Assessment of the Phenotype and Functionality of Porcine CD8 T CellResponses following Vaccination with Live Attenuated Classical SwineFever Virus (CSFV) and Virulent CSFV Challenge

Giulia Franzoni,a,b Nitin V. Kurkure,a,c Daniel S. Edgar,a,b Helen E. Everett,a Wilhelm Gerner,d Kikki B. Bodman-Smith,b

Helen R. Crooke,a Simon P. Grahama

Virology Department, Animal Health and Veterinary Laboratories Agency, Addlestone, United Kingdoma; Department of Microbial and Cellular Sciences, University ofSurrey, Guildford, United Kingdomb; Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur, Indiac; Department of Pathobiology,Institute of Immunology, University of Veterinary Medicine Vienna, Vienna, Austriad

Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has beenassociated with virus-specific T cell gamma interferon (IFN-�) responses. In this study, we employed flow cytometry to charac-terize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3� CD4� CD8hi Tcell population was the first and major source of CSFV-specific IFN-�. A proportion of these cells showed evidence for cytotoxic-ity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-�). To assess the durability andrecall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV af-ter 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3� CD4� CD8��) responses were detected, thedominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hi CD62L�

and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority ofvirus-specific IFN-�� CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface,and subpopulations that coexpressed TNF-� and interleukin 2 (IL-2) were identified. While it is hoped that these data will aidthe rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developedshould also be of value in the study of porcine T cell responses to other pathogens/vaccines.

Classical swine fever (CSF) is one of the most important viralinfectious diseases of domestic pigs and wild boars. CSF is

caused by the classical swine fever virus (CSFV), a highly conta-gious, small, enveloped, single-stranded RNA virus belonging tothe family Flaviviridae (1). Since 1990, outbreaks of CSF in theEuropean Union have been controlled through a stamping-outslaughter policy, epidemiological and virological investigations,and movement restriction for pigs and pig products (1, 2). Owingto the economic losses caused by the stamping-out policy, there ispressure to develop alternative strategies to control CSF outbreaksand to minimize the need for mass culling (2).

Live attenuated CSF vaccines, such as C-strain viruses, providea rapid onset of complete protection but pose problems in dis-criminating infected from vaccinated animals. Many studies haveaimed to develop marker subunit vaccines, but they frequently failto show an appropriate level of efficacy for use under emergencyoutbreak conditions (1, 3, 4). An understanding of the immuno-logical basis of rapid protection afforded by live attenuated C-strain vaccine would aid the development of the next generation ofmarker CSFV vaccines, both through the identification of vaccinecandidate antigens and through informing the selection of appro-priate delivery systems/adjuvants to trigger protective responses.While the immunological effector mechanisms are not well de-fined, C-strain-induced protection may precede the appearance ofneutralizing antibody but not gamma interferon (IFN-�)-secret-ing cells in peripheral blood, suggesting that cellular immunity isresponsible (5). Moreover, it has recently been shown that there isa close temporal correlation between the induction of CSFV-spe-cific T cell IFN-� responses and rapid protection induced by a

C-strain vaccine (6). In addition to the secretion of IFN-�, whichhas been shown to exert direct antiviral effects on CSFV in vitro(7), vaccine-induced CSFV-specific T cells have also been shownto have the capacity to lyse infected cells with specificities mappedto the structural protein E2 and the nonstructural protein NS3(8–10). Flow cytometric studies have identified both CD4 andCD8 T cells as the cellular source of CSFV-specific IFN-�, with thelatter coexpressing the cytolytic molecule perforin (7, 11). Furtherevidence for a protective role for T cells stems from recent subunitvaccine studies which have shown that the protective capacity of aCSFV E2 DNA vaccine was associated with T cell IFN-� ratherthan neutralizing antibody responses (12) and that inclusion of adefined NS3 T cell epitope improved the immunogenicity and thedegree of protection afforded by a peptide-based CSF vaccine(13).

The goal of an efficient vaccine is to generate memory CD4and/or CD8 T cells capable of recognizing and rapidly expandingto combat infection (14). While challenging, it has been proposed

Received 27 June 2013 Returned for modification 26 July 2013Accepted 12 August 2013

Published ahead of print 21 August 2013

Address correspondence to Simon P. Graham, Simon.Graham@ahvla.gsi.gov.uk.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00415-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00415-13

