Researchers in New York City and Guangzhou, China applied the rapidly-developing carbohydrate microarray technology to study an inactivated SARS-coronavirus (SARS-CoV) vaccine and discovered autoimmunogenic activity of this newly identified human viral pathogen.
Using glycan microarrays, the researchers characterized the carbohydrate binding activity of SARS-CoV neutralizing antibodies elicited by an inactivated SARS viral vaccine and found:
- An undesired autoantibody reactivity is present in SARS-CoV neutralization antibodies
- the autoimmune reactivity is directed toward the complex carbohydrate of an abundant human serum glycoprotein, ASOR (asialo-orosomucoid)
- lectin PHA-L is identified as a specific immunologic probe to detect this complex carbohydrate
- this lectin stains the SARS-CoV-infected cells specifically and intensively.
The authors said that based on these findings they "have sufficient immunologic evidence that a viral-expressed carbohydrate structure is responsible for the induction of the anti-ASOR autoimmunity in vaccinated animals. These observations raise concerns on human use of the whole virus-based SARS vaccine that is produced by the monkey Vero E6 cell."
They consider that it is too risky to introduce a whole-viral SARS vaccine to human subjects since its immunological property remains largely uncharacterized. They said: "It is necessary to eliminate the undesired autoimmunogenic activity of this preparation of inactivated SARS-CoV. It is possible to identify an alternative cell line or to genetically modify the Vero E6 cell line by altering its glycosylation pathway, thereby producing vaccines with enhanced efficacy without autoimmunogenic activity."
Wang and Lu note that the experimental approaches developed in their research are likely applicable for the immunologic characterization of other viral pathogens.
Research done at Columbia University and Sun Yat-sen University
The co-equal authors of the paper "Glycan arrays lead to the discovery of autoimmunogenic activities of SARS-CoV" are Denong Wang, who was head of the functional genomics division of Columbia University Genome Center, College of Physicians & Surgeons, New York, New York; and Jiahai Lu, Associate Professor at the School of Public Health at Sun Yat-sen University, Guangzhou, China.
Lu also is in charge of the SARS-CoV vaccine program for Guangdong Province.
Wang recently moved his carbohydrate microarray laboratory to the Departments of Genetics, Neurology and Neurological Sciences, Stanford University School of Medicine, Palo Alto, California.
The research was first published online in the American Physiological Society's Articles in PresS May 25 and appears in the July 2004 issue of Physiological Genomics, one of 14 journals containing almost 4,000 articles annually, published by APS.
Experiments and results
In 2002, Wang's laboratory developed a practical bioarray platform, which utilizes nitrocellulose-coated glass slides as substrate to immobilize carbohydrate antigens. This procedure is suitable for high-throughput construction of carbohydrate arrays using existing microarray spotting devices. His group reported that a range of carbohydrate-containing macromolecules of distinct structural configurations, including polysaccharides, natural glycoconjugates, and the mono- and oligosaccharides coupled to carrier molecules, are applicable for this bioarray platform.
Using this technology, Wang's group constructed a glycan array to display a collection of carbohydrate antigens, including microbial and plant-derived polysaccharides, as well as cellular glycan complex carbohydrates. To extend the repertoire of diverse carbohydrate structures on an array, they especially included blood group substances A, B, O, Lewis, I, and i antigens, their precursors and structural derivatives that were fostered by the late Columbia Professor Elvin A. Kabat and others.
Wang and Lu believed that scanning the antibody "fingerprints" of immunized or infected subjects using a broad-range glycan array is a specific immunologic approach to exploring the evidence of viral expression of corresponding complex carbohydrates. With this novel experimental strategy, they characterized SARS-CoV neutralization antibodies and detected significant levels of IgG antibodies to a human serum glycoprotein ASOR in horse anti-SARS-CoV antibodies. However, there was no detectable antibody reactivity to agalacto-orosomucoid (AGOR), a derivative of ASOR, which differs from ASOR solely by the absence of the terminal galactosyl sugar residue.
Therefore, the researchers considered that the anti-ASOR antibody reactivity is likely specific for the sugar moieties of ASOR, and that the terminal galactose (Gal) contributes significantly to the carbohydrate binding reactivity. These "chip-hits" led the way to rapid identification of specific immunologic probes that were subsequently applied to determine whether SARS-CoV-infected monkey cells expresses antigenic structures that mimic the complex carbohydrates on human glycoprotein.
Question about autoimmune responses in SARS pathogenesis
Expression of ASOR-like complex carbohydrates by coronaviruses is previously unrecognized. ASOR is an abundant human serum glycoprotein and the ASOR-type complex carbohydrates are also expressed by other host glycoproteins. Thus the human immune system is generally non-responsive to these "self" carbohydrate structures. However, when similar sugar moieties are expressed by a viral glycoprotein, their cluster configuration could differ significantly from those displayed by a cellular glycan, thereby generating a novel "non-self" antigenic structure.