NoV-specific Antibody Research Articles

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.

Development of T cell immunity to norovirus and rotavirus in children under five years of age

Most of the research effort to understand protective immunity against norovirus (NoV) has focused on humoral immunity, whereas immunity against another major pediatric enteric virus, rotavirus (RV), has been studied more thoroughly. The aim of this study was to investigate development of cell-mediated immunity to NoV in early childhood. Immune responses to NoV GI.3 and GII.4 virus-like particles and RV VP6 were determined in longitudinal blood samples of 10 healthy children from three months to four years of age. Serum IgG antibodies were measured using enzyme-linked immunosorbent assay and production of interferon-gamma by peripheral blood T cells was analyzed by enzyme-linked immunospot assay. NoV-specific T cells were detected in eight of 10 children by the age of four, with some individual variation. T cell responses to NoV GII.4 were higher than those to GI.3, but these responses were generally lower than responses to RV VP6. In contrast to NoV-specific antibodies, T cell responses were transient in nature. No correlation between cell-mediated and antibody responses was observed. NoV exposure induces vigorous T cell responses in children under five years of age, similar to RV. A role of T cells in protection from NoV infection in early childhood warrants further investigation.

Norovirus-specific mucosal antibodies correlate to systemic antibodies and block norovirus virus-like particles binding to histo-blood group antigens

The best acknowledged correlate of protection from norovirus (NoV) infection is the ability of serum antibodies to block binding of NoV virus-like particles (VLPs) to histo-blood group antigens (HBGAs). We investigated mucosal NoV-specific antibody levels in adult volunteers and used saliva from a single donor to determine whether purified saliva antibodies confer blocking. NoV-specific IgG and IgA levels in saliva and plasma samples were measured against four NoV genotype VLPs. NoV-specific IgG and IgA titers in saliva and plasma samples correlated significantly. Antibodies were detected against all VLPs with the highest level of antibodies directed against ancestral GII.4 99 genotype. Affinity chromatography purified salivary IgA and IgG blocked binding of GII.4 99 VLPs to HBGAs. Saliva sampling is a non-invasive alternative to blood drawing and an excellent biological fluid to study NoV-specific immune responses. Mucosal anti-NoV antibodies block binding of NoV VLPs to HBGAs, and may therefore be protective.

Functionality and avidity of norovirus-specific antibodies and T cells induced by GII.4 virus-like particles alone or co-administered with different genotypes

Norovirus (NoV) is the main cause of acute gastroenteritis worldwide across all age groups. Current NoV vaccine candidates are based on non-infectious highly immunogenic virus-like particles (VLPs) produced in cell cultures in vitro. As NoVs infecting human population are highly divergent, it is proposed that the vaccine should contain at least two different NoV genotypes, potentially affecting the immunogenicity of each other. We investigated the immunogenicity of NoV GII.4 VLPs administered by intramuscular (IM) or intradermal (ID) injections to BALB/c mice either alone or co-delivered with genogroup I (GI) and other genogroup GII VLPs. Serum NoV-specific IgG binding antibody titers and antibody functionality in terms of avidity and blocking potential were assessed. Furthermore, the specificity and functional avidity of CD4+ and CD8+ T cell responses were analyzed using synthetic peptides previously identified to contain NoV VP1 P2 domain-specific H-2d epitopes. The results showed that IM and ID immunization induced comparable GII.4-specific antibodies and T cell responses. Similar magnitude and functionality of antibodies and interferon-gamma producing T cells were developed using monovalent GII.4 VLPs or different genotype combinations. For the first time, degranulation assay using multicolor flow cytometry showed that NoV GII.4-specific CD8+ T cells had cytotoxic T lymphocyte phenotype. To conclude, our results demonstrate that there is no immunological interference even if up to five different NoV VLP genotypes were co-administered at the same time. Furthermore, no inhibition of NoV-specific antibody functionality or the magnitude, specificity and affinity of T cell responses was observed in any of the immunized animals, observations relevant for the development of a multivalent NoV VLP vaccine.

