INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 18, no. 3, 525-530 (2005)

T-CELL RESPONSE PROFILING TO BIOLOGICAL THREAT AGENTS

INCLUDING THE SARS CORONAVIRUS

C. GIOIA, D. HOREJSH1, C. AGRATI, F. MARTINI, M.R. CAPOBIANCHP,

G. IPPOLIT02 and F. POCCIA

Unit ofCellular Immunology, 'Laboratory ofVirology, 'Department ofEpidemiology,National Institute for Infectious Diseases (I.NM.I.) "Lazzaro Spallanzani" I.R.C Cs., Rome, Italy

Received April 21, 2004 - Accepted November 24, 2004

The emergence of pathogens such as SARS and the increased threat of bioterrorism has stimulatedthe development of novel diagnostic assays for differential diagnosis. Rather than focusing on thedetection of an individual pathogen component, we have developed a T cell profiling system to monitorresponses to the pathogens in an array format. Using a matrix of antigens specific for differentpathogens, a specific T cell profile was generated for each individual by monitoring the intracellularproduction of interferon-gamma by flow cytometry. This assay allows for the testing of multiple proteinsor peptides at a single time and provides a quantitative and phenotypic assessment of CD4(+) and CD8(+)responding cells. We present profiling examples for several positive individuals, including thosevaccinated with the smallpox and anthrax vaccines. We also show antigen optimization for the SARS­hCoV, as studies revealed that these proteins contain peptides which cross-react with more commoncoronaviruses, a cause ofthe common cold. The T cell array is an early and sensitive multiplex measureof active infection, exposure to a pathogen, or effective, recent vaccination.

With the continual emergence of new pathogensand the increased threat of bioterrorism attacks, thedifferential diagnosis or identification of etiologicalagents in infection is the first important step forcontrolling the diffusion of a disease. The SARScoronavirus (SARS-hCoV) tested the ability of thescientific community to develop methods to isolate,identify, and characterize an emerging virus. Theanthrax scare after September 11th highlighted theneed to develop assays that also detect the intentionalrelease of pathogens intended to "terrorize" thepublic. The key to an effective public health responseis the early diagnosis of infection and theidentification of the biological agents to curboutbreaks and secondary spread of these diseases.

For the differential diagnosis of infection, there are

several methods currently in use. Pathogen, isolation,PCR detection ofnucleic acids, and antibody detectionby ELISA, immunofluorescence, or neutralizationhavebeen used to confirm infection (1-3). Practicallaboratory preparedness problems limit the use ofisolation of infectious agents in many institutions,especially for high-risk pathogens. Therefore, morerapid a PCR-based and antibody detection methodshave been developed as commercial products fordiagnosis. During the acute phase of infection, PCR­based nucleic acid detection method may be the mosteffective, but the results are not always reliable due toindividual variability, sampling time, and sample type(4). Antibody detection by ELISA is a more precisemethod and offers the advantage of detection inexposed, uninfected individuals, but an effective

Keywords: antigen stimulation, array, interferon-gamma, activation profile

Mailing address: Dr. Douglas HorejshNational Institute for Infectious Diseases "1. Spallanzani",LR.C.C.S., Via Portuense, 29200149 Rome, ITALYTel +390655170907 - FAX +390655170918e-mail: horejsh@inmi.it

525

0394-6320 (2005)Copyright© by BIOLIFE,s.a.s.

This publicationand/orarticle is for individualuse only and may not be furtherreproducedwithoutwrittenpermissionfromthe copyrightholder.

Unauthorizedreproductionmay results in fmancialand other penalties

526 C. GIOIA ET AL.

antibody response can take weeks to develop (theSARS-hCoV exclusion criteria used by the CDCsuggests that this response can take up to 28 days todevelop).

The optimal assay should incorporate thedetection at an earlier time point, with the sensitivityof an immune-based assay to detect exposedindividuals. In the absence of a detectable serology,antigen-specific T cell responses could be detected inexposed, but uninfected persons, as shown for HIVand HCV contacts (5-6). Also, the initialdevelopment of delayed hypersensitivity, an index ofcell-mediated immunity, occurs as early as two daysafter a smallpox vaccination (7). Thus, monitoring aT cell response profile to a diverse panel of antigensmay allow an earlier identification of the infectingagent. In addition, this assay also may be used totestassa the robustness of specific immunity aftervaccination (8). We evaluated the feasibility of aneasy, rapid, and sensitive assay to monitor T cellresponses to a composite-diverse panel ofpathogens.

