JGV Papers in Press. Published April 24, 2014 as doi:10.1099/vir.0.064832-0
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Trimeric knob protein specifically distinguishes neutralizing
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antibodies to different human adenovirus species: potential
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application for adenovirus seroepidemiology
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Bin Yu,1, 2 Jianing Dong,1 Chu Wang,1 Zhen Wang, 1 Lei Gao, 1 Haihong
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Zhang,1, 2 Jiaxin Wu,1, 2 Wei Kong,1, 2, * and Xianghui Yu1, 2, *
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1
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University, Changchun, China
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin
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2
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School of Life Sciences, Jilin University, Changchun, China
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*
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2699 Qianjin Street, Changchun 130012, China. Phone: +86 431 85167826. Fax: +86 431
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85167751. E-mail: [email protected] (W. Kong), [email protected] (XH. Yu)
Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education,
Corresponding author. Mailing address: School of Life Sciences, Jilin University, No.
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Running Title: Trimeric knob for adenovirus NAbs serotyping
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Word count: 5201; 3 tables and 4 figures
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GenBank accession numbers: AB361388 (knob of Ad12), AB361380 (knob of Ad3),
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AB361378 (knob of Ad1), AB361379 (knob of Ad2), AB361382 (knob of Ad5),
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AB361383 (knob of Ad6), AB361413 (knob of Ad37), AB361381 (knob of Ad4),
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AB361417 (knob of Ad41)
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Summary
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Adenoviruses (Ads) are nonenveloped DNA viruses that have been extensively studied
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and used as vectors for gene therapy and several potential vaccines. There are 57 Ad
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serotypes in seven species (A-G), and Ad neutralizing antibodies (NAbs) titers can vary
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by serotypes and geographic location. Until now serotype- and species-specific antibodies
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have been detected by neutralization or hemagglutination inhibition assays. These
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expensive and cumbersome methods of adenovirus typing have mainly been used in
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epidemiological studies. Our prior work demonstrated that NAbs against the fiber protein
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are commonly generated during natural Ad infection in humans and the trimeric knob is
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preferentially recognized by fiber-induced NAbs. In this study, we expressed nine
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trimeric knob proteins from representative Ad serotypes of human Ad (HAdV)-A to -F in
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Escherichia coli and found no cross-reactivity of these recombinant proteins with rabbit
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hyperimmune sera (among HAdV-A to -F or within HAdV-C). Results of the ELISA
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based on Ad2 and Ad5 (both HAdV-C) knob proteins were consistent with those of
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neutralization assays, indicating that the trimeric knob protein would be a good candidate
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antigen for detecting Ad serotype-specific NAbs in sera from naturally infected subjects.
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We also demonstrated the primary seroepidemiology of nine Ad serotypes in 274
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children samples by the knob-based ELISA. These results will have potential
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implications for epidemiology of Ad serotypes and future development of Ad-based
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vaccines and gene therapy.
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Keywords: Adenovirus; neutralizing antibody; knob; cross-immunity; seroepidemiology
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Introduction
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Human adenoviruses (HAdVs) are non-enveloped DNA viruses that have been
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extensively studied and used as vectors for vaccine and gene delivery. The major
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limitation of the most well-studied adenovirus type 5 (Ad5) vaccine vector is the high
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titers of naturally occurring Ad5 neutralizing antibodies (NAbs) in human populations,
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particularly in the developing world (Mast et al., 2010; Yu et al., 2012b). Baseline Ad5
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NAbs have been shown to suppress the immunogenicity of recombinant Ad5 (rAd5)
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vector-based vaccines for HIV-1 in both preclinical studies (Casimiro et al., 2003;
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Roberts et al., 2006) and clinical trials (Buchbinder et al., 2008; Kolavic-Gray et al.,
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2002; Priddy et al., 2008). To address this and other problems with rAd5 vectors,
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alternative human serotype rAd vectors (Abbink et al., 2007; Barouch et al., 2004;
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Vogels et al., 2003), hexon-chimeric rAd vectors (Roberts et al., 2006) and rAd vectors
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derived from other species (Farina et al., 2001; Fitzgerald et al., 2003) have been
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constructed. All of these studies highlight the importance of understanding differences in
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virulence and seroepidemiology among the various Ad serotypes.
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Currently, 57 human Ad serotypes are defined and distributed into seven species (or
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subgroups) from A to G (Walsh et al., 2011). Rare serotypes such as Ad14 and Ad55
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have been considered to be responsible for most of the outbreaks of acute respiratory
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disease among school and military recruits, and an emerging variant (HAdV-14p1) was
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associated with the high rates of illness and death in United States and Europe (Carr et al.,
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2011; Zhang et al., 2012). Diagnosis of Ad infections is currently based on virus isolation
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in cell culture and genus-specific antibody and antigen detection by enzyme
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immunoassays (Weinberg et al., 1989), as well as Ad DNA detection by PCR (Berk, 3
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2007). Infection-induced humoral immunity to a particular serotype does not provide
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cross-immunity to an Ad serotype of another subgroup (Hierholzer et al., 1991). Thus,
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Ad serotypes are determined based on inhibition by specific immune sera and
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phylogenetic analysis of DNA sequences (Takeuchi et al., 1999), while serotype-specific
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NAbs are detected by viral vector-based neutralization assays (Aste-Amezaga et al., 2004;
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Sprangers et al., 2003). These Ad typing methods have been used in epidemiological
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studies on the prevalence of pre-existing NAbs to several conceptually interesting vaccine
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vector candidate types (Mast et al., 2010; Pereira, 1959). Although neutralization assays
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are efficient means for detecting specific antibodies against each Ad serotype, they are
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cumbersome and expensive to perform. Therefore, more convenient typing methods for
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NAbs are needed to analyze Ad serotype infections.
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There are three major capsid proteins on Ad particles: fiber, penton base and hexon. Both
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the fiber and hexon proteins contain group-specific as well as type-specific epitopes
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(Norrby, 1969; Rux & Burnett, 2000). The fiber protein is composed of three portions, a
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tail anchoring the fiber into the vertex capsomer, a shaft and a knob containing the
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domain responsible for interaction with the host cell receptor (Khare et al., 2011). The
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globular knob domain in the carboxy-terminal segment of the fiber protein includes
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175–220 amino acid residues, which vary among different serotypes. Our prior studies
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have emphasized the contribution of anti-fiber antibodies to Ad5 neutralization responses
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generated during natural viral infection and rAd5 immunizations, and other group also
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showed that the trimeric knob was preferentially recognized by fiber induced NAbs
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(Gahery-Segard et al., 1998; Yu et al., 2013). We demonstrated that NAbs against fiber
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or knob are commonly generated in natural Ad infection in humans and observed no
4
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cross-immunity of the sera naturally infected with Ad2 and Ad5 (both species C) by
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reactivity with two trimeric knob antigens in Western blotting assays (Yu et al., 2013).
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In this study, we further analyzed the cross-reactivity of the trimeric knob with rabbit
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hyperimmune sera among HAdV-A to -F and attempted to establish a knob-based ELISA
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for Ad epidemiological studies. We showed that the stable trimeric knob protein was
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specifically recognized by Ad NAbs in all six species especially in HAdV-C, indicating
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that it would be a good candidate antigen to detect serotype-specific NAbs in natural Ad
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infection and rAd vaccination.
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Results
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Expression of trimeric knob proteins used for serotype-specific NAb
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detection
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Prior phylogenetic analyses of fiber knob amino acid sequences derived from 45
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serotypes of HAdV-A to -F showed a good correlation with the classification based on
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classical subtyping assays (i.e., HA inhibition and cross-neutralization assays) (Havenga
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et al., 2002). While the identities between the knob sequence of Ad4 in species E and the
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serotypes of HAdV-C are very high, the genome sequence of Ad4 is more closely related
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to Ads of species B. In order to analyze the species (HAdV-A to-F) and serotype
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cross-reactivity of the knob proteins with rabbit hyperimmune sera or Ad NAbs, nine
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representative Ad serotypes of each species (HAdV-A; Ad12, HAdV-B; Ad3, HAdV-C;
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Ad1, 2, 5 and 6, HAdV-D; Ad37, HAdV-E; Ad4, HAdV-F; Ad41) were selected to
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derive the knob antigens (only the long fiber knob of Ad41 was used for the analysis as it
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has an analogous function with other types) and rabbit polyclonal sera. To obtain the
5
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trimeric knob protein, the knob domains with the last repeat in the shaft of the fiber were
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expressed as His-tagged fusion proteins in E. coli. As shown in Fig. 1(a), all of the knob
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proteins of the indicated types migrated as trimers in the native gel, and these trimeric
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proteins were stable even in the presence of 1% SDS in this study. The trimerization of
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the purified proteins was also confirmed by size-exclusion chromatography (data not
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shown). After immunization with these purified trimeric proteins in rabbits, NAbs against
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Ad2 and Ad5 were found (we only have rAd2-luc and rAd5-luc) in the corresponding
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hyperimmune sera and showed no cross-neutralization among these Ad serotypes even
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within the same species (Fig. 1b and c). These results further confirmed that trimeric
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knob proteins can induce serotype-specific Ad NAbs as we have previously described
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(Yu et al., 2013).
