Understanding the T cell immune response in SARS coronavirus infection

Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

There had been numerous attempts to screen for CTL epitopes in the N protein through the use of overlapping peptides spanning the entire N protein. One such study that used PBMCs from recovered SARS patients 2 years post-infection has revealed that the major dominant antigenic site of the N protein lies in the C-terminal region (amino acids 331 to 362). At least 2 different T cell epitopes (N331-347 and N346-362) have been found in this region when the PBMCs were stimulated with a pool of 57 overlapping N peptides in vitro, followed by IFNc Enzyme-linked immunosorbent spot (ELISPOT) assay. 28 Using the same approach, another group identified 2 potential CTL epitopes at positions N211-235 and N330-354 in the N protein. 32 More recently, we also identified the same dominant response (N216-230) in SARS-recovered patients 6 years post-infection. 33 This response was observed in 19% [3/16] of our cohort of recovered SARS patients. Similarly, a comprehensive study of T cell responses against all the SARS-CoV proteins conducted by Li et al. showed that 11% of their SARS subjects gave positive T cell responses against peptide N211-225, and it was identified as the most recognized epitope in the N protein. 34 Exact epitope mapping by our group further indicated that the CTL epitope was a 10 mer (N216-225) restricted by HLA-B*40:01 and that PBMCs from healthy individuals can be transduced to become N peptide-specific T cells. 33 In one of the first animal studies conducted in monkeys, adenoviralbased vectors were used to test the efficacy of the S, M and N proteins. 35 The monkeys were injected intramuscularly with adenoviral-based vectors that expressed codon-optimized S1 domain, M and N proteins. The S1 domain of the S protein was found to induce strong humoral response, while the N protein elicited high frequency of IFNc-producing T cells as determined using N peptides as the antigen in the ELISPOT assay. This was the first indication that the N protein could be a good vaccine candidate for cell-mediated T cell response. This phenomenon was also found in mice where DNA vaccines encoding the N protein elicited good T cell responses. 36-39 C3H/ He mice intramuscularly immunized with N protein pcDNA-fn vector showed both high antibody titre and CTL activity after 3 injections; 36 and using Balb/c mice, two other groups showed that DNA vaccines encoding N protein alone could elicit T cell proliferation, IFNc release, delayed-type hypersensitivity (DTH) and in vivo cytotoxic T cell activity. 37, 38 Further experiments reported enhanced T cell response when calreticulin (CRT)-linked DNA vaccine was used 39 or DNA vaccination was performed with the addition of a chemical adjuvant levamisole. 38 Synthetic N peptides coupled to the surface of liposomes were also reported to enhance T cell response. 40 These synthetic N peptides not only induced CTL response, but the mice were also able to clear vaccinia virus-expressing SARS-CoV epitopes when challenged. 40 In summary, several different studies have identified immunogenic regions in amino acids 211 to 362 of the N protein to contain T cell epitopes. However, to date, the only epitope characterized in detail is the 10-mer epitope (N216-225) which is restricted by HLA-B*40:01. 33