T Cell Responses to Whole SARS Coronavirus in Humans1

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-け ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8+ T cell responses were more frequent and of a greater magnitude than CD4+ T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27+/CD45RO+) with a significantly higher frequency of polyfunctional CD4+ T cells producing IFN-け, TNF-g, and IL-2, and CD8+ T cells producing IFN-け, TNF-g, and CD107a (degranulation), as compared with the mildmoderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation. 1This work was support by Beijing Municipal Government, National Science Fund for Distinguished Young Scholars, Medical Research Council U.K., and the Euro-Asian SARS-DTV Network (SP22-CT-2004–511064) from the European Commission specific research and technological development Program “Integrating and strengthening the European Research area.” 3Address correspondence and reprint requests to: Dr. Xiao-Ning Xu, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, U.K. E-mail address: E-mail: [email protected] or Dr. Jin-Lin Hou, Department of Infectious Diseases, Nan-fan Hospital, Guangzhou, China. E-mail address: E-mail: [email protected]. 2C.K.L., H.W., H.Y., and S.M. contributed equally to this work. Disclosures The authors have no financial conflict of interest. NIH Public Access Author Manuscript J Immunol. Author manuscript; available in PMC 2009 May 18. Published in final edited form as: J Immunol. 2008 October 15; 181(8): 5490–5500. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Although a huge public health initiative successfully contained the original severe acute respiratory syndrome (SARS)4 outbreak of 2002–2003 caused by a novel coronavirus (SARSCoV), many concerns remain over the possibility of its re-emergence, either naturally or accidentally, as is evidenced by sporadic SARS cases in late 2003/early 2004 and several laboratory-acquired infections after the outbreak. Phylogenetic analysis indicates that SARSCoV is a zoonotic virus that crossed the species barrier and evolved in palm civets and humans (1). However, the failure to isolate SARS-CoV from wild civets or farmed civets from nonepidemic areas argues against the civets being the natural reservoir of the virus (2). Recently, several SARS-CoV-like viruses have been isolated from wild bats, and if the bats are the natural reservoir, it is unlikely that we can prevent further spread of this virus to the human population (3). Given that SARS has a significant impact on health and economics, there is an urgent need to develop effective treatments as well as prophylactic vaccines against any future outbreak of SARS. The clinical outcomes of SARS infection were highly variable. So far, there has been no consensus regarding whether any treatment benefited SARS patients during the outbreak (4). In addition, it is not clear what role host immunity against SARS-CoV played in viral clearance or tissue damage. High initial viral load was shown to be independently associated with severity of the disease, and may be influenced by host immune responses (5). However, recent studies have suggested that type I IFN played a key role in the switch from innate immunity to adaptive immunity during the acute phase of SARS, and patients with poor outcomes showed type I IFN-mediated immunopathological events and deficient adaptive immune responses (6,7). Several studies have shown that most recovered SARS patients have higher and sustainable levels of neutralizing Ab responses, whereas patients with a longer illness showed a lower neutralizing Ab activity than patients with a shorter illness duration (8,9), suggesting that Ab responses are likely to play an important role in determining the ultimate disease outcome of SARS-CoV infection. Several forms of possible vaccines, such as attenuated or inactivated SARS-CoV, DNA, and viral vector-based vaccines have been evaluated in a number of animal models, including nonhuman primates (10). Neutralizing Abs to SARS-CoV spike protein are the major components of protective immunity (11,12). However, these animal models, including nonhuman primates, lack the severe clinical disease features observed in humans (13). Hence, it is difficult to evaluate whether these vaccines will prevent the disease in humans. It is possible that a vaccine could be harmful, because immune-mediated enhancement of pathology has been reported in other animal coronavirus infections (14) as well as in animals vaccinated with a modified vaccinia virus expressing SARS-CoV spike protein (15). Some variants of SARS-CoV were resistant to Ab neutralization, and the infection was enhanced by the Abs (16). Thus, without full understanding of the mechanism underlying protective immunity, many fear that some vaccines might