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Reactivity of rabbit hyperimmune sera raised against trimeric knob
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antigens or whole virions displaying knob antigens
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To confirm that the trimeric knob protein can serve as an antigen for distinguishing
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serotype specific responses, rabbit hyperimmune sera directed against various knob
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antigens were tested for cross-reactivity. The rabbit Ad5 knob antiserum recognized the
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monomeric knob antigens of all nine serotypes but only strongly reacted to the trimeric
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form of Ad5 knob protein, and this reaction pattern was similar to that of rabbit and
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human antisera raised against Ad5 virions (Fig. 2a). Furthermore, no or very weak
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cross-reactivity of the trimeric knob protein of Ad2 and Ad5 was shown with antisera
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raised against the trimeric knob antigens or whole virions of other Ad serotypes (Fig. 2b).
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The reactivities of sera raised against recombinant knob antigens or against purified
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virions with trimeric knob proteins of nine Ad serotypes are summarized in Table 1. Only 6
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a few serum samples raised against recombinant proteins weakly cross-reacted with
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trimeric knob from other serotypes in the same Ad species at serum dilutions of 1:102 to
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1:104. By contrast, antisera raised against whole virions showed stronger and more
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specific levels of reactivity with the corresponding trimeric knob proteins. Overall, these
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results indicated no cross-immunity in rabbit antisera raised against HAdV-A to -F
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species with trimeric knob proteins of HAdV-C and the representative antigens of other
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species.
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Use of knob proteins for detection by ELISA of NAbs in human sera
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after natural Ad infection
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After demonstrating the specificity of the trimeric knob proteins using the Ad rabbit
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hyperimmune sera above, we set out to establish an ELISA system for typing Ad NAbs in
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human sera. To determine the reliability of the ELISA method using knob as the antigen
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for detecting Ad NAbs, a total of 496 serum samples from healthy donors in China were
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first screened for the prevalence of Ad2 and Ad5 NAbs by a luciferase-based
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neutralization assay. As shown in Fig. 3(a), Ad2 and Ad5 seroprevalence rates were
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69.39% and 57.25% in all subjects, respectively. It was found that 54.06% of participants
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had baseline Ad2 NAb titers > 200, compared with 43.64% for Ad5. To determine the
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optimal serum dilution for the knob-based ELISA, 24 serum samples were selected at
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random from each Ad5 NAb titer group and diluted at 1:20, 1:100 and 1:1000. The
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dilution of 1:100 was found to be suitable for detecting NAbs in human sera by ELISA,
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since OD values of the sera showed a relatively good correlation with different NAb titer
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groups (1000) (Fig. 3b). Subsequently, all human sera were
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tested by ELISA with Ad2 and Ad5 knob proteins. As shown in Table 2, the ELISA 7
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results were compared with those of the neutralization assays using different cut-off
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values. When using the neutralization titer of < 20 as the cut-off value for seronegative
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samples, the seropositivity rate was slightly lower by ELISA, with the coincidence rate of
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over 80% compared with the neutralization assay. Meanwhile, the false negative rates
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(FNR) were 14.1% for Ad2 and 14.9% for Ad5. When using the baseline neutralization
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titer of 200 as the cut-off value, we found coincidence rates of nearly 90% for both of the
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Ad types. These results indicated that the trimeric knob protein could be used for the
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detection of serotype-specific NAbs.
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Analysis of knob-based ELISA data does not to correlate with
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neutralizing assay
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To determine the cause of false positive results in the Ad5 knob ELISA, the knob
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proteins of Ad1, 2, 5 and 6 in HAdV-C were used for Western blot analysis of the serum
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samples. In the > 200 titer group, the relatively high false positive rate (FPR) of 7.3%
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was mainly attributed to the low Ad5 NAb titer (20–200) of sera infected with either Ad5
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alone or multiple serotypes (samples 11-12 and 2848). However, in the > 20 NAb titer
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group, we found that very few samples with high infection levels of other serotypes such
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as Ad1 in HAdV-C seemingly could cause the positive responses to Ad2, 5 and 6 (sample
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2746) (Fig. 4a). To determine the extent of this cross-reactivity, a total of 100 Ad2
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seropositive samples (not tested in neutralization assays with other rAds) with NAb
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titers > 20 or >200 were tested with the Ad5 knob protein by ELISA and Ad5
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neutralization assay. The results showed that 46 of serum samples with NAb titers > 20
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were detected by the Ad5 knob protein ELISA, three of which were not detected by the
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neutralization assay (Fig. 4c). Meanwhile, 40 serum samples with NAb titers >200 were 8
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detected by the Ad5 knob protein ELISA, nine of which were not detected by the
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neutralization assay (Fig. 4b). Further analysis by Western blotting showed that only 3 of
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the 12 samples which had not been detected by the Ad5 neutralization assays were false
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positives in the Ad2 neutralization assay (data not shown). To analyze the detection
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efficiency of the knob-based ELISA, all of the false negative samples (10, 2%) in the
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NAb titer >200 group were analyzed by Western blot using native knob, HVR and virions
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for analysis of fiber- and hexon-specific antibodies as described previously (Yu et al.,
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2013). Results showed that all of these samples were anti-knob and anti-HVR negative
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but were anti-fiber positive, and three of them were anti-hexon positive (Fig. 4e). Nearly
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all of the samples had NAb titers 20 and >200, respectively (Fig. 4d and f). These results
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indicated that some serum samples with relatively low Ad5 NAb titers did not have NAbs
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against fiber (knob) and hexon (HVR) and could not be detected by trimeric Ad5 knob.
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Prevalence of Ads detected by knob-based ELISA
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As the participants from children are less likely to have multiple Ad infections, a total of
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274 children sera from Jilin province (northeast China) were screened for the prevalence
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of Ad antibodies by ELISA using knob proteins of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad12,
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Ad37 and Ad41 (Table 3). For Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41, 36 serum
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samples from 7- to 12-month-old infants were considered as negative controls and used to
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determine the cut-off value (removal of very high OD values), since the seroprevalence of
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Ad2 and Ad5 were very low in this age group and prior studies have also reported that
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children in this age group have the lowest levels of Ad NAbs (Appaiahgari et al., 2007;
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Mast et al., 2010). Results of the ELISA showed that 44.2–57.5% and 27.4–41.3% 9
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seropositivity in these children against in HAdV-C types with NAb titer cut-off values
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of >20 and >200, respectively. By contrast, this infant population showed lower infection
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levels with the other several serotypes especially Ad12 in HAdV-A which was detected in
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12% of the samples were seropositive (titer >20) and only 4.4% showing moderate to
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high NAb titers (titer >200). However, Ad37 in HAdV-D also showed a relatively high
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level of NAb seropositivity.
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Discussion
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Ads are the cause of common upper respiratory infections, and thus most individuals
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have circulating antibodies to these viruses. Pre-existing immunity to Ad is an important
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issue in clinical trials with rAd vector-based vaccines as it can limit in vivo delivery of
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the vectors and gene transfer (Roberts et al., 2006). Thus, understanding the nature of
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anti-Ad immunity and mapping the targets of dominant Ad-specific NAbs in human sera
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is important for the development of vaccines against HIV-1 and other infections or
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diseases. Our prior studies showed that NAbs against Ad5 fiber or knob are commonly
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generated in natural Ad infection and preferentially recognize the trimeric knob protein
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(Yu et al., 2013). We assumed that the knob protein from different Ad serotypes would
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specifically recognize NAbs of the same Ad serotype since NAbs induced by different Ad
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serotypes typically do not show cross-immunity. Although we previously showed that the
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pattern of reactivity of human sera indicated no cross-immunity against trimeric knob
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proteins of Ad2 and Ad5 of the HAdV-C species, more knob proteins from other Ad
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serotypes still needed to be examined to evaluate the cross-immunity of sera raised
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against viruses in different human Ad species as the widely used classification is based
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on hemagglutinating antigens (Berk, 2007).
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In this study, we expressed four knob proteins from HAdV-C and other five
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representative knob proteins of HAdV-A, -B, -D, -E and -F in E. coli to analyze the
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reactivity and cross-reactivity of NAbs to knob antigens produced in rabbit hyperimmune
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sera, which may be used to guide investigations in Ad antibodies generated in humans.
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The pattern of reactivity of the rabbit and two types of human sera (Yu et al., 2013)
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indicated there was no cross-immunity against trimeric knob proteins of the HAdV-A to
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-F species. In Fig. 2 and Table 2, Anti-K5, Anti-Ad5 and hAnti-Ad5 were all at dilution
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of 1:100 to 1:10000 and showed differently binding activities with these knob proteins in
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SDS and NDS westerns. We tend to think it is related with the fact that when the
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immunogens were the trimeric knob proteins (with adjuvant and other factors), it
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produced much of binding antibodies (IgG) resulting in cross-reactivity in SDS western.
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However, antisera raised against whole virions or human sera with natural adenovirus
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infection produced much more NAbs and less binding antibodies, thence showed weaker
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binding with the denatured proteins but stronger and more specific reactivities with the
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corresponding trimeric knob proteins, as the trimeric knob proteins are preferentially
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recognized by NAbs against Ad fiber. These results indicated that trimer knob protein
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would be very suitable for the detection of serotype-specific NAbs in sera of
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immunocompetent population after natural Ad infection and rAd vaccination. The ELISA
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results of human sera with Ad2 and Ad5 knob were consistent with those of the
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neutralization assays and the different levels of antibodies to each knob in the same
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serum sample, further confirming our above hypothesis on knob recognition among Ad
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serotypes, although it would be more convincing if we could do more comparisons
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among different Ad serotypes.
