Antibody responses to individual proteins of SARS coronavirus and their neutralization activities

Abstract

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.

The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

“…The immunogenic properties of ORF3a in SARS-CoV-infected patients has been shown to vary depending on: (i) whether or not the sera was collected from convalescent or recovered patients; (ii) whether or not full-length or truncated fusion proteins were used to detect immunoglobulin G (IgGs) to ORF3a; and (iii) the method used to detect these antibodies to ORF3a, namely western blot, ELISA or protein microarrays. To date, in general, IgGs to ORF3a could be detected in only ~60–73% of patient sera screened [35,57,58,59]. This could be due to frame-shift mutations within the 3a gene, resulting in a heterogeneous population of sgRNA3 transcripts in patients with acute SARS-CoV infection containing copies of both wild-type and mutant 3a genes.…”

“…The sera from rabbits immunized with recombinant ORF3a (a.a. 125–274) does not exhibit any significant neutralizing activity on SARS-CoV infected Vero E6 cells [59]. In another study, however, sera from rabbits immunized with a different recombinant ORF3a (a.a. 15–28) contains neutralizing antibodies that do inhibit SARS-CoV infection in Vero E6 cells [61].…”

“…The antigenicity of ORF3b has also come into question as IgGs to ORF3b are detectable in SARS patient sera in some studies [69,70], but not in others [59]. To date, the presence of ORF3b in SARS-CoV-infected Vero E6 cells is the only evidence for the expression of this protein [37].…”

“…Protein microarrays fail to detect antibodies to ORF7a in SARS patient sera [59] and SARS patient sera has low to non-specific antibody responses to ORF7a epitopes when tested on a peptide chip platform [70]. The selected ORF7a epitopes are therefore poorly immunogenic, and so ORF7a is not likely to serve as a potential marker for SARS-CoV infection or be important for vaccine development.…”

Label-free, arrayed sensing of immune response to influenza antigens

“…Where necessary, the presence of neutralizing antibodies could be subsequently confirmed for strong-responder samples by HAI or MN. Proteome profiling via protein microarrays has proven useful in many studies focused on understanding basic biochemical processes [xviii, xix, xx], but, more germane to the research reported herein, microarrays have also been used to discover antigenic proteins and monitor immunological responses to them [xxi, xxii, xxiii]. Thus, antigen arrays would seem to be ideal for the development of influenza surveillance and immunity screening tools.…”