MATERIALS AND METHODS

AntigensAntigen preparations are fully described in Table I,

including the commercial or academic source and the quantityused in these analyses.

AntibodiesUnconjugated mouse-anti-human CD28 (IgGl, clone

CD28.2); unconjugated mouse-anti-human CD49d (IgGl,clone 9FIO); fluorescein (FITC)-conjugated mouse-anti­human IFN (IgGl, clone B27); PE-cyanine-5 (Cy-5)­conjugated mouse-anti-human CD3 (lgGl, clone RPA-T3);AlloPhycoCyanin (APhC)-conjugated mouse-anti-humanCD8 (lgGl, clone RPA-T8) monoclonal antibodies, andFITC-conjugated IgG1 isotype-matched control (lgG 1 cloneMOPC-21) were obtained from Becton DickinsonImmunocytometry Systems (San Jose, CA).

Cell stimulationPeripheral blood mononuclear cells (PBMC) were

obtained using standard Ficoll-Hypaque (Pharmacia,Uppsala, Sweden) density centrifugation. Stimulation wasperformed as already described with minor modifications (8).

lx106 freshly or lx106 lived thawed PBMC in 1 ml ofcomplete RPM! 1640, 10% vlv heat-inactivated FCS, 2 mML-Glutamine, 10 Vlml penicillin/streptomycin, were

incubated with 1 ug each of anti-CD28 and CD49dmonoclonal antibodies and the antigenic preparations listedin Table I. To control the spontaneous cytokine production,cells incubated with only anti-CD28 and -CD49d wereincluded in each experiment. The IFN-y release induced byPMA (50 ng/ml) plus ionomycin (10 mg/ml) was used as apositive control. The cultures were incubated at 37°C in a 5%C02 incubator for 1 h, followed by an additional 5 hours of

incubation with 10ug/ml ofthe secretion inhibitor Brefeldin­A (Sigma, St. Louis, MO).

Immunofluorescent stainingAntigen-stimulated and control cultures were washed in

cold Dulbecco's phosphate-buffered saline (dPBS) containing1% bovine serum albumin and 0.1% sodium azide. Cells werewashed twice in PBS, 1% BSA, and 0.1% sodium azide, andthen stained with monoclonal antibodies specific for themembrane antigens described above for 15 min at 4°C.Samples were fixed in 1% paraformaldehyde for 10 min at4°C, incubated with anti-interferon (IFN)-y monoclonalantibody diluted in PBS IX, BSA 1% and saponin 0.5%. Thecells were finally washed twice in PBS IX, BSA 1%, 0.1%saponin and resuspended in FACS FLOW prior to acquisitionusing a FACScalibur cytometer (Becton Dickinson). Controlsfor non-specific staining were monitored using an isotype­matched monoclonal antibody and non-specific staining wasalways subtracted from specific results.

Flow cytometric analysisSix-parameter flow cytometric analysis was performed on

a FACScalibur flow cytometer (Becton DickinsonImmunocytometry Systems), using FITC, PECy-5 andAPhCas the fluorescent parameters. At least 100,000 live eventswere acquired, gated on small viable lymphocytes. Data fileswere analyzed using CellQuest software (Becton Dickinson).Data were compiled in a Microsoft Excel spreadsheet(Microsoft Corporation, Seattle, WA) for array analyses.

Statistical analysisGrouped T cell response data are presented as means ±

standard deviations (SD) of the mean.

RESULTS

T cell response profiling of individuals using theantigen array

A T cell response profile was developed forseveral individuals (Fig. la). There was a wideindividual variability, but sample duplicatesconfirmed specificity. A marked, specific responseto CMV antigens was seen in each of the healthy

Int. J. Immunopathol. Pharmacol. 527

a) Representative T-cell response profile of a healthy individual.