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Of the more than 50 human Ad serotypes occurring worldwide, the most commonly used
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rAd vector is Ad5, although its high seroprevalence in the human population has
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warranted investigations of other serotypes. The evaluation of serotype-specific NAbs as
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a means of selecting the adequate serotype adenovector(s) is important for both clinical
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applications and epidemiological studies (Piedra et al., 1998; Sanchez et al., 2001).
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However, the lower replication and difficult construction of some rAds will limit the
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application of the quantitative neutralization assay based on rAds carrying a reporter gene
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for additional Ad serotypes. In this study, we developed a knob-based ELISA to
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determine Ad serotype-specific antibody responses. By using this assay, we detected the
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NAbs seroprevalence of two serotypes (Ad2 and Ad5) from the same species and found
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that the results had a high correlation with those of the standard neutralization assays
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although still had low FPR and FNR caused by dilutions of sera and other reasons. A
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problem noted with this assay was that it did not detect approximately 10% of the human
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serum samples in this study determined to be seropositive by the neutralization assay, as
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the sera did not recognize either the native knob or HVR antigens, which is consistent
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with previous studies (Cheng et al., 2009). Further analysis showed that nearly all of
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these undetected samples had low (20 < titer < 200) or moderate (200 < titer < 1000)
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NAb titers. This indeed partially limits the application of the knob based ELISA for
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adenovirus seroepidemiology and clinical diagnosis. Nevertheless, since some previous
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studies showed no influence of low levels of Ad5 NAb on the immunogenicity and
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protective efficacy of rAd5 vectors in humans and mice (Appaiahgari et al., 2006;
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Buchbinder et al., 2008), our new method still could be suitable for testing moderate and
275
high level Ad infections to guide the application of rAd-based vaccine and gene therapy.
12
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Non-capsid specific humoral immune response in human sera from natural Ad infection
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are still needed to be studied to find a detection antigen for the Ad seropositive serum
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samples without NAbs against the capsid protein.
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The Ad fiber protein is primarily responsible for the high affinity attachment of the virus
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to its receptor expressed on the surface of target cells. The fiber knob domain confers
281
specificity for cellular receptors. Most Ad serotypes including all those within HAdV-C
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use the coxsackie and Ad receptor (CAR) as the primary attachment receptor (Bergelson
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et al., 1997; Berk, 2007). However, the fiber protein of species D Ad37 can bind to CAR,
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CD46 and sialic acid, but it appears only able to use sialic acid as a receptor for infection
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(Arnberg et al., 2000; Wang et al., 2011). This effect may be due to the very short length
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of species D fibers with only eight β-spiral repeats. Despite the differences in structure
287
and function, we also found that the trimeric Ad37 knob protein was strongly recognized
288
by serum NAbs induced by Ad37 but not those by other selected Ads. This result was
289
consistent with our previous speculation and prior studies determining that the receptor
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binding regions in knob are relatively conservative and that the epitopes are
291
hypervariable and located in the outer loops or uppermost β-sheets in the structure
292
(Marttila et al., 2005; Zhang & Bergelson, 2005). Therefore, these results indicated that
293
our knob-based ELISA method may be expanded for detection of additional Ad serotypes.
294
Previous studies showed that Ad1, 2, 5 and 6 are ubiquitous, particularly in young
295
children. Antibodies to Ad1, 2, and 5 are most common and are present in 40–60% of
296
children (Badger et al., 1956; Brandt et al., 1969), while the incidence of NAbs to Ad26,
297
35 and 48 is low in the same age group (Barouch et al., 2011; Pereira, 1959). In this study,
298
we showed an analogous trend in Chinese children with apparently higher seroprevalence
13
299
of Ad1, 2, 5, 6 and 37 than those of Ad3, 4, 12and Ad41.
300
In conclusion, this study highlights the utility of trimeric knob proteins to analyze
301
serotype-specific NAbs in HAdV-C and other species. Furthermore, we have shown that
302
the knob-based ELISA would be used to test the prevalence of several Ad serotypes. Our
303
study contributes to the understanding of natural Ad capsid-specific humoral immune
304
responses and serotype-specific NAbs in humans and may have implications in
305
seroepidemiology of Ads.
306
Methods
307
Cell lines and serum. Human embryonic kidney (HEK) 293 cells were obtained from the
308
American Type Culture Collection (ATCC; Manassas, VA). All cells were cultured in
309
Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA) containing
310
10% fetal bovine serum, (FBS; Hyclone; Logan, UT), 2 mM L-glutamine, 100 U/ml
311
penicillin and 100 μg/ml streptomycin (Invitrogen).
312
Recombinant Ad antigens. The sequence of Ad knob including the last repeat in the
313
shaft of the fiber (Seiradake et al., 2006; Yeh et al., 1994) was cloned into the pRSET-B
314
plasmid (Invitrogen) at the Nco Ι and Xho Ι restriction sites. The plasmid containing an
315
N-terminal His tag was expressed in BL21 cells overnight at 16°C. The pellet from a
316
2-liter culture was treated with 1 mg of lysozyme/ml on ice and sonicated 10 times for 10
317
s each time. After centrifugation at 40,000 × g for 30 min, 10 mM imidazole was added
318
to the soluble fraction at 4°C with gentle stirring. The resin was filled in a small
319
polyethylene column and washed with 20 mM imidazole in a buffer containing 25 mM
320
Tris-HCl (pH 7.5) and 150 mM NaCl. The protein was eluted with 500 mM imidazole in
14
321
the same buffer and analyzed by SDS-PAGE. The Ad5 hexon hypervariable regions
322
(HVR) protein was expressed as described previously (Yu et al., 2012a).
323
Hyperimmune sera. Rabbit polyclonal sera directed against the knob proteins of Ad1,
324
Ad2, Ad3, Ad4, Ad5, Ad6, Ad12, Ad37 and Ad41 were produced in rabbits given three
325
immunizations with purified proteins (300 μg per injection) together with Freund’s
326
complete and incomplete adjuvants. All animal studies were approved by the University
327
Committee on the Use and Care of Animals of Jilin University. Anti-Ad1, Ad2, Ad3, Ad4,
328
Ad5, Ad6, Ad12, Ad37 and Ad41 rabbit polyclonal antibody sera were gifts from
329
Panyong Mao (Beijing 302 Hospital, Beijing, China).
330
Human serum. Human serum samples were collected randomly during 2010–2011 in
331
China by the Provincial Centers for Disease Control and Prevention. Donors of both
332
sexes ranged in age from 1 day to 78 years and had no history of febrile illness during the
333
few weeks preceding sample collection. Informed consent was obtained from all
334
participants or their guardians in written form to utilize the serum samples in Ad
335
neutralization assays, as approved by the Human Ethical Committee Institutional Review
336
Board, Jilin University, China. All human sera were inactivated at 56°C for 60 min prior
337
to testing.
338
Adenovirus infection assay. HEK293 cells were grown in 6-well plates and were
339
infected with the Ad vectors at an MOI of 10 with or without rabbit anti-knob sera. After
340
incubation at 37°C for 48 h, the cytopathic effect (CPE) of 293 cells was observed at a
341
low magnification (200×) by microscopy.
342
Virus neutralization assays. Ad-specific NAb titers in human serum samples and rabbit
343
hyperimmune sera were assessed by luciferase-based assays as described previously
15
344
(Sprangers et al., 2003). HEK293 cells were plated at a density of 1 × 104 cells per well
345
in 96-well plates and infected with replication-incompetent rAd-Luc reporter constructs
346
at a multiplicity of infection (MOI) of 500 with 2-fold serial dilutions of serum in 200 µl
347
reaction volumes. Following 24-hour incubation, luciferase activity in the cells was
348
measured using the Steady-Glo Luc reagent system (Promega, Madison, WI) with a
349
Victor 1420 Multilabel Counter (Perkin Elmer, Wellesley, MA). Neutralization titers
350
were defined as the maximum serum dilution that inhibited 90% of luciferase activity.
351
Western blotting analysis. SDS-PAGE was performed on 12% SDS-polyacrylamide gels.
352
Samples were denatured by boiling at 98°C in Laemmli buffer (62.5 mM Tris-HCl, 2%
353
SDS, 1% β-mercaptoethanol, 25% glycerol, 0.01% bromophenol blue) prior to
354
electrophoresis. NDS-PAGE was similar to SDS-PAGE, except that the samples were
355
neither boiled nor exposed to β-mercaptoethanol in order to retain stable trimeric proteins.
356
Native-PAGE differed from SDS-PAGE in that the proteins were not denatured by boiling
357
prior to electrophoresis on SDS-free 6-8% Tris-acetate gels (Invitrogen) and running
358
buffer, and the samples were diluted in native buffer (62.5 mM Tris-HCl, 40% glycerol,
359
0.01% bromophenol blue).
