-secreting cell responses. This is the first demonstration of dose-dependent, functional B(M) responses in humans immunized intranasally with a NV-VLP vaccine.

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations.

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can be established for processing virus-like particles (VLPs) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.

Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus.

A hemagglutination inhibition (HAI) assay to assess serum antibody responses following Norwalk virus (NV) infection was developed. HAI activity increased significantly in individuals experimentally infected with NV (n = 18) and correlated with antibody levels measured in a histo-blood group antigen (HBGA) blocking assay. Prechallenge HAI antibody levels also correlated with protection from the development of gastroenteritis (Mann-Whitney test, P = 0.02). The HAI assay is another assay suitable for the detection of antibody that correlates with protection from Norwalk virus-associated disease.

Serological responses to experimental Norwalk virus infection measured using a quantitative duplex time-resolved fluorescence immunoassay.

A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA).

Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond.

In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages.

An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles.

Norwalk virus (NV) is an enteric pathogen from the genus Norovirus and a major cause of nonbacterial gastroenteritis in humans. NV virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered nasally; however, the correlates of immune protection are unknown, and codelivery with a safe and immunogenic mucosal adjuvant may enhance protective anti-NV immune responses. Resiquimod (R848), an imidazoquinoline-based Toll-like receptor 7 and/or 8 (TLR7/8) agonist, is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. As such, we evaluated the adjuvant activity of two imidazoquinoline-based TLR7 and TLR7/8 agonists when codelivered intranasally with plant-derived NV VLPs. We also compared the activity of these agonists to the gold standard mucosal adjuvant, cholera toxin (CT). Our results indicate that codelivery with the TLR7 agonist, gardiquimod (GARD), induces NV VLP-specific serum IgG and IgG isotype responses and mucosal IgA responses in the gastrointestinal, respiratory, and reproductive tracts that are superior to those induced by R848 and comparable to those induced by the mucosal adjuvant CT. This study supports the continued investigation of GARD as a mucosal adjuvant for NV VLPs and possible use for other VLP-based vaccines for which immune responses at distal mucosal sites (e.g., respiratory and reproductive tracts) are desired.

Adjuvanted intranasal Norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues.

BACKGROUND: Noroviruses cause significant morbidity and mortality from acute gastroenteritis in all age groups worldwide. METHODS: We conducted 2 phase 1 double-blind, controlled studies of a virus-like particle (VLP) vaccine derived from norovirus GI.1 genotype adjuvanted with monophosphoryl lipid A (MPL) and the mucoadherent chitosan. Healthy subjects 18-49 years of age were randomized to 2 doses of intranasal Norwalk VLP vaccine or controls 21 days apart. Study 1 evaluated 5-, 15-, and 50-µg dosages of Norwalk antigen, and study 2 evaluated 50- and 100-µg dosages. Volunteers recorded symptoms for 7 days after dosing, and safety was followed up for 180 days. Blood samples were collected for serological profile, antibody secreting cells (ASCs), and analysis of ASC homing receptors. RESULTS: The most common symptoms were nasal stuffiness, discharge, and sneezing. No vaccine-related serious adverse events occurred. Norwalk VLP-specific immunoglobulin G and immunoglobulin A antibodies increased 4.8- and 9.1-fold, respectively, for the 100-µg dosage level. All subjects tested who received the 50- or 100-µg vaccine dose developed immunoglobulin A ASCs. These cells expressed molecules associated with homing to mucosal and peripheral lymphoid tissues. CONCLUSIONS: The intranasal monovalent adjuvanted Norwalk VLP vaccine was well tolerated and highly immunogenic and is a candidate for additional study.

Serological correlate of protection against norovirus-induced gastroenteritis.

BACKGROUND: Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure. METHODS: We developed an HBGA blocking assay to examine the ability of human serum to block the interaction of NV viruslike particles with H type 1 and H type 3 glycans. Serum samples from persons who were experimentally challenged with NV were evaluated. RESULTS: There was a high correlation between the H type 1 and H type 3 synthetic glycan assays (r = 0.977; P < .001); the H type 1 assay had higher quantitative sensitivity (P < .001). Among 18 infected secretor-positive individuals, blocking titers peaked by day 28 after challenge and were higher for individuals who did not develop gastroenteritis than for those who developed gastroenteritis on days 0, 14, 28, and 180 (P < .05 for each). In addition, 6 of 6 subjects without gastroenteritis had measurable prechallenge blocking titers (>25), compared with 2 of 12 subjects with gastroenteritis (P = .002). CONCLUSIONS: Blocking antibodies correlate with protection against clinical NV gastroenteritis. This knowledge will help guide the evaluation of new vaccine strategies and the elucidation of the nature of immunity to the virus. Trial registration. ClinicalTrials.gov identifier: NCT00138476.