NS - no stimulation

CMV+ - CMV-inf. cell lysate

CMV- - mock-inf ocell lysate

VV+ - vaccin ia proteins

VV- - contro l proteins '

HIV - Gag peptide pool

SARS-hCoV - viral proteins

Ortho - Orthomyxovirus HA

B.anth. - Anthra x proteins

inac. B.anth - control

500 3000

400:s 2000.s'" 300alil 200C'" 1000>'" 100 •0 0

NS CMV+ CMV- VV+ VV- HIV SARS Ortho B.anth. inac. PMAhCoV B.anth.

b) T-cell response examples for infected or vaccinated individuals.

HIV-infected

NS gagpool

anthrax-vaccinee100

75 I::~

NS SA

smallpo x-vaccinee150

HAI

NSo

1000

orthomyxo-infected3000

2000 ,-

_~Io

200

2 400c~'"

800

:s.s 600 ~--

'"a

Fig. 1. T cell response profiling measures recent infection or vaccination response. a) The T cell response profile ofthisrepresentative, healthy individual indicates exposure to orthomyxovirus and CMV. Non-stimulated and PMA-stimulatedcontrols are shown at the typically expected levels. b) The T cell responses of infected or vaccinated individuals arepositive for the expected, corresponding pathogen.

donor panels. In several individuals, a response wasalso detected to influenza virus, albeit at low levelsin subjects neither recently vaccinated, nor recentlyinfected. Neither of these results were unexpected, asthe prevalence of sero-positivity for CMV in Italy isquite high and the response levels were expected tovary, depending on the individual. Also, influenzavirus is recurrent on a yearly basis, so virtually allindividuals have been exposed to this virus andrespond to various strains due to antigenic sin.Pathogen-infected or recently vaccinated individualswere used as controls to confirm the reactivity of theantigen mixes for the response panel. As shown inFig. Ib, a robust response was observed inrepresentative examples of infected or vaccinatedindividuals for their respective pathogens.

Development of the SARS-Co V antigen for theantigen mix

A small, but reproducible response was seen to therecombinant SARS CoV protein pool in a number ofhealthy donors (Fig. 2a). The SARS-CoV epitopes inour preparation are not unique to group IV

coronavirus, but are instead conserved among theother classes of coronaviruses that cause the commoncold or gastroenteritis (Fig. 2b). It is, therefore, notunexpected that the recombinant proteins for SARS­CoV E and N2 contain cross-reactive epitopes, asthese proteins stimulated a response abovebackground in donors not exposed to the SARS CoV(9). Peptide design and optimization will further tunethe SARS-CoV component of the assay as morereactive peptides are defined as being specific forSARS-CoV without cross-reaction to the morecommon and less dangerous coronaviruses.

DISCUSSION

An importantaspect in diagnosticassay developmentis the availability ofa rapid and easilyautomatedsystem.that works on virtually all persons who carry thedisease.Cultivationofhigh-risk pathogens is impracticalfor biosafety and/or technical reasons in the routinelaboratory. Also, early detection using PCR or pathogenprotein ELISA can be difficult due to individualhost andpathogenacutephase variability. Hostantibodydetection

528 C. GIOIA ET AL.

(a) T-cell response to coronavirus proteins for a group of healthy individuals .

80

60roB'"0

40sE~ no stimulation" 20

o - __

1 234567 .

heatthy donor #

recombinant SARS pro tein pool

1234567 .

healthy donor #

(b) T-cell responses of a healthy individual to a panel of coronav irus proteins.

50

40g

-1___-s 30'"0;;;

20E~

" 10

0NS CoY CoY SARS SARS SARS SARS

protein pept ide E M N1 N2pool

Fig. 2. Cross-reaction between phylo genetically-defined classes is identified through the use ofdifferent coronavirus ant igens.a) The T cell response projile ofseven healthy controls to coronavirus protein reveals the presence of cross-reactive ep itopes.Column "x " is the mean ofthe group . with the indicated standard deviation. b) The T cell responses ofa healthy individual toa panel of coronavirus proteins indicated that coronavirus proteins E and N2 contain ep itopes that are conserved amongcoronaviruses . Coronavirus proteins that tend to be more class-specific were seen to be negative in the healthy donor group.

gives the added advantage of detection in exposed,uninfected individuals, buttheantibody response cantakeweeks to develop. As cell-mediated immunity isstimulated almost immediately after infection orvaccination, we chose to focus our detection methods onthe immune cells that directly respond to the pathogenantigens. Intracellular T cell cytokine staining by flowcytometry presents several advantages compared withother techniques such as tetramerstaining and ELISpot.In fact, flowcytometry allows fortesting multiple antigenssimultaneously in array formatand provides at the sametimea quantitative andphenotypic assessment ofCD8(+)and CD4(+) respondingT cells (10-11). We have nowbeenableto showthat it ispossible to develop an arrayofthese antigens to screen for exposure, infection, oreffective vaccination by a givenpathogen.