360
Protein samples were electrophoresed by SDS-PAGE, NDS-PAGE or Native-PAGE and
361
transferred to nitrocellulose membranes for Western blotting. SDS was not added into the
362
transfer buffer (39 mM glycine, 48 mM Tris-HCl, 20% methanol, pH 9.2) when proteins
363
were separated by Native-PAGE. Membranes were incubated with different human,
364
rabbit and mouse sera overnight at 4°C in PBS containing 1.0% milk and then
365
subsequently incubated with ImmunoPure alkaline phosphatase-conjugated goat
366
anti-rabbit IgG secondary Abs (Jackson ImmunoResearch Laboratories) at a 1/10,000
16
367
dilution for 1 h. The color reaction was developed with 0.1 M Tris-HCl (0.1 M NaCl, 5
368
mM MgCl2, pH 9.5) containing 0.66% NBT solution and 0.33% BCIP solution.
369
Enzyme-linked immunosorbent assay (ELISA). Serum antibody titers were measured
370
by ELISA using a horseradish peroxidase (HRP) detection system. Each well of the
371
96-well polyvinylchloride microtiter plates was coated with knob proteins (200 ng/ 100
372
μl) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. The plates were then
373
washed three times with PBS-T (20 mM PBS containing 0.05% Tween-20) and blocked
374
with 2% BSA/PBS-T at 37°C. After three washes, different dilutions of serum were
375
added to the plate (100 μl per well) and incubated for 1 h at 37°C. After another three
376
washes, a 1/100,000 dilution of HRP-conjugated goat anti-human IgG (Proteintech Group,
377
Chicago, IL) was added to each well and incubated for 1 h at 37°C. After the final three
378
washes, TMB peroxidase substrate was added to the plates, incubated for 15 min in a
379
darkroom, stopped with 50 μl of 2 M H2SO4 and analyzed using dual wavelengths of
380
450/630 nm with an iMark Microplate reader (Bio-Rad, Hercules, CA).
381
Acknowledgments
382
This study was supported by the National Nature Science Foundation of China (No.
383
30872396, 31300756) and the Key Projects in the National Science & Technology Pillar
384
Program in the Twelfth Five-year Plan Period (Grant 2012ZX10001-009). We gratefully
385
acknowledge Panyong Mao (Beijing 302 Hospital, Beijing, China) for providing the
386
anti-Ad rabbit polyclonal sera and acknowledge Ling Chen (Guangzhou Institute of
387
Biomedicine and Health, Guangzhou, China) for providing the rAd2 vectors. The authors
388
also wish to acknowledge Thi Sarkis for editorial support in the preparation of this
389
manuscript. 17
390
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20
522
Figure legends
523
Fig. 1. Expression of trimeric knob proteins from Ad serotypes in HAdV-A to -F and
524
analysis of NAb titers in rabbit hyperimmune sera against knob. (a) Knob proteins were
525
analyzed by NDS-PAGE and SDS-PAGE. (b) Neutralizing activity of anti-knob
526
polyclonal sera was analyzed between Ad2 and Ad5 following infection of 293 cells. 293
527
cells were infected with the indicated Ad vectors with or without anti-knob polyclonal
528
sera at an MOI of 10 PFU/cell. ‘‘Anti-K2’’ and ‘‘Anti-K5’’ are serums obtained from
529
rabbits with knob proteins of Ad2 and Ad5 immunizations and used at a dilution of 1:200.
530
(c) Ad NAbs induced by knob proteins were determined by luciferase-based rAd2 and
531
rAd5 neutralization assays. Knob proteins derived from human Ad species A to F
532
(HAdV-A; Ad12, HAdV-B; Ad3, HAdV-C; Ad1, 2, 5 and 6, HAdV-D; Ad37, HAdV-E;
533
Ad4, HAdV-F; Ad41).
534
Fig. 2. Reactivity of rabbit hyperimmune sera raised against the trimeric knob antigens or
535
whole virions with knob antigens by Western blotting. (a) Reactivity of rabbit anti-K5
536
and anti-Ad5 rabbit polyclonal sera and human anti-Ad5 (hAnti-Ad5, NAb positive for
537
Ad5 alone) serum with knob antigens of nine Ad serotypes was analyzed by
538
SDS-denatured or NDS Western blotting. (b) Reactivity of anti-knob and anti-Ad
539
polyclonal sera with knob of Ad2 and Ad5 was analyzed by NDS-denatured Western
540
blotting. Anti-K12, 3, 1, 2, 5, 6, 37, 4 and 41 represent polyclonal anti-knob sera.
541
Anti-Ad12, 3, 1, 2, 5, 6, 37, 4 and 41 represent rabbit polyclonal anti-Ad sera. All sera
542
were tested at the dilution of 1:100.
543
Fig. 3. Seroprevalence of NAbs to Ad2 and Ad5 in healthy populations in China. (a) Ad2
21
544
and Ad5 NAb titers were detected in 496 human serum samples and were stratified by the
545
following titers: 1000 (high).
546
(b) Ad5 seronegative sera (light blue) and sera with low (green), medium (orange) or high
547
(red) NAb titers were used to determine the appropriate serum dilutions for Ad5
548
knob-based ELISA. Sera used at dilutions of 1:20, 1:100 and 1:1000 are indicated.
549
Fig. 4. Analysis of Ad5 knob-based ELISA results does not correlate with neutralizing
550
assay titers. (a) False positive serum samples were assessed against knob proteins of Ad1,
551
Ad2, Ad5 and Ad6 by NDS Western blotting. The > 200 and > 20 NAb titer groups and
552
serial number of human serum samples are indicated on the left. Serum containing both
553
baseline Ad2 and Ad5 NAbs as the positive control is shown at the bottom. Number
554
11-12 represent serum samples collected in 2011, and 2848, etc were collected in 2010.
555
(b) One hundred serum samples with baseline Ad2 NAbs were analyzed by Ad5
556
knob-based ELISA and Ad5 neutralizing assays. (c) One hundred Ad2 seropositive serum
557
samples were analyzed by Ad5 knob-based ELISA and Ad5 neutralizing assays. (d) Ad5
558
NAb titers were detected in all of the false negative samples (10, 2%) in the >200 NAb
559
titer group by neutralizing assay. (e) All of the false negative samples in the >200 NAb
560
titer group were analyzed by Western blotting against fiber, hexon, native knob and HVR.
561
(f) Median Ad5 NAb titers of the false negative sera in the > 20 and >200 NAb titer
562
groups are shown.
22
Table 1. Reactivity of rabbit hyperimmune sera against trimeric knob proteins detected by NDS-Western blotting Reaction pattern of sera directed against knob proteins or against purified virions† Antigen
AdK12/Ad12 [A] AdK3/Ad3 [B]
AdK1/Ad1 [C]
AdK2/Ad2 [C]
AdK5/Ad5 [C]
AdK6/Ad6 [C]
AdK37/Ad37 [D] AdK4/Ad4 [E]
AdK41/Ad41 [F]
knob12 [A]
+++/+++
-/-
-/-
-/-
-/-
-/-
-/-
-/-
-/-
knob3 [B]
-/-
+++/+++
-/-
-/-
-/-
-/-
-/-
-/-
-/-
knob1 [C]
-/-
-/-
+++/+++
-/-
-/-
-/+
-/-
-/-
-/-
knob2 [C]
-/-
-/-
-/-
+++/+++
-/+
+/++
+/-
-/+
-/-
knob5 [C]
-/-
+/-
+/+
-/-
++/+++
-/+
+/-
-/-
-/-
knob6 [C]
-/-
-/-
-/-
+/+
-/-
++/+++
+/-
-/-
-/-
knob37 [D]
-/-
-/-
-/-
-/-
-/-
-/-
+++/+++
-/-
-/-
knob4 [E]
-/-
-/-
-/-
-/-
-/-
-/-
-/-
++/+++
-/-
knob41 [F]
-/-
-/-
-/-
-/-
-/-
-/-
+/-
-/-
++/+++
†
Abbreviations for reaction intensity: -, no reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction.
All sera were tested at dilutions of 1:102 to 1:104.
23
Table 2. Comparison of Ad2 and Ad5 knob-based ELISA with neutralization assay
Prevalence
Cut-off value†
Seropositive rate
Coincidence rate
FPR
FNR
Ad2
0.253
64.9%
84.5%
1.4%
14.1%
Ad5
0.261
46.7%
82.3%
2.8%
14.9%
Ad2
0.302
56.7%
89.1%
7.5%
3.4%
Ad5
0.305
41.2%
90.7%
7.3%
2.0%
/ Serotypes Ad titer > 20
Ad titer > 200
†
OD values of serum samples with NAb titers 20
12.0%
31.4%
46.7%
57.5%
44.2%
47.4%
43.8%
19.7%
35.8%
NAb titer >200
4.4%
13.9%
36.5%
41.3%
27.4%
29.2%
24.8%
10.2%
16.2%
†
Percentages of Ad NAb titers >20 or >200 in all 274 children serum samples.
§Ad2
and Ad5 cut-off values were calculated as in Table 2 (based on Ad2 and Ad5 neutralization assays). For Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41, 36 sera samples from 7- to 12-month-old infants were considered as negative controls and used to determine the cut-off value. Data of NAb titer >200 for Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41 were calculated by the color reaction relative to that of Ad2 and Ad5.
25
1
Trimeric knob protein specifically distinguishes neutralizing
2
antibodies to different human adenovirus species: potential
3
application for adenovirus seroepidemiology
4 5
Bin Yu,1, 2 Jianing Dong,1 Chu Wang,1 Zhen Wang, 1 Lei Gao, 1 Haihong
6
Zhang,1, 2 Jiaxin Wu,1, 2 Wei Kong,1, 2, * and Xianghui Yu1, 2, *
7 8
1
9
University, Changchun, China
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin
10
2
11
School of Life Sciences, Jilin University, Changchun, China
12 13
*
14
2699 Qianjin Street, Changchun 130012, China. Phone: +86 431 85167826. Fax: +86 431
15
85167751. E-mail: [email protected] (W. Kong), [email protected] (XH. Yu)
Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education,
Corresponding author. Mailing address: School of Life Sciences, Jilin University, No.
16 17
Running Title: Trimeric knob for adenovirus NAbs serotyping
18
Word count: 5201; 3 tables and 4 figures
19
GenBank accession numbers: AB361388 (knob of Ad12), AB361380 (knob of Ad3),
20
AB361378 (knob of Ad1), AB361379 (knob of Ad2), AB361382 (knob of Ad5),
21
AB361383 (knob of Ad6), AB361413 (knob of Ad37), AB361381 (knob of Ad4),
22
AB361417 (knob of Ad41)
23 1
24
Summary
25
Adenoviruses (Ads) are nonenveloped DNA viruses that have been extensively studied
26
and used as vectors for gene therapy and several potential vaccines. There are 57 Ad
27
serotypes in seven species (A-G), and Ad neutralizing antibodies (NAbs) titers can vary
28
by serotypes and geographic location. Until now serotype- and species-specific antibodies
29
have been detected by neutralization or hemagglutination inhibition assays. These
30
expensive and cumbersome methods of adenovirus typing have mainly been used in
31
epidemiological studies. Our prior work demonstrated that NAbs against the fiber protein
32
are commonly generated during natural Ad infection in humans and the trimeric knob is
33
preferentially recognized by fiber-induced NAbs. In this study, we expressed nine
34
trimeric knob proteins from representative Ad serotypes of human Ad (HAdV)-A to -F in
35
Escherichia coli and found no cross-reactivity of these recombinant proteins with rabbit
36
hyperimmune sera (among HAdV-A to -F or within HAdV-C). Results of the ELISA
37
based on Ad2 and Ad5 (both HAdV-C) knob proteins were consistent with those of
38
neutralization assays, indicating that the trimeric knob protein would be a good candidate
39
antigen for detecting Ad serotype-specific NAbs in sera from naturally infected subjects.
40
We also demonstrated the primary seroepidemiology of nine Ad serotypes in 274
41
children samples by the knob-based ELISA. These results will have potential
42
implications for epidemiology of Ad serotypes and future development of Ad-based
43
vaccines and gene therapy.
44 45
Keywords: Adenovirus; neutralizing antibody; knob; cross-immunity; seroepidemiology
46
2
47
Introduction
48
Human adenoviruses (HAdVs) are non-enveloped DNA viruses that have been
49
extensively studied and used as vectors for vaccine and gene delivery. The major
50
limitation of the most well-studied adenovirus type 5 (Ad5) vaccine vector is the high
51
titers of naturally occurring Ad5 neutralizing antibodies (NAbs) in human populations,
52
particularly in the developing world (Mast et al., 2010; Yu et al., 2012b). Baseline Ad5
53
NAbs have been shown to suppress the immunogenicity of recombinant Ad5 (rAd5)
54
vector-based vaccines for HIV-1 in both preclinical studies (Casimiro et al., 2003;
55
Roberts et al., 2006) and clinical trials (Buchbinder et al., 2008; Kolavic-Gray et al.,
56
2002; Priddy et al., 2008). To address this and other problems with rAd5 vectors,
57
alternative human serotype rAd vectors (Abbink et al., 2007; Barouch et al., 2004;
58
Vogels et al., 2003), hexon-chimeric rAd vectors (Roberts et al., 2006) and rAd vectors
59
derived from other species (Farina et al., 2001; Fitzgerald et al., 2003) have been
60
constructed. All of these studies highlight the importance of understanding differences in
61
virulence and seroepidemiology among the various Ad serotypes.
62
Currently, 57 human Ad serotypes are defined and distributed into seven species (or
63
subgroups) from A to G (Walsh et al., 2011). Rare serotypes such as Ad14 and Ad55
64
have been considered to be responsible for most of the outbreaks of acute respiratory
65
disease among school and military recruits, and an emerging variant (HAdV-14p1) was
66
associated with the high rates of illness and death in United States and Europe (Carr et al.,
67
2011; Zhang et al., 2012). Diagnosis of Ad infections is currently based on virus isolation
68
in cell culture and genus-specific antibody and antigen detection by enzyme
69
immunoassays (Weinberg et al., 1989), as well as Ad DNA detection by PCR (Berk, 3
70
2007). Infection-induced humoral immunity to a particular serotype does not provide
71
cross-immunity to an Ad serotype of another subgroup (Hierholzer et al., 1991). Thus,
72
Ad serotypes are determined based on inhibition by specific immune sera and
73
phylogenetic analysis of DNA sequences (Takeuchi et al., 1999), while serotype-specific
74
NAbs are detected by viral vector-based neutralization assays (Aste-Amezaga et al., 2004;
75
Sprangers et al., 2003). These Ad typing methods have been used in epidemiological
76
studies on the prevalence of pre-existing NAbs to several conceptually interesting vaccine
77
vector candidate types (Mast et al., 2010; Pereira, 1959). Although neutralization assays
78
are efficient means for detecting specific antibodies against each Ad serotype, they are
79
cumbersome and expensive to perform. Therefore, more convenient typing methods for
80
NAbs are needed to analyze Ad serotype infections.
81
There are three major capsid proteins on Ad particles: fiber, penton base and hexon. Both
82
the fiber and hexon proteins contain group-specific as well as type-specific epitopes
83
(Norrby, 1969; Rux & Burnett, 2000). The fiber protein is composed of three portions, a
84
tail anchoring the fiber into the vertex capsomer, a shaft and a knob containing the
85
domain responsible for interaction with the host cell receptor (Khare et al., 2011). The
86
globular knob domain in the carboxy-terminal segment of the fiber protein includes
87
175–220 amino acid residues, which vary among different serotypes. Our prior studies
88
have emphasized the contribution of anti-fiber antibodies to Ad5 neutralization responses
89
generated during natural viral infection and rAd5 immunizations, and other group also
90
showed that the trimeric knob was preferentially recognized by fiber induced NAbs
91
(Gahery-Segard et al., 1998; Yu et al., 2013). We demonstrated that NAbs against fiber
92
or knob are commonly generated in natural Ad infection in humans and observed no
4
93
cross-immunity of the sera naturally infected with Ad2 and Ad5 (both species C) by
94
reactivity with two trimeric knob antigens in Western blotting assays (Yu et al., 2013).
95
In this study, we further analyzed the cross-reactivity of the trimeric knob with rabbit
96
hyperimmune sera among HAdV-A to -F and attempted to establish a knob-based ELISA
97
for Ad epidemiological studies. We showed that the stable trimeric knob protein was
98
specifically recognized by Ad NAbs in all six species especially in HAdV-C, indicating
99
that it would be a good candidate antigen to detect serotype-specific NAbs in natural Ad
100
infection and rAd vaccination.
101
Results
102
Expression of trimeric knob proteins used for serotype-specific NAb
103
detection
104
Prior phylogenetic analyses of fiber knob amino acid sequences derived from 45
105
serotypes of HAdV-A to -F showed a good correlation with the classification based on
106
classical subtyping assays (i.e., HA inhibition and cross-neutralization assays) (Havenga
107
et al., 2002). While the identities between the knob sequence of Ad4 in species E and the
108
serotypes of HAdV-C are very high, the genome sequence of Ad4 is more closely related
109
to Ads of species B. In order to analyze the species (HAdV-A to-F) and serotype
110
cross-reactivity of the knob proteins with rabbit hyperimmune sera or Ad NAbs, nine
111
representative Ad serotypes of each species (HAdV-A; Ad12, HAdV-B; Ad3, HAdV-C;
112
Ad1, 2, 5 and 6, HAdV-D; Ad37, HAdV-E; Ad4, HAdV-F; Ad41) were selected to
113
derive the knob antigens (only the long fiber knob of Ad41 was used for the analysis as it
114
has an analogous function with other types) and rabbit polyclonal sera. To obtain the
5
115
trimeric knob protein, the knob domains with the last repeat in the shaft of the fiber were
116
expressed as His-tagged fusion proteins in E. coli. As shown in Fig. 1(a), all of the knob
117
proteins of the indicated types migrated as trimers in the native gel, and these trimeric
118
proteins were stable even in the presence of 1% SDS in this study. The trimerization of
119
the purified proteins was also confirmed by size-exclusion chromatography (data not
120
shown). After immunization with these purified trimeric proteins in rabbits, NAbs against
121
Ad2 and Ad5 were found (we only have rAd2-luc and rAd5-luc) in the corresponding
122
hyperimmune sera and showed no cross-neutralization among these Ad serotypes even
123
within the same species (Fig. 1b and c). These results further confirmed that trimeric
124
knob proteins can induce serotype-specific Ad NAbs as we have previously described
125
(Yu et al., 2013).
126
Reactivity of rabbit hyperimmune sera raised against trimeric knob
127
antigens or whole virions displaying knob antigens
128
To confirm that the trimeric knob protein can serve as an antigen for distinguishing
129
serotype specific responses, rabbit hyperimmune sera directed against various knob
130
antigens were tested for cross-reactivity. The rabbit Ad5 knob antiserum recognized the
131
monomeric knob antigens of all nine serotypes but only strongly reacted to the trimeric
132
form of Ad5 knob protein, and this reaction pattern was similar to that of rabbit and
133
human antisera raised against Ad5 virions (Fig. 2a). Furthermore, no or very weak
134
cross-reactivity of the trimeric knob protein of Ad2 and Ad5 was shown with antisera
135
raised against the trimeric knob antigens or whole virions of other Ad serotypes (Fig. 2b).
136
The reactivities of sera raised against recombinant knob antigens or against purified
137
virions with trimeric knob proteins of nine Ad serotypes are summarized in Table 1. Only 6
138
a few serum samples raised against recombinant proteins weakly cross-reacted with
139
trimeric knob from other serotypes in the same Ad species at serum dilutions of 1:102 to
140
1:104. By contrast, antisera raised against whole virions showed stronger and more
141
specific levels of reactivity with the corresponding trimeric knob proteins. Overall, these
142
results indicated no cross-immunity in rabbit antisera raised against HAdV-A to -F
143
species with trimeric knob proteins of HAdV-C and the representative antigens of other
144
species.
145
Use of knob proteins for detection by ELISA of NAbs in human sera
146
after natural Ad infection
147
After demonstrating the specificity of the trimeric knob proteins using the Ad rabbit
148
hyperimmune sera above, we set out to establish an ELISA system for typing Ad NAbs in
149
human sera. To determine the reliability of the ELISA method using knob as the antigen
150
for detecting Ad NAbs, a total of 496 serum samples from healthy donors in China were
151
first screened for the prevalence of Ad2 and Ad5 NAbs by a luciferase-based
152
neutralization assay. As shown in Fig. 3(a), Ad2 and Ad5 seroprevalence rates were
153
69.39% and 57.25% in all subjects, respectively. It was found that 54.06% of participants
154
had baseline Ad2 NAb titers > 200, compared with 43.64% for Ad5. To determine the
155
optimal serum dilution for the knob-based ELISA, 24 serum samples were selected at
156
random from each Ad5 NAb titer group and diluted at 1:20, 1:100 and 1:1000. The
157
dilution of 1:100 was found to be suitable for detecting NAbs in human sera by ELISA,
158
since OD values of the sera showed a relatively good correlation with different NAb titer
159
groups (1000) (Fig. 3b). Subsequently, all human sera were
160
tested by ELISA with Ad2 and Ad5 knob proteins. As shown in Table 2, the ELISA 7
161
results were compared with those of the neutralization assays using different cut-off
162
values. When using the neutralization titer of < 20 as the cut-off value for seronegative
163
samples, the seropositivity rate was slightly lower by ELISA, with the coincidence rate of
164
over 80% compared with the neutralization assay. Meanwhile, the false negative rates
165
(FNR) were 14.1% for Ad2 and 14.9% for Ad5. When using the baseline neutralization
166
titer of 200 as the cut-off value, we found coincidence rates of nearly 90% for both of the
167
Ad types. These results indicated that the trimeric knob protein could be used for the
168
detection of serotype-specific NAbs.
169
Analysis of knob-based ELISA data does not to correlate with
170
neutralizing assay
171
To determine the cause of false positive results in the Ad5 knob ELISA, the knob
172
proteins of Ad1, 2, 5 and 6 in HAdV-C were used for Western blot analysis of the serum
173
samples. In the > 200 titer group, the relatively high false positive rate (FPR) of 7.3%
174
was mainly attributed to the low Ad5 NAb titer (20–200) of sera infected with either Ad5
175
alone or multiple serotypes (samples 11-12 and 2848). However, in the > 20 NAb titer
176
group, we found that very few samples with high infection levels of other serotypes such
177
as Ad1 in HAdV-C seemingly could cause the positive responses to Ad2, 5 and 6 (sample
178
2746) (Fig. 4a). To determine the extent of this cross-reactivity, a total of 100 Ad2
179
seropositive samples (not tested in neutralization assays with other rAds) with NAb
180
titers > 20 or >200 were tested with the Ad5 knob protein by ELISA and Ad5
181
neutralization assay. The results showed that 46 of serum samples with NAb titers > 20
182
were detected by the Ad5 knob protein ELISA, three of which were not detected by the
183
neutralization assay (Fig. 4c). Meanwhile, 40 serum samples with NAb titers >200 were 8
184
detected by the Ad5 knob protein ELISA, nine of which were not detected by the
185
neutralization assay (Fig. 4b). Further analysis by Western blotting showed that only 3 of
186
the 12 samples which had not been detected by the Ad5 neutralization assays were false
187
positives in the Ad2 neutralization assay (data not shown). To analyze the detection
188
efficiency of the knob-based ELISA, all of the false negative samples (10, 2%) in the
189
NAb titer >200 group were analyzed by Western blot using native knob, HVR and virions
190
for analysis of fiber- and hexon-specific antibodies as described previously (Yu et al.,
191
2013). Results showed that all of these samples were anti-knob and anti-HVR negative
192
but were anti-fiber positive, and three of them were anti-hexon positive (Fig. 4e). Nearly
193
all of the samples had NAb titers 20 and >200, respectively (Fig. 4d and f). These results
195
indicated that some serum samples with relatively low Ad5 NAb titers did not have NAbs
196
against fiber (knob) and hexon (HVR) and could not be detected by trimeric Ad5 knob.
197
Prevalence of Ads detected by knob-based ELISA
198
As the participants from children are less likely to have multiple Ad infections, a total of
199
274 children sera from Jilin province (northeast China) were screened for the prevalence
200
of Ad antibodies by ELISA using knob proteins of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad12,
201
Ad37 and Ad41 (Table 3). For Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41, 36 serum
202
samples from 7- to 12-month-old infants were considered as negative controls and used to
203
determine the cut-off value (removal of very high OD values), since the seroprevalence of
204
Ad2 and Ad5 were very low in this age group and prior studies have also reported that
205
children in this age group have the lowest levels of Ad NAbs (Appaiahgari et al., 2007;
206
Mast et al., 2010). Results of the ELISA showed that 44.2–57.5% and 27.4–41.3% 9
207
seropositivity in these children against in HAdV-C types with NAb titer cut-off values
208
of >20 and >200, respectively. By contrast, this infant population showed lower infection
209
levels with the other several serotypes especially Ad12 in HAdV-A which was detected in
210
12% of the samples were seropositive (titer >20) and only 4.4% showing moderate to
211
high NAb titers (titer >200). However, Ad37 in HAdV-D also showed a relatively high
212
level of NAb seropositivity.
213
Discussion
214
Ads are the cause of common upper respiratory infections, and thus most individuals
215
have circulating antibodies to these viruses. Pre-existing immunity to Ad is an important
216
issue in clinical trials with rAd vector-based vaccines as it can limit in vivo delivery of
217
the vectors and gene transfer (Roberts et al., 2006). Thus, understanding the nature of
218
anti-Ad immunity and mapping the targets of dominant Ad-specific NAbs in human sera
219
is important for the development of vaccines against HIV-1 and other infections or
220
diseases. Our prior studies showed that NAbs against Ad5 fiber or knob are commonly
221
generated in natural Ad infection and preferentially recognize the trimeric knob protein
222
(Yu et al., 2013). We assumed that the knob protein from different Ad serotypes would
223
specifically recognize NAbs of the same Ad serotype since NAbs induced by different Ad
224
serotypes typically do not show cross-immunity. Although we previously showed that the
225
pattern of reactivity of human sera indicated no cross-immunity against trimeric knob
226
proteins of Ad2 and Ad5 of the HAdV-C species, more knob proteins from other Ad
227
serotypes still needed to be examined to evaluate the cross-immunity of sera raised
228
against viruses in different human Ad species as the widely used classification is based
229
on hemagglutinating antigens (Berk, 2007).
10
230
In this study, we expressed four knob proteins from HAdV-C and other five
231
representative knob proteins of HAdV-A, -B, -D, -E and -F in E. coli to analyze the
232
reactivity and cross-reactivity of NAbs to knob antigens produced in rabbit hyperimmune
233
sera, which may be used to guide investigations in Ad antibodies generated in humans.
234
The pattern of reactivity of the rabbit and two types of human sera (Yu et al., 2013)
235
indicated there was no cross-immunity against trimeric knob proteins of the HAdV-A to
236
-F species. In Fig. 2 and Table 2, Anti-K5, Anti-Ad5 and hAnti-Ad5 were all at dilution
237
of 1:100 to 1:10000 and showed differently binding activities with these knob proteins in
238
SDS and NDS westerns. We tend to think it is related with the fact that when the
239
immunogens were the trimeric knob proteins (with adjuvant and other factors), it
240
produced much of binding antibodies (IgG) resulting in cross-reactivity in SDS western.
241
However, antisera raised against whole virions or human sera with natural adenovirus
242
infection produced much more NAbs and less binding antibodies, thence showed weaker
243
binding with the denatured proteins but stronger and more specific reactivities with the
244
corresponding trimeric knob proteins, as the trimeric knob proteins are preferentially
245
recognized by NAbs against Ad fiber. These results indicated that trimer knob protein
246
would be very suitable for the detection of serotype-specific NAbs in sera of
247
immunocompetent population after natural Ad infection and rAd vaccination. The ELISA
248
results of human sera with Ad2 and Ad5 knob were consistent with those of the
249
neutralization assays and the different levels of antibodies to each knob in the same
250
serum sample, further confirming our above hypothesis on knob recognition among Ad
251
serotypes, although it would be more convincing if we could do more comparisons
252
among different Ad serotypes.
11
253
Of the more than 50 human Ad serotypes occurring worldwide, the most commonly used
254
rAd vector is Ad5, although its high seroprevalence in the human population has
255
warranted investigations of other serotypes. The evaluation of serotype-specific NAbs as
256
a means of selecting the adequate serotype adenovector(s) is important for both clinical
257
applications and epidemiological studies (Piedra et al., 1998; Sanchez et al., 2001).
258
However, the lower replication and difficult construction of some rAds will limit the
259
application of the quantitative neutralization assay based on rAds carrying a reporter gene
260
for additional Ad serotypes. In this study, we developed a knob-based ELISA to
261
determine Ad serotype-specific antibody responses. By using this assay, we detected the
262
NAbs seroprevalence of two serotypes (Ad2 and Ad5) from the same species and found
263
that the results had a high correlation with those of the standard neutralization assays
264
although still had low FPR and FNR caused by dilutions of sera and other reasons. A
265
problem noted with this assay was that it did not detect approximately 10% of the human
266
serum samples in this study determined to be seropositive by the neutralization assay, as
267
the sera did not recognize either the native knob or HVR antigens, which is consistent
268
with previous studies (Cheng et al., 2009). Further analysis showed that nearly all of
269
these undetected samples had low (20 < titer < 200) or moderate (200 < titer < 1000)
270
NAb titers. This indeed partially limits the application of the knob based ELISA for
271
adenovirus seroepidemiology and clinical diagnosis. Nevertheless, since some previous
272
studies showed no influence of low levels of Ad5 NAb on the immunogenicity and
273
protective efficacy of rAd5 vectors in humans and mice (Appaiahgari et al., 2006;
274
Buchbinder et al., 2008), our new method still could be suitable for testing moderate and
275
high level Ad infections to guide the application of rAd-based vaccine and gene therapy.
12
276
Non-capsid specific humoral immune response in human sera from natural Ad infection
277
are still needed to be studied to find a detection antigen for the Ad seropositive serum
278
samples without NAbs against the capsid protein.
279
The Ad fiber protein is primarily responsible for the high affinity attachment of the virus
280
to its receptor expressed on the surface of target cells. The fiber knob domain confers
281
specificity for cellular receptors. Most Ad serotypes including all those within HAdV-C
282
use the coxsackie and Ad receptor (CAR) as the primary attachment receptor (Bergelson
283
et al., 1997; Berk, 2007). However, the fiber protein of species D Ad37 can bind to CAR,
284
CD46 and sialic acid, but it appears only able to use sialic acid as a receptor for infection
285
(Arnberg et al., 2000; Wang et al., 2011). This effect may be due to the very short length
286
of species D fibers with only eight β-spiral repeats. Despite the differences in structure
287
and function, we also found that the trimeric Ad37 knob protein was strongly recognized
288
by serum NAbs induced by Ad37 but not those by other selected Ads. This result was
289
consistent with our previous speculation and prior studies determining that the receptor
290
binding regions in knob are relatively conservative and that the epitopes are
291
hypervariable and located in the outer loops or uppermost β-sheets in the structure
292
(Marttila et al., 2005; Zhang & Bergelson, 2005). Therefore, these results indicated that
293
our knob-based ELISA method may be expanded for detection of additional Ad serotypes.
294
Previous studies showed that Ad1, 2, 5 and 6 are ubiquitous, particularly in young
295
children. Antibodies to Ad1, 2, and 5 are most common and are present in 40–60% of
296
children (Badger et al., 1956; Brandt et al., 1969), while the incidence of NAbs to Ad26,
297
35 and 48 is low in the same age group (Barouch et al., 2011; Pereira, 1959). In this study,
298
we showed an analogous trend in Chinese children with apparently higher seroprevalence
13
299
of Ad1, 2, 5, 6 and 37 than those of Ad3, 4, 12and Ad41.
300
In conclusion, this study highlights the utility of trimeric knob proteins to analyze
301
serotype-specific NAbs in HAdV-C and other species. Furthermore, we have shown that
302
the knob-based ELISA would be used to test the prevalence of several Ad serotypes. Our
303
study contributes to the understanding of natural Ad capsid-specific humoral immune
304
responses and serotype-specific NAbs in humans and may have implications in
305
seroepidemiology of Ads.
306
Methods
307
Cell lines and serum. Human embryonic kidney (HEK) 293 cells were obtained from the
308
American Type Culture Collection (ATCC; Manassas, VA). All cells were cultured in
309
Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA) containing
310
10% fetal bovine serum, (FBS; Hyclone; Logan, UT), 2 mM L-glutamine, 100 U/ml
311
penicillin and 100 μg/ml streptomycin (Invitrogen).
312
Recombinant Ad antigens. The sequence of Ad knob including the last repeat in the
313
shaft of the fiber (Seiradake et al., 2006; Yeh et al., 1994) was cloned into the pRSET-B
314
plasmid (Invitrogen) at the Nco Ι and Xho Ι restriction sites. The plasmid containing an
315
N-terminal His tag was expressed in BL21 cells overnight at 16°C. The pellet from a
316
2-liter culture was treated with 1 mg of lysozyme/ml on ice and sonicated 10 times for 10
317
s each time. After centrifugation at 40,000 × g for 30 min, 10 mM imidazole was added
318
to the soluble fraction at 4°C with gentle stirring. The resin was filled in a small
319
polyethylene column and washed with 20 mM imidazole in a buffer containing 25 mM
320
Tris-HCl (pH 7.5) and 150 mM NaCl. The protein was eluted with 500 mM imidazole in
14
321
the same buffer and analyzed by SDS-PAGE. The Ad5 hexon hypervariable regions
322
(HVR) protein was expressed as described previously (Yu et al., 2012a).
323
Hyperimmune sera. Rabbit polyclonal sera directed against the knob proteins of Ad1,
324
Ad2, Ad3, Ad4, Ad5, Ad6, Ad12, Ad37 and Ad41 were produced in rabbits given three
325
immunizations with purified proteins (300 μg per injection) together with Freund’s
326
complete and incomplete adjuvants. All animal studies were approved by the University
327
Committee on the Use and Care of Animals of Jilin University. Anti-Ad1, Ad2, Ad3, Ad4,
328
Ad5, Ad6, Ad12, Ad37 and Ad41 rabbit polyclonal antibody sera were gifts from
329
Panyong Mao (Beijing 302 Hospital, Beijing, China).
330
Human serum. Human serum samples were collected randomly during 2010–2011 in
331
China by the Provincial Centers for Disease Control and Prevention. Donors of both
332
sexes ranged in age from 1 day to 78 years and had no history of febrile illness during the
333
few weeks preceding sample collection. Informed consent was obtained from all
334
participants or their guardians in written form to utilize the serum samples in Ad
335
neutralization assays, as approved by the Human Ethical Committee Institutional Review
336
Board, Jilin University, China. All human sera were inactivated at 56°C for 60 min prior
337
to testing.
338
Adenovirus infection assay. HEK293 cells were grown in 6-well plates and were
339
infected with the Ad vectors at an MOI of 10 with or without rabbit anti-knob sera. After
340
incubation at 37°C for 48 h, the cytopathic effect (CPE) of 293 cells was observed at a
341
low magnification (200×) by microscopy.
342
Virus neutralization assays. Ad-specific NAb titers in human serum samples and rabbit
343
hyperimmune sera were assessed by luciferase-based assays as described previously
15
344
(Sprangers et al., 2003). HEK293 cells were plated at a density of 1 × 104 cells per well
345
in 96-well plates and infected with replication-incompetent rAd-Luc reporter constructs
346
at a multiplicity of infection (MOI) of 500 with 2-fold serial dilutions of serum in 200 µl
347
reaction volumes. Following 24-hour incubation, luciferase activity in the cells was
348
measured using the Steady-Glo Luc reagent system (Promega, Madison, WI) with a
349
Victor 1420 Multilabel Counter (Perkin Elmer, Wellesley, MA). Neutralization titers
350
were defined as the maximum serum dilution that inhibited 90% of luciferase activity.
351
Western blotting analysis. SDS-PAGE was performed on 12% SDS-polyacrylamide gels.
352
Samples were denatured by boiling at 98°C in Laemmli buffer (62.5 mM Tris-HCl, 2%
353
SDS, 1% β-mercaptoethanol, 25% glycerol, 0.01% bromophenol blue) prior to
354
electrophoresis. NDS-PAGE was similar to SDS-PAGE, except that the samples were
355
neither boiled nor exposed to β-mercaptoethanol in order to retain stable trimeric proteins.
356
Native-PAGE differed from SDS-PAGE in that the proteins were not denatured by boiling
357
prior to electrophoresis on SDS-free 6-8% Tris-acetate gels (Invitrogen) and running
358
buffer, and the samples were diluted in native buffer (62.5 mM Tris-HCl, 40% glycerol,
359
0.01% bromophenol blue).