Development and maturation of norovirus antibodies in childhood

The burden of norovirus (NoV) gastroenteritis is substantial in young children. Maternal antibodies are thought to protect a child from NoV infection in early infancy but subsequent development of NoV-specific protective immunity in children is still largely unexplored.We have determined NoV-specific antibody seroconversion to GII.4 virus-like particles as an indicator of NoV infection in two children prospectively followed from birth to eight years of age. Blocking activity and affinity maturation of maternal and serum IgG antibodies were evaluated.Our results show that multiple infections occur in children up to eight years of age. The titer, blocking activity and avidity of maternal antibodies determined susceptibility of an infant to NoV infection. NoV GII.4-specific antibodies with high blocking potential and avidity were developed at two to three years of age and were retained throughout the follow-up. Subsequent NoV infections may have contributed to the duration of protective NoV-specific immune responses that lasted for several years.This study adds to current understanding of the duration of passive protection by maternal antibodies and the duration and quality of acquired immunity following primary and subsequent NoV infections in infants and young children, who are the main target group for NoV vaccine development.

Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine

ABSTRACTRotavirus (RV) and norovirus (NoV) are the 2 leading causes of acute viral gastroenteritis worldwide. We have developed a non-live NoV and RV vaccine candidate consisting of NoV virus-like particles (VLPs) and recombinant polymeric RV VP6 protein produced in baculovirus-insect cell expression system. Both components have been shown to induce strong potentially protective immune responses. As VP6 nanotubes are highly immunogenic, we investigated here a possible adjuvant effect of these structures on NoV-specific immune responses in vivo. BALB/c mice were immunized intramuscularly with a suboptimal dose (0.3 μg) of GII.4 or GI.3 VLPs either alone or in a combination with 10 μg dose of VP6 and induction of NoV-specific antibodies in sera of experimental animals were measured. Blocking assay using human saliva or synthetic histo-blood group antigens was employed to test NoV blocking antibodies. Suboptimal doses of the VLPs alone did not induce substantial anti-NoV antibodies. When co-administered with the VP6, considerable titers of not only type-specific but also cross-reactive IgG antibodies against NoV VLP genotypes not included in the vaccine composition were induced. Most importantly, NoV-specific blocking antibodies, a surrogate for neutralizing antibodies, were generated. Our results show that RV VP6 protein has an in vivo adjuvant effect on NoV-specific antibody responses and support the use of VP6 protein as a part of the NoV-RV combination vaccine, especially when addition of external adjuvants is not desirable.

Seroepidemiology of Predominant Norovirus Strains Circulating in Korea by Using Recombinant Virus-Like Particle Antigens

An epidemiological survey on human norovirus (NoV)-associated gastroenteritis was conducted to clarify the prevalence of NoV infections in children and adults in Korea. Recombinant capsid proteins from three major NoV genotypes (GI-4, GII-3, and GII-4) were expressed using a baculovirus expression system, and the morphology and antigenicity of self-assembled virus-like particles were then confirmed by electron microscopy and Western blotting with a NoV-specific antibody. To determine seroprevalence, an enzyme-linked immunosorbent assay was performed to detect antibodies against virus-like particles antigen in 346 serum specimens collected from persons who visited five public heath care centers for regular physical examination in Jeollanam-do, Korea, between 2005 and 2006. The seroprevalence of immunoglobulin G antibodies against the GI-4, GII-3, and GII-4 NoV genotypes was 84.1%, 76.3%, and 94.5%, respectively. A rapid decrease in seroprevalence occurred after birth, with the lowest levels observed in the <23-month age group, and a steep increase in seroprevalence occurred in early childhood, reaching 60.5% for GI-4, 65.1% for GII-3, and 90.7% for GII-4 at age 2-5 years, and over 80% for all three genotypes in subjects aged 20 years or older. The seroprevalence of different NoV genotypes statistically differed across the age groups (p<0.01).

