Elsevier

Clinical Immunology

Volume 197, December 2018, Pages 110-117
Clinical Immunology

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

https://doi.org/10.1016/j.clim.2018.09.009Get rights and content

Highlights

  • NoV-specific mucosal IgG and IgA antibody titers correlate to systemic titers.

  • Purified saliva NoV antibodies confer blocking in a surrogate neutralization assay.

  • The highest level of antibodies were directed against ancestral GII.4 99 genotype.

Abstract

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.

Introduction

Human noroviruses (NoVs) are major causative agents of acute gastroenteritis (AGE) globally [1]. NoV outbreaks and wintertime sporadic infections cause morbidity and mortality particularly in vulnerable populations [[2], [3], [4]] and put a huge burden on healthcare [5], as no preventive vaccine is available [6]. Genetically and antigenically divergent NoVs are divided into seven genogroups (GI- GVII) of which GI and GII comprise at least 28 different human infecting genotypes [7]. Over two decades, GII.4 has been recognized as the most prevalent genotype, and its variants escape herd immunity in 3–5 year intervals causing worldwide pandemics [8,9]. Last pandemic GII.4 variant, GII.4 Sydney, emerged in 2012 [10] and its recombination subvariants still persist as the most prevalent NoV genotype [11].

Humans from an early childhood have a very high prevalence of NoV antibodies and repetitive NoV exposures increase the magnitude of pre-existing NoV antibodies with age [[12], [13], [14]]. Serum NoV antibodies are broadly cross-reactive [[15], [16], [17]] and cross-reactivity is highest among closely related NoV variants and decreases gradually in relation to amino acid sequence divergence [[18], [19], [20], [21]]. NoV seropositivity itself is not associated with protection in adults [15,22,23]. The protection against a certain NoV genotype is short-lived and natural cross-protection between different genotypes is controversial [24]. The best-recognized correlate of protection is the ability of serum antibodies to block NoV virus-like particles (VLPs) binding to histo-blood group antigens (HBGAs) in a surrogate neutralization assay [16,23,25]. HBGAs are expressed i.e. in bodily secretions and on mucosal surfaces where they are thought to facilitate NoV entry and infection [26,27]. The biosynthesis of complex HBGAs on mucosa and secretions is dependent on fucosyltransferase 2 (FUT2) enzyme activity and individuals with dysfunctional FUT2-gene (non-secretors) have reduced risk of NoV-infection [28,29].

Saliva is as an intriguing non-invasive alternative to blood to be used in serological assays [30]. It is readily and abundantly available, the collection is painless and the drawing does not require an authorized technician. Salivary samples can be used as a representative mucosal fluid in enteric infections [[31], [32], [33]], however, there are some drawbacks in using saliva that should be taken into the consideration; i.e. the viscosity, proteolytic degradation of antibodies and temporal variability of individual-specific antibody levels [34,35]. Also in the case of severe dehydration, saliva sample could be hard to obtain. Despite these disadvantages, there are several validated diagnostic applications using saliva as biological fluid [35,36]. Saliva is widely used as a biomarker to certain diseases such as the coeliac disease [[37], [38], [39]] and saliva-based serological assays have been utilized in the research of many viruses including NoV [[40], [41], [42], [43], [44], [45], [46]]. New immunological tools based on luminescence were recently published for the detection of NoV infection from saliva samples [41].

In the present study we used salivary samples from adult volunteers in qualification of an ELISA-based method for the detection of mucosal NoV antibodies. The results demonstrate, that affinity chromatography purified saliva anti-NoV antibodies block binding of NoV VLPs to HBGAs in a surrogate neutralization assay.

Section snippets

Volunteers

Saliva samples were collected in 2016 from 23 healthy adults (age-range 26–56 years) and a blood sample was obtained from ten of these volunteers (laboratory personnel) originally used to study NoV-specific T-cell and humoral immune responses [47]. Additional saliva samples were collected from two donors within 9 months follow-up period to study saliva antibody level fluctuations. An informed consent was obtained from each donor prior sample collection and all procedures were conducted in

Qualification of NoV-specific saliva ELISA

Intra-assay precision was assessed by calculating CVs between positive OD values of triplicate wells of three serially diluted saliva samples. Intra-assay CVs were on average 4.1 ± 3.6% (range 0.07% to 12.7%) for IgG assay, and 2.9 ± 2.6% (range 0.27% to 9.24%) for IgA assay. Inter-assay precision was determined by calculating CV of three serially diluted saliva samples positive OD values from three consecutive assays. Inter-assay CVs were on average 11.8 ± 5.5% (range 5.5% to 21.3%) for IgG