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that the efficacy of T-cell-based vaccines could be improved bymanipulating the generation and maintenance of distinct memoryT cell subsets which in recent years have been identified in humansand mice through the application of multiparameter flow cytom-etry (15). The two main categories of memory T cells are classifiedas effector memory (TEM) and central memory (TCM) (16) andcan be distinguished with the lymph node homing markersCD62L and CCR7, with CD62L� CCR7� and CD62L� CCR7�

cells representing the TCM and TEM, respectively (17). The adhe-sion molecules CD44 and CD11a are further used to differentiatenaive from effector/memory CD8 T cells (17). Moreover, the si-multaneous detection of cytokines (e.g., IFN-�, tumor necrosisfactor alpha [TNF-�], interleukin 2 [IL-2]) and other effectormolecules/markers (e.g., CD107a mobilization) has enabled as-sessment of T cell polyfunctionality and has been attributed tocells capable of exerting multiple functions with greater protectivecapacity (18). Recent studies using such approaches have sug-gested that effector memory and/or polyfunctional CD8 T cellsplay an important role in immunity to other viruses belonging tothe family Flaviviridae, such as hepatitis C virus (HCV) (19–21)and yellow fever virus (YFV) (22). To provide an improved un-derstanding of the role that T cells may play in immunity to CSFV,we deployed novel flow cytometric antibody panels to quantita-tively and qualitatively assess the T cell response induced follow-ing C-strain vaccination and subsequent CSFV challenge infec-tion. We additionally assessed whether the T cell responses ofvaccinated and challenged animals were sustained and/or readilyrecalled upon subsequent rechallenge infection and the impactthat this rechallenge had on their phenotype/function.

MATERIALS AND METHODSEthics statement. All animal work was approved by the Animal Healthand Veterinary Laboratories Agency Ethics Committee, and all proce-dures were conducted in accordance with the United Kingdom Animals(Scientific Procedures) Act 1986 under project license permit numbersPPL 70/6559 and 70/7403. To minimize animal suffering, careful comple-tion of clinical score sheets and regular observation were conducted whichinformed euthanasia decisions based on a predefined humane endpoint(clinical score of �15 or if temperature was �41°C and more than 2individual scores [other than the temperature score] had a value of 3).However, none of the animals experienced clinical signs necessitating eu-thanasia. All animals were euthanized on predetermined days by intra-muscular (i.m.) administration of ketamine-xylazine (Rompun) sedativefollowed by intravenous administration of 20% sodium pentobarbitonesolution.

Viruses. A commercially available live attenuated C-strain CSFV (ACRiemser Schweinepestvakzine; Riemser Arzneimittel AG, Riems, Ger-

many) was used for vaccinations. This CSFV C-strain vaccine was atten-uated by multiple passages through rabbits followed by adaptation toculture in porcine kidney cells (23) and had previously been shown able toprotect pigs against CSFV challenge after only 5 days (6). For challengeinfections, the CSFV Brescia stain (kindly provided by Alexandra Meindl-Böhmer, University of Veterinary Medicine, Hannover, Germany) wasselected as it had been shown in recent studies to be a highly virulent strain(24–26). The virulent CSFV Alfort 187 strain (AHVLA CSF ReferenceLaboratory Virus Archive, Addlestone, United Kingdom) was selected foruse in in vitro restimulation assays, since this virus is of the same subgeno-group (1.1) as C-strain and Brescia and, importantly, readily grows tohigh titers in PK15 cells, enabling its use in restimulation assays at a mul-tiplicity of infection (MOI) of 1, which we had previously shown to inducea better recall of T cell responses than lower MOIs (7). All three viruseswere propagated in vitro by inoculation of subconfluent PK15 cell mono-layers. PK15 cells were maintained in Eagle’s minimum essential medium(E-MEM) (Invitrogen, Paisley, United Kingdom) supplemented with 100IU/ml penicillin, 100 �g/ml streptomycin (Invitrogen), and 10% fetalbovine serum (FBS) (Autogen Bioclear, Calne, United Kingdom). After 4days, the supernatant was collected and pooled with a freeze-thawed celllysate. The resultant pool was clarified by centrifugation at 524 � g for 10min, aliquoted, and stored at �80°C. Virus titers were obtained by serialdilution of the virus suspension on PK15 cells followed by immunoper-oxidase staining to identify infection rates as described previously (27).Mock virus supernatant was prepared in an identical manner from unin-fected PK15 cells.

Vaccination and challenge of pigs with CSFV. In the first experiment(experiment 1), 42 Large White/Landrace crossbreed pigs of 8 to 10 weeksof age were randomly assigned to four groups. Numbers of animals pergroup, the treatments administered, and the days that animals were sam-pled and responses were analyzed are summarized in Table 1. Five daysprior to challenge, animals in groups 1 (n � 6) and 2 (n � 12) wereinoculated intranasally (i.n.) with 105 50% tissue culture infective doses(TCID50) of C-strain CSFV (1 ml divided equally between each nostril andadministered using a mucosal atomization device [MAD-300; Wolfe ToryMedical, USA]), those in group 3 (n � 12) were inoculated with 2 ml ofreconstituted vaccine into the brachiocephalic muscle (as recommendedby the manufacturer, Riemser Arzneimittel AG), and those in group 4(n � 12) were intranasally inoculated with 1 ml of mock virus supernatantas described for groups 1 and 2. On day 0 postchallenge, groups 2 to 4 wereinoculated intranasally with 105 TCID50 of CSFV Brescia strain as de-scribed above and group 1 received a similar inoculation of mock virussupernatant. On days �2 and 0 postchallenge, three animals were eutha-nized from groups 2 to 4, and on days 3 and 5 postchallenge, three animalswere euthanized from all four groups and blood samples were collectedfor analysis of CSFV-specific T cell responses. A second experiment (ex-periment 2) utilized 11 Large White/Landrace pigs, 6 months of age. Eightanimals were vaccinated at day �5 postchallenge by intramuscular inoc-ulation of 105 TCID50 of C-strain CSFV and challenged at day 0 and day 28by intranasal inoculation of 105 and 106 TCID50 of CSFV Brescia, respec-

TABLE 1 Experimental design of first vaccination/challenge experiment

Exptl group

Procedure(s) at day postchallenge:

�5 �2 0 3 5

1, vaccine (i.n.) only (n � 6) Vaccination with C-strain,105 TCID50, i.n.