Characterization and immunogenicity of norovirus capsid-derived virus-like particles purified by anion exchange chromatography

Recombinant baculovirus (BV) expression systems are widely applied in the production of viral capsid proteins and virus-like particles (VLPs) for use as immunogens and vaccine candidates. Traditional density gradient purification of VLPs does not enable complete elimination of BV-derived impurities, including live viruses, envelope glycoprotein gp64 and baculoviral DNA. We used an additional purification system based on ionic strength to purify norovirus (NoV) GII-4 capsid-derived VLPs. The anion exchange chromatography purification led to highly purified VLPs free from BV impurities with intact morphology. In addition, highly purified VLPs induced strong NoV-specific antibody responses in BALB/c mice. Here, we describe a method for NoV VLP purification and several methods for determining their purity, including quantitative PCR for BV DNA detection.

Evaluation of anti-norovirus IgY from egg yolk of chickens immunized with norovirus P particles

Noroviruses (NoVs) are a leading cause of epidemic acute gastroenteritis affecting millions of people worldwide. Understanding of NoV remains limited due to the lack of a cell culture system and small animal models. Currently, there are no effective vaccines or antivirals against NoVs. In this study, an approach for large-scale production of anti-NoV antibodies for use as a potential treatment for NoV disease using passive immunization was evaluated. NoV-specific immunoglobulins (IgYs) were produced by immunizing chickens with NoV P particles. The birds continuously produced high titers of antibodies in their eggs for at least 3months, in which NoV-specific antibody levels reached 4.7-9.2mg/egg yolk. The egg yolk antibodies strongly reacted with NoV P particles by both ELISA and Western blot and blocked NoV virus-like particle (VLP) and P particle binding to the histo-blood group antigen (HBGA) receptors with a BT(50) of about 1:800. The blocking activity of the chicken IgY remained after an incubation at 70°C for 30min or treatments at pH 4-9 for 3h. These data suggested that chicken IgY could be a practical strategy for large-scale production of anti-NoV antibodies for potential use as passive immunization against NoV infection, as well as for diagnostic purposes.

A comparison of immunogenicity of norovirus GII‐4 virus‐like particles and P‐particles

Norovirus (NoV) -derived virus-like particles (VLPs) resemble empty shells of the virus and NoV P-particles contain only protruding domains of the NoV capsid. Both NoV-derived subviral particles show similar functionality and antigenicity in vitro and are considered to be potential vaccine candidates against NoV gastroenteritis. BALB/c mice were immunized with baculovirus-produced GII-4 VLPs or the corresponding Escherichia coli-produced P-particles by the intramuscular or intradermal route and the NoV-specific antibody and T-cell immune responses were compared. Elevated antibody levels were induced with a single VLP immunization, whereas P-particle immunization required a boost. High avidity antibodies were raised only by VLP immunization. VLP immunization resulted in a balanced T helper type 1/type 2 immune response whereas P-particles induced a T helper type 2-biased response. Only VLP immunization primed T cells for interferon-γ production. Most importantly, cross-reactive B and T cells were induced solely by VLP immunization. In addition, VLP antiserum blocked the binding of heterotypic VLPs to human histo-blood group antigen receptor and saliva. The findings in this study are relevant for the development of NoV vaccines.

Detection of Antibodies against Norovirus Genogroup GIV in Carnivores

Noroviruses (NoVs) resembling human NoV genotype GIV (Alphatron-like) have recently been detected in carnivores. By using an enzyme-linked immunosorbent assay based on baculovirus-expressed capsid protein VP1 of lion strain GGIV.2/Pistoia/387/06/ITA, NoV-specific antibodies were detected in cats (16.11%) and dogs (4.8%), demonstrating that these animals are exposed to infections caused by NoVs.

Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression.

AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological...