Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C

<div><p>Background</p><p>Human rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects.</p><p>Methods</p><p>Recombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults.</p><p>Results</p><p>Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (<i>P</i><0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies.</p><p>Conclusions</p><p>High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.</p></div

Inhibition of IgG1 binding to HRV-C3 VP1 by HRV-A or HRV-B and HPV Sabin.

<p>Inhibition by lysate containing HRV-A, or a mixture of HRV-B and HPV Sabin in 30 subjects with high total IgG1 titres to HRV-C3. Percentage of cross-reactivity to the inhibiting species was calculated by (Titre after absorption/Total IgG1)×100. <i>P</i><0.0001 between inhibition by HRV-A and inhibition by HRV-B and HPV Sabin. Three negative control sera and a titration of reference sera were included on every plate to assess reproducibility and quantitation of IgG1 binding.</p

Total IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults.

<p>The geometric mean and 95% confidence interval are indicated. Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay.</p

Antibody responses to HRV-C VP1 with an N-terminal truncation in 30 adult subjects.

<p>(A) IgG1 binding (ng/ml) to truncated HRV-C3 VP1 expressed from residue 14 to 275, in comparison to full-length VP1. <i>P</i><0.0001 between full-length and truncated HRV-C3 VP1. (B) Correlation of IgG1 binding (ng/ml) between full-length HRV-C3 VP1 pre-absorbed with HRV-A to remove cross-reactivity to HRV-A, and truncated HRV-C3 VP1. A reference sera and three negative sera that did not have IgG1 binding to HRV-C3, as determined by the immunoassays and immunoabsorptions, were included on every plate for quantitation of binding.</p

Amino acid sequence identity for six HRV VP1 and HPV Sabin 1 VP1.

<p>Amino acid sequence identity for six HRV VP1 and HPV Sabin 1 VP1.</p

Cross-reactivity between HRV-A and HRV-C.

<p>(A) An example of strong cross-inhibition in HRVA34 and HRV-C3 double-positive sera. (B) An example of poor cross-inhibition between HRV-A34 and HRV-C3 observed in a minority of subjects who had high IgG1 titres to both HRV-A and HRV-C. The inhibition of binding to HRV-A34 and HRV-C3 by an irrelevant recombinant antigen (bacterial protein, Psp-C), used as a negative control, are also indicated.</p

Species-specific IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults.

<p>Each sera was absorbed with lysates containing the VP1 of the other HRV species, as well as HPV Sabin VP1. IgG1 binding to HRV-C (HRV-C3 and HRV-C5) antigens were significantly lower than HRV-A, HRV-B and HPV Sabin titres (<i>P</i><0.0001) as indicated by **. Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay.</p

An example of the level of cross-reactivity found between HRV species and HPV Sabin VP1 in an individual’s serum.

<p>(A) HRV-A VP1 antigen inhibited by HRV-B, HRV-C and HPV Sabin antigens. (B) HRV-B VP1 antigen inhibited by HRV-A, HRV-C and HPV Sabin antigens. (C) HRV-C VP1 antigen inhibited by HRV-A, HRV-B and HPV Sabin antigens. (D) HPV Sabin antigen inhibited by antigens representing the three HRV species. An irrelevant recombinant antigen (Fel d 3) was used as a negative control for each competitive inhibition assay (data not shown).</p

Percentages of the secondary structure of the circular dichroism (CD) spectra of HRV VP1 antigens following subtraction of GST^*.

*<p>Analysed using DiChroWeb Server: CDSSTR algorithm, reference set 4.</p

Purification and SDS-PAGE analysis of recombinant HRV-C3 VP1 pGEX-2T.

<p>(A) Chromatograph of HRS300 size exclusion chromatography immediately following affinity chromatography (Run 1). The void volume and the peak at 660 kDa are indicated above each of the main peaks. Each fraction has a 5 ml elution volume. Fractions containing the 660 kDa peak from Run 1 were pooled and concentrated for a second run and third run on the HRS300. The bar indicates fractions used for subsequent runs and for binding analysis. (B) A 10 µL aliquot of the pooled fractions containing the 660 kDa peak collected from the first run of gel filtration (as indicated by *) was analysed by SDS-PAGE. The MW of the protein standard is indicated.</p