360
Protein samples were electrophoresed by SDS-PAGE, NDS-PAGE or Native-PAGE and
361
transferred to nitrocellulose membranes for Western blotting. SDS was not added into the
362
transfer buffer (39 mM glycine, 48 mM Tris-HCl, 20% methanol, pH 9.2) when proteins
363
were separated by Native-PAGE. Membranes were incubated with different human,
364
rabbit and mouse sera overnight at 4°C in PBS containing 1.0% milk and then
365
subsequently incubated with ImmunoPure alkaline phosphatase-conjugated goat
366
anti-rabbit IgG secondary Abs (Jackson ImmunoResearch Laboratories) at a 1/10,000
16
367
dilution for 1 h. The color reaction was developed with 0.1 M Tris-HCl (0.1 M NaCl, 5
368
mM MgCl2, pH 9.5) containing 0.66% NBT solution and 0.33% BCIP solution.
369
Enzyme-linked immunosorbent assay (ELISA). Serum antibody titers were measured
370
by ELISA using a horseradish peroxidase (HRP) detection system. Each well of the
371
96-well polyvinylchloride microtiter plates was coated with knob proteins (200 ng/ 100
372
μl) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. The plates were then
373
washed three times with PBS-T (20 mM PBS containing 0.05% Tween-20) and blocked
374
with 2% BSA/PBS-T at 37°C. After three washes, different dilutions of serum were
375
added to the plate (100 μl per well) and incubated for 1 h at 37°C. After another three
376
washes, a 1/100,000 dilution of HRP-conjugated goat anti-human IgG (Proteintech Group,
377
Chicago, IL) was added to each well and incubated for 1 h at 37°C. After the final three
378
washes, TMB peroxidase substrate was added to the plates, incubated for 15 min in a
379
darkroom, stopped with 50 μl of 2 M H2SO4 and analyzed using dual wavelengths of
380
450/630 nm with an iMark Microplate reader (Bio-Rad, Hercules, CA).
381
Acknowledgments
382
This study was supported by the National Nature Science Foundation of China (No.
383
30872396, 31300756) and the Key Projects in the National Science & Technology Pillar
384
Program in the Twelfth Five-year Plan Period (Grant 2012ZX10001-009). We gratefully
385
acknowledge Panyong Mao (Beijing 302 Hospital, Beijing, China) for providing the
386
anti-Ad rabbit polyclonal sera and acknowledge Ling Chen (Guangzhou Institute of
387
Biomedicine and Health, Guangzhou, China) for providing the rAd2 vectors. The authors
388
also wish to acknowledge Thi Sarkis for editorial support in the preparation of this
389
manuscript. 17
390
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20
522
Figure legends
523
Fig. 1. Expression of trimeric knob proteins from Ad serotypes in HAdV-A to -F and
524
analysis of NAb titers in rabbit hyperimmune sera against knob. (a) Knob proteins were
525
analyzed by NDS-PAGE and SDS-PAGE. (b) Neutralizing activity of anti-knob
526
polyclonal sera was analyzed between Ad2 and Ad5 following infection of 293 cells. 293
527
cells were infected with the indicated Ad vectors with or without anti-knob polyclonal
528
sera at an MOI of 10 PFU/cell. ‘‘Anti-K2’’ and ‘‘Anti-K5’’ are serums obtained from
529
rabbits with knob proteins of Ad2 and Ad5 immunizations and used at a dilution of 1:200.
530
(c) Ad NAbs induced by knob proteins were determined by luciferase-based rAd2 and
531
rAd5 neutralization assays. Knob proteins derived from human Ad species A to F
532
(HAdV-A; Ad12, HAdV-B; Ad3, HAdV-C; Ad1, 2, 5 and 6, HAdV-D; Ad37, HAdV-E;
533
Ad4, HAdV-F; Ad41).
534
Fig. 2. Reactivity of rabbit hyperimmune sera raised against the trimeric knob antigens or
535
whole virions with knob antigens by Western blotting. (a) Reactivity of rabbit anti-K5
536
and anti-Ad5 rabbit polyclonal sera and human anti-Ad5 (hAnti-Ad5, NAb positive for
537
Ad5 alone) serum with knob antigens of nine Ad serotypes was analyzed by
538
SDS-denatured or NDS Western blotting. (b) Reactivity of anti-knob and anti-Ad
539
polyclonal sera with knob of Ad2 and Ad5 was analyzed by NDS-denatured Western
540
blotting. Anti-K12, 3, 1, 2, 5, 6, 37, 4 and 41 represent polyclonal anti-knob sera.
541
Anti-Ad12, 3, 1, 2, 5, 6, 37, 4 and 41 represent rabbit polyclonal anti-Ad sera. All sera
542
were tested at the dilution of 1:100.
543
Fig. 3. Seroprevalence of NAbs to Ad2 and Ad5 in healthy populations in China. (a) Ad2
21
544
and Ad5 NAb titers were detected in 496 human serum samples and were stratified by the
545
following titers: 1000 (high).
546
(b) Ad5 seronegative sera (light blue) and sera with low (green), medium (orange) or high
547
(red) NAb titers were used to determine the appropriate serum dilutions for Ad5
548
knob-based ELISA. Sera used at dilutions of 1:20, 1:100 and 1:1000 are indicated.
549
Fig. 4. Analysis of Ad5 knob-based ELISA results does not correlate with neutralizing
550
assay titers. (a) False positive serum samples were assessed against knob proteins of Ad1,
551
Ad2, Ad5 and Ad6 by NDS Western blotting. The > 200 and > 20 NAb titer groups and
552
serial number of human serum samples are indicated on the left. Serum containing both
553
baseline Ad2 and Ad5 NAbs as the positive control is shown at the bottom. Number
554
11-12 represent serum samples collected in 2011, and 2848, etc were collected in 2010.
555
(b) One hundred serum samples with baseline Ad2 NAbs were analyzed by Ad5
556
knob-based ELISA and Ad5 neutralizing assays. (c) One hundred Ad2 seropositive serum
557
samples were analyzed by Ad5 knob-based ELISA and Ad5 neutralizing assays. (d) Ad5
558
NAb titers were detected in all of the false negative samples (10, 2%) in the >200 NAb
559
titer group by neutralizing assay. (e) All of the false negative samples in the >200 NAb
560
titer group were analyzed by Western blotting against fiber, hexon, native knob and HVR.
561
(f) Median Ad5 NAb titers of the false negative sera in the > 20 and >200 NAb titer
562
groups are shown.
22
Table 1. Reactivity of rabbit hyperimmune sera against trimeric knob proteins detected by NDS-Western blotting Reaction pattern of sera directed against knob proteins or against purified virions† Antigen
AdK12/Ad12 [A] AdK3/Ad3 [B]
AdK1/Ad1 [C]
AdK2/Ad2 [C]
AdK5/Ad5 [C]
AdK6/Ad6 [C]
AdK37/Ad37 [D] AdK4/Ad4 [E]
AdK41/Ad41 [F]
knob12 [A]
+++/+++
-/-
-/-
-/-
-/-
-/-
-/-
-/-
-/-
knob3 [B]
-/-
+++/+++
-/-
-/-
-/-
-/-
-/-
-/-
-/-
knob1 [C]
-/-
-/-
+++/+++
-/-
-/-
-/+
-/-
-/-
-/-
knob2 [C]
-/-
-/-
-/-
+++/+++
-/+
+/++
+/-
-/+
-/-
knob5 [C]
-/-
+/-
+/+
-/-
++/+++
-/+
+/-
-/-
-/-
knob6 [C]
-/-
-/-
-/-
+/+
-/-
++/+++
+/-
-/-
-/-
knob37 [D]
-/-
-/-
-/-
-/-
-/-
-/-
+++/+++
-/-
-/-
knob4 [E]
-/-
-/-
-/-
-/-
-/-
-/-
-/-
++/+++
-/-
knob41 [F]
-/-
-/-
-/-
-/-
-/-
-/-
+/-
-/-
++/+++
†
Abbreviations for reaction intensity: -, no reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction.
All sera were tested at dilutions of 1:102 to 1:104.
23
Table 2. Comparison of Ad2 and Ad5 knob-based ELISA with neutralization assay
Prevalence
Cut-off value†
Seropositive rate
Coincidence rate
FPR
FNR
Ad2
0.253
64.9%
84.5%
1.4%
14.1%
Ad5
0.261
46.7%
82.3%
2.8%
14.9%
Ad2
0.302
56.7%
89.1%
7.5%
3.4%
Ad5
0.305
41.2%
90.7%
7.3%
2.0%
/ Serotypes Ad titer > 20
Ad titer > 200
†
OD values of serum samples with NAb titers 20
12.0%
31.4%
46.7%
57.5%
44.2%
47.4%
43.8%
19.7%
35.8%
NAb titer >200
4.4%
13.9%
36.5%
41.3%
27.4%
29.2%
24.8%
10.2%
16.2%
†
Percentages of Ad NAb titers >20 or >200 in all 274 children serum samples.
§Ad2
and Ad5 cut-off values were calculated as in Table 2 (based on Ad2 and Ad5 neutralization assays). For Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41, 36 sera samples from 7- to 12-month-old infants were considered as negative controls and used to determine the cut-off value. Data of NAb titer >200 for Ad1, Ad3, Ad4, Ad6, Ad12, Ad37 and Ad41 were calculated by the color reaction relative to that of Ad2 and Ad5.
25