worsen the disease rather than prevent it, echoing the respiratory syncytial virus vaccine disaster between 1960 and 1970 (17). A major obstacle to accurate and rapid development of vaccines for SARS is the scarcity of basic information about epitopes recognized by adaptive immune responses to SARS-CoV infection in humans. This lack of knowledge also hampers studies to determine what types of immune responses are involved in protection or pathology during the course of the infection, with a view to designing an effective vaccine to stimulate only protective immunity. In this study, we aimed to characterize the entire SARS-CoV-specific T cell response in a cohort of patients (n = 128) who had recovered from SARS-CoV infection during the 2003 outbreak in China, using a matrix of 1843 overlapping peptides spanning the whole SARS-CoV proteome and fresh PBMC in IFN-け ELISPOT assays. We also used polychromatic flow cytometry to 4Abbreviations used in this paper: SARS, severe acute respiratory syndrome; CoV, coronavirus; PBMC, peripheral blood mononuclear cell; ICS, intracellular cytokine staining; SFC, spot forming cell; Nab, neutralizing Ab. Li et al. Page 2 J Immunol. Author manuscript; available in PMC 2009 May 18. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript evaluate T cell effector functions at the single-cell level (18). Additionally, we investigated a similar number of high-risk health care workers who were in close contact with SARS infected patients during the outbreak. Epidemiological data suggested SARS had its origins in Guangdong province, China. To investigate the change of immune response following the evolution of SARS-CoV, we collected samples from South China (Guangdong province) for the early outbreak and North China (Beijing) for the later, more severe outbreak. In addition, we analyzed the serum cytokine profile in another cohort of the acute phase infection from the early phase of the outbreak. Our study provides the first roadmap of the immunogenicity of SARS-CoV in humans and identifies types of immune responses that may be, at least partially, responsible for virus clearance or disease progression. The results may serve as a benchmark for SARS-CoV vaccine design and evaluation. Materials and Methods Demographics of study populations Two cohorts of SARS infection containing a total of 226 SARS-infected patients were used in the present study: a convalescent cohort to study protective immune responses and an acute cohort to study acute infection through serum cytokine responses. This study was approved by the ethical research committees of the Medical School of Oxford University (U.K.), Beijing Youan Hospital, and Guangzhou Nan-Fang Hospital. The convalescent cohort contained a total of 128 recovered SARS patients (12 mo postinfection, 79% female and 21% male). It included 49 individuals from South China (Guangdong) and 79 from North China (Beijing). In control groups, 113 high-risk health care workers from South China (n = 62) and North China (n = 51) during the 2003 outbreak were also included. The illness onset time of the patients recruited was between March and April 2003 during the peak of the SARS outbreak, and the blood sampling time of convalescent samples was between April and September 2004, a year after the infection. In brief, 61 and 89% of patients were treated with steroids in South and North China, respectively. In the acute cohort, a total of 98 patients from South China (Guangdong) were admitted between January 30 and April 27, 2003. In the control groups, 21 high-risk healthy individuals and 32 non-SARS pneumonia patients were included. The non-SARS pneumonia group was patients who had x-ray evidence of pneumonia, and they all responded to treatment with antibiotics, suggesting that most or all were suffering from bacterial pneumonia rather than viral pneumonia. The average time of sera collection following disease onset (fever and respiratory symptoms) was 7.5 days ± 4.4 SD. For the acute cohort, most of the sera (>90%) were collected within 2 days of hospital admission, before any specific drug treatment, and stored at −80°C. None of the patients received steroid therapy before sample collection. The clinical diagnosis of SARS was made according to the World Health Organization case definitions (19), and disease severity was further classified according to Centre for Disease Control and Prevention criteria (20). In mild-to-moderate illness, the cases were defined by fever (>38°C), one or more clinical findings of lower respiratory illness (e.g., cough, shortness of breath, difficulty in breathing), and radiographic evidence of pneumonia. In addition to the above clinical features of mild-to-moderate illness, the severe cases were defined by evidence of respiratory failure (pO2 < 60 mmHg and pCO2 > 40 mmHg). All patients had a history of exposure to SARS patients. During hospital admission, all patients had evidence of SARSCoV infection detected by ELISA for anti-SARS-CoV-specific IgG Ab in the serum and/or RT-PCR for the SARS virus, as described previously (21). In the convalescent cohort study, PBMC were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation for an immediate IFN-け ELISPOT assay. Paired serum samples were collected for determining SARS-specific Ab responses. DNA from the blood was extracted from each patient sample for in-house HLA typing by PCR with sequence-specific primers. In the acute cohort study, serum Li et al. Page 3 J Immunol. Author manuscript; available in PMC 2009 May 18. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript for cytokine analysis was collected from 5 ml blood samples taken for routine diagnostic purposes. A flow diagram illustrating how the studies were performed is presented in the Fig. 2.5 SARS-IgG ELISA and virus neutralization assays SARS-specific IgG in the serum samples was determined by ELISA based on purified whole virus lysates (S200300004, Hua Da Company). A positive result was defined as OD ≥ 0.14 (0.13 + mean of negatives (0.01)) as described previously (22). Neutralizing Ab against spike protein was measured by a pseudotype retrovirus-based neutralization assay as described previously (8). In brief, serum samples were heat inactivated at 56°C for 30 min, 2-fold serially diluted from 1/10 in culture medium, and mixed with murine leukemia virus pseudotyped with SARS spike protein, murine leukemia virus (SARS), and virions (≈100 IU) at a 1:1 v/v ratio. After incubation at 37°C for 1 h, 100 たl of each dilution was added to QT6/ACE2 cells seeded at 1 × 104 cells per well in 96-well flat-bottom tissue culture plates seeded 24 h previously. GFP-positive cells were counted 48 h later by fluorescence microscopy. Neutralizing Ab titers are presented as geometric mean titers of assays performed in triplicate. IC90 was used as the end-point titer, and titers ≥1:10 were considered to be positive, as described previously. Ex vivo IFN-け ELISPOT To identify T cell epitopes from SARS-CoV in the cohort, we used overlapping synthetic peptides (total 1843 peptides, 15–18mers overlapping by 10 amino acid residues) spanning the whole proteome of SARS-CoV (Tor-2, Accession number AY274119). The peptides were manufactured by New England Peptides and the quality of each peptide was checked by MALDI-TOF mass spectrometry, identifying the correct mass at >75% of purity. As previously described (23), we simultaneously tested all 1,843 overlapping peptides in each individual using 2-dimensional matrices with a total of 88 pools (1st D = 43 pools; 2nd D = 44 pools; up to 45 peptides/pool) so that each peptide was present in two different pools. Peptides were used at a final concentration of 2 たM each. The internal negative control was no peptide in triplicates, and positive controls were FEC (a mixture of Flu, EBV, and CMV T cell epitope peptides), PPD, or PHA. Fresh PBMCs were isolated from 50 ml of blood collected in heparin containing tubes (BD Vacutainer) by venipunture and added into 96-well plates at 300,000 cells/well for overnight incubation. All ELISPOT assays were performed using the human IFN-け ELISPOT kit (Mabtech) according to the manufacturer’s instructions. The spots on each well were counted using an AID-ELISPOT reader (Autoimmune Diagnostika). Any true positive response was determined by two separated ELISPOT assays: the matrix screening (first ELISPOT) and a confirmatory test examining the individual peptide (second ELISPOT). Wells containing spot numbers greater than the mean + 3 SD of three negative control wells (no peptide) were regarded as positives in each individual, provided that the total was greater than 18 spot forming cells (SFC)/million PBMC, to rule out false positives where background was very low. In all assays, values of no peptide control wells were 6.7 ± 0.9 SFC/million PBMC for healthy subjects and 8.3 ± 0.7 SFC/million PBMC for SARS patients. To determine whether T cells were CD4 or CD8, in the second ELISPOT assay, cell depletion was also conducted by Dynal CD8 beads, as described in the manufacturer’s instructions (Invitrogen), before the ELISPOT assay. Undepleted PBMC were used as positive controls. Intracellular cytokine staining (ICS) T cell lines were generated as effector cells to confirm SARS peptides and the CD4 or CD8+ property of each peptide by ICS and flow cytometry, as described previously (24). In brief, frozen samples of PBMC were thawed and stimulated with 100 たM of each peptide for 1 h. 5The online version of this article contains supplemental data. Li et al. Page 4 J Immunol. Author manuscript; available in PMC 2009 May 18. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Cells were cultured in RPMI 1640 supplemented with 10% human serum and 25 ng/ml IL-7 (PeproTech) for 3 days, and then 100 U of IL-2/ml was added every 3 to 4 days thereafter. In assay, T cell lines were stimulated with 100 たM peptide-pulsed EBV-transformed autologous B cell lines in the presence of anti-CD28 and anti-CD49d mAbs (each at 1 たg/ml; BD Biosciences) for 1 h at 37°C and before the addition of brefeldin A (10 たg/ml, Sigma-Aldrich). After a further 5-h incubation, cells were washed in PBS with 1% FCS and sodium azide, then fixed and permeabilized in permeabilizing buffer (BD Biosciences). Cells were stained with mAbs against human IFN-け (BD Biosciences), CD4 (DakoCytomation), and CD8 (BD Biosciences). Lymphocytes were gated on a FACSCalibur flow cytometer (BD Biosciences), and cells stimulated with medium alone were used as negative controls. Polychromatic flow cytometric analysis Eleven-parameter flow cytometric analysis was performed using a LSRII flow cytometer (BD Biosciences). FITC, PE, Cy7PE, Cy5.5PE, allophycocyanin, Cy7 allophycocyanin, Texas Red PE, violet amine reactive dye, and Quantum-dot 705 (QD705) were used as the fluorophores. At least 300,000 live lymphocytes were collected. The list-mode data files were analyzed using FlowJo (Tree Star). Functional capacity was determined after Boolean gating and subsequent analysis was performed using Simplified Presentation of Incredibly Complex Evaluations (SPICE, version 2.9, Mario Roederer, VRC, NIAID, National Institutes of Health). All values used for analyzing proportionate representation of responses are background-subtracted. To study ex vivo peptide-specific polyfunctional responses of memory T lymphocytes from recovered SARS patients, a 6-h short-term stimulation was performed on fresh or frozen lymphocytes as described elsewhere (25). Freshly isolated or thawed lymphocytes were resuspended at 106/ml in R10 supplemented with 1 たg/ml anti-CD28 and anti-CD49d Abs. Peptides 15 amino acids in length, overlapping by 11 amino acids, and encompassing SARS spike protein, were used to stimulate SARS-specific T cells in the presence of brefeldin-A (1 たg/ml, Sigma-Aldrich) for 6 h at 37°C. All cells were surface-stained for phenotypic markers of interest and intracellularly stained for cytokines (ICS). mAbs used for phenotypic and functional characterization of T cell subsets were anti-CD3 Cy7 allophycocyanin, anti-CD45RO Texas Red PE, anti-CD27 Cy5PE, anti-CD4 Cy5.5PE, antiCD8 QD705, anti-IFN-け FITC, anti-IL-4 PE and anti-TNF-g Cy7PE, and anti-IL-2 allophycocyanin (BD Pharmingen). As naive T cells do not express CCR5, and as SARSspecific T cells are not detectable in the naive T cell pool, we report these data as percentages of memory T cells. We first gated for memory CD4 and CD8 T cells based upon characteristic expression patterns of CD45RO and CD27. Cytokine cytometric bead array Serum concentrations of the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFNけ, and TNF-g in acute samples (50 たl) were measured using cytometric bead-array assays (BD Biosciences) according to the manufacturer’s instructions. The sensitivities of cytometric bead array for each cytokine were 7.2 pg/ml (IL-1), 2.6 pg/ml (IL-2), 2.6 pg/ml (IL-4), 2.4 pg/ml (IL-5), 2.5 pg/ml (IL-6), 3.6 pg/ml (IL-8), 2.8 pg/ml (IL-10), 1.9 pg/ml (IL-12), 7.1 pg/ml (IFNけ), and 2.8 pg/ml (TNF-g). Statistical analysis Graphs were presented by Prism 4 (GraphPad) software. All statistical analysis was conducted independently by a statistician using SPSS 12.0 software. The T cell response (breadth and magnitude) was analyzed as described previously (23). Categorical variables were compared using Fisher’s exact test, and group comparison of numerical data was analyzed by MannWhitney U test. To compare the immune response in different disease groups, multiple Li et al. Page 5 J Immunol. Author manuscript; available in PMC 2009 May 18. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript regression analysis was conducted for each response measure separately, all adjusted for age, sex, and location. In the regression model, natural logarithm transformation was performed for each response variable, and a t test was used to test the difference of the log-transformed mean immune response between the mild/moderate group and the severe group. A two-tailed t test was used to compare two different groups taking into account unequal variances, and a p value <0.05 was considered to be significant. For acute cytokine analysis, continuous variables were compared using the independent samples t test. Recovered and fatal SARS groups were described using the geometric mean ratio and 95% confidence intervals. ANOVA with age as a covariate was used for comparison of variables between groups of patients.