Discussion

NoV-specific serum antibody responses, namely blocking antibodies, are the first established correlate of protection [12,25,54]. In addition to serum blocking activity, pre-challenge salivary IgA [46] and an early rise in NoV-specific salivary IgA post infection [28] have been identified as mucosal correlates of protection. Fecal NoV-specific IgA have also been shown to reduce the viral load [46]. The goal of this study was to employ a simple ELISA-based method for measuring mucosal antibodies

Conflict of interest

None of the authors have any conflicts of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgements

The volunteers participating in this study are warmly acknowledged. The laboratory personnel of Vaccine Research Center are thanked for their valuable technical assistance and help during the study.

References (70)

  • D. Malamud

    Saliva as a diagnostic fluid

    Dent. Clin. N. Am.

    (2011)
  • S.M. Griffin et al.

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

    J. Immunol. Methods

    (2015)
  • V. Blazevic et al.

    Norovirus VLPs and rotavirus VP6 protein as combined vaccine for childhood gastroenteritis

    Vaccine

    (2011)
  • M. Yoshida et al.

    Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells

    Immunity

    (2004)
  • B.A. Lopman et al.

    The Vast and Varied Global Burden of Norovirus: prospects for Prevention and Control

    PLoS Med.

    (2016)
  • K. Bok et al.

    Norovirus gastroenteritis in immunocompromised patients

    N. Engl. J. Med.

    (2012)
  • G.I. Parra et al.

    Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity

    PLoS Pathog.

    (2017)
  • K. Bok et al.

    Evolutionary dynamics of GII.4 noroviruses over a 34-year period

    J. Virol.

    (2009)
  • K. Debbink et al.

    Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity

    J. Virol.

    (2012)
  • J. van Beek et al.

    Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012

    Euro Surveill

    (2013)
  • K. Nurminen et al.

    Prevalence of norovirus GII-4 antibodies in Finnish children

    J. Med. Virol.

    (2011)
  • J. van Beek et al.

    Comparison of norovirus genogroup I, II and IV seroprevalence among children in the Netherlands, 1963, 1983 and 2006

    J. Gen. Virol.

    (2016)
  • V.K. Menon et al.

    Comparison of age-stratified seroprevalence of antibodies against norovirus GII in India and the United Kingdom

    PLoS One

    (2013)
  • L.C. Lindesmith et al.

    Heterotypic humoral and cellular immune responses following Norwalk virus infection

    J. Virol.

    (2010)
  • M. Malm et al.

    High serum levels of norovirus genotype-specific blocking antibodies correlate with protection from infection in children

    J. Infect. Dis.

    (2014)
  • B. Rockx et al.

    Characterization of the homo- and heterotypic immune responses after natural norovirus infection

    J. Med. Virol.

    (2005)
  • M. Malm et al.

    Genotype considerations for virus-like particle-based bivalent norovirus vaccine composition

    Clin. Vaccine Immunol.

    (2015)
  • G.S. Hansman et al.

    Genetic and antigenic diversity among noroviruses

    J. Gen. Virol.

    (2006)
  • V. Blazevic et al.

    Induction of homologous and cross-reactive GII.4-specific blocking antibodies in children after GII.4 New Orleans norovirus infection

    J. Med. Virol.

    (2015)
  • L. Lindesmith et al.

    Cellular and humoral immunity following Snow Mountain virus challenge

    J. Virol.

    (2005)
  • R.L. Atmar et al.

    Serological Correlates of Protection against a GII.4 Norovirus

    Clin. Vaccine Immunol.

    (2015)
  • G.I. Parra et al.

    Sequential gastroenteritis episodes caused by 2 norovirus genotypes

    Emerg. Infect. Dis.

    (2014)
  • A. Reeck et al.

    Serological correlate of protection against norovirus-induced gastroenteritis

    J. Infect. Dis.

    (2010)
  • P.R. Harrington et al.

    Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice

    J. Virol.

    (2002)
  • L. Lindesmith et al.

    Human susceptibility and resistance to Norwalk virus infection

    Nat. Med.

    (2003)
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