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