Moreover, optimization of the antigen preparationswith peptide pools designed to be pathogen-specific,highly conserved, and independent of HLA haplotypes,may allowfor the development of a second generation ofmore sensitive flowcytometric T cellassays, extending the

possibility also to perform retrospective studies usingcryopreserved samples (12). Accurate monitoring of thesecells is crucial in differentialdiagnosis or in determiningthe effects of'HfVtherapy andvaccine efficacy. Usinganintracellular cytokine staining basedassay, we are abletodirectly quantify fimctional antigen-specific CD8+T cells.This assay is highlyreproducible, and can be performedusingbothfresh andcryopreserved peripheral bloodcells.Importantly, this assay can be used to examinemultiplepeptide epitopes simultaneously, and can be designed tobe independentof patientHLA haplotype. We fmd thatwhen usingmixes of multiple peptides, the CD8+T cellresponse to the mixture is equivalent to the sum of theresponses to the individual peptides contained therein.Although somepatients sharing HLAalleles occasionallyrecognize commonpeptides, rarelyareresponses to thosepeptides dominant within the same group of patients.These results confirm our previous findings that theresponses tosingleHIV-peptides are rarely representativeof the entire Hl'V response.

The technique couldbe easilyautomated through the

Int. J. ImmunopathoI. PharmacoI.

Table I. Antigenic preparations used in the T-cell response profile.

Microbe Antigens Source Amount/ml

CMV Ag (AD 169) Infected Cell lysate BioWhittaker (Walkersville, 20f!1 (Zug)

USA)

CMV negative Control Ag BioWhittaker (Walkersville, 20f!1 (Zug)

control USA)

VacciniaAg Infected Cell extract Main Biotechnology 1Of!1 (dil

Service, Inc 1:100)

(portland, USA)

Vaccinia negative Control Ag Main Biotechnology 1Of!1 (dil

control Service, Inc 1:100)

(portland, USA)

HIV-l GAG Peptide Pool Sigma-Genosys Iul/pept

(Cambridge, UK) (Iug/pept)

Bacillus anthracis Toxin Kindly provided by 20f!1 (Sug)

Dr. A. Fasanella

(Bari, Italy)

Coronavirus, SARS Recombinant Protein Biodesign International 1Of!1 (Zug)

assoc., M Protein (Saco, USA)

Coronavirus, SARS Recombinant Protein Biodesign International 1Of!1 (Zug)

assoc., E Protein (Saco, USA)

Coronavirus, SARS Recombinant Protein Biodesign International 10ul (2ug)

assoc., N Protein (Saco, USA)

(aa.I-49)

Coronavirus, SARS Recombinant Protein Biodesign International 1Of!1 (Zug)

assoc., N Protein (Saco, USA)

(aa.192-220)

Coronavirus Peptide Pool Adaltis 1Of!1 (Zug)

(Montreal, Canada)

Orthomyxovirus Viral lysate L. Spallanzani - Virology 1OI-ll (dil

Lab 1:100)

529

530 C. GIOIA ET AL.

use of analytical instruments already available in mostclinical laboratories (13) that use flow cytometry. Therecent availability of mobile flow cytometer units mayeven allow use of this assay for field diagnostic andepidemiologic investigation (14). More T cell responsepanels are being completed on healthy, vaccinated, orinfected subjects to continue our evaluation anddevelopmentofthis assay.

ACKNOWLEDGEMENTS

This work was supported by funding from the"Ministero della Salute" of the Italian government.The authors also wish to thank Dr. M. Houde(Adaltis Inc., Montreal, Canada) and Dr. P. K. S.Chan (Department of Microbiology, The ChineseUniversity of Hong Kong) for their continuedcollaboration and advice in the development of theSARS response test.

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