Mock challenge i.n. PM/bleed (n � 3) PM/bleed (n � 3)

2, vaccine (i.n.) pluschallenge (n � 12)

Vaccination with C-strain,105 TCID50, i.n.

PM/bleeda (n � 3) Challenge with CSFV Brescia, 105

TCID50, i.n.; PM/bleed (n � 3)PM/bleed (n � 3) PM/bleed (n � 3)

3, vaccine (i.m.) pluschallenge (n � 12)

Vaccination with C-strain,102 TCID50, i.m.

PM/bleed (n � 3) Challenge with CSFV Brescia, 105

TCID50, i.n.; PM/bleed (n � 3)PM/bleed (n � 3) PM/bleed (n � 3)

4, challenge only (n � 12) Mock vaccination i.n. PM/bleed (n � 3) Challenge with CSFV Brescia, 105

TCID50, i.n.; PM/bleed (n � 3)PM/bleed (n � 3) PM/bleed (n � 3)

a PM/bleed, euthanization and collection of blood samples.

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tively, as described above. Three negative-control animals received similarinoculations of mock virus supernatant on each occasion. A third animalexperiment (experiment 3) was performed, and the experimental designand procedures were described previously (28). In brief, 6 Large White/Landrace cross male pigs, 8 to 10 weeks of age, were utilized. Four pigswere inoculated with 1 ml (100 50% protective doses [PD50]) of the re-constituted C-strain vaccine into the brachiocephalic muscle (as recom-mended by the manufacturer, Riemser Arzneimittel AG), and two unvac-cinated pigs were used as negative controls and housed in a separate pen.

Clinical, hematological, and virological methods. Animals were in-spected twice daily (a.m. and p.m.), and 9 parameters relevant for indica-tion of CSF (liveliness, body tension, body shape, breathing, walking, skin,eye/conjunctiva, appetite/leftovers at feedings, and defecation) were ex-amined and scored as 0 (normal), 1 (slightly altered), 2 (distinct clinicalsign), or 3 (CSF symptoms) (29). A total clinical score for each animal wasassigned twice daily, and temperatures were monitored by rectal ther-mometer readings and recorded once daily. For experiment 1, tempera-ture and clinical score monitoring commenced on day �11 postchallengeand continued until the termination of the experiment (day 5 postchal-lenge), and for experiment 2, temperature and clinical score were moni-tored for 7 days before and after each of the CSFV Brescia inoculations.Peripheral blood leukocytes were enumerated every 3 or 7 days duringexperiment 1 or 2, respectively, using flow cytometry (29). In brief, 50 �lEDTA blood was incubated with 5 �l of anti-porcine CD45-fluoresceinisothiocyanate (FITC) monoclonal antibody (MAb) (0.1 mg/ml, K252-1E4; AbD Serotec, Oxford, United Kingdom) for 10 min at room temper-ature (RT) in the dark (details of all the antibodies used in this work arereported in Table 2). Fluorescence-activated cell sorting (FACS) lysingsolution (945 �l) (BD Biosciences, Oxford, United Kingdom) was addedfor 10 min at RT to lyse erythrocytes and fix leukocytes. Cell counts wereobtained on a volumetric flow cytometer (MACSQuant Analyzer; Milte-nyi Biotec, Bisley, United Kingdom) by gating FITC-positive events, andleukocyte counts per �l of blood were obtained by multiplying the leuko-cyte density by the dilution factor (�20). For experiment 1, CSFV RNAwas detected in blood samples collected every 3 days. Total RNA wasextracted from EDTA blood using the QIAamp viral RNA mini-extrac-tion kit, according to the manufacturer’s protocols (Qiagen, Crawley,United Kingdom). CSFV viral copy numbers were assessed by quantita-tive reverse transcription-PCR (RT-PCR), using the One-Step Super-script III Platinum kit (Invitrogen) (29). Primers CSF100-F andCSF192-R were used at a concentration of 600 nM, and the 6-carboxy-fluorescein (FAM)-labeled CSF probe (30) was used at 200 nM. The viralgenome copy number was determined by comparison to a 10-fold serial

dilution of a standard, which consisted of RNA transcribed from plasmidpCRXLV234-6, containing the 5= untranscribed region (UTR) of CSFVstrain Alfort 187 (29).

Purification and cryopreservation of PBMC. Heparinized venousblood was collected on days �5, �2, 0, 2, and 5 postchallenge duringexperiment 1; on days �5 and 0 and every 7 days until day 56 postchal-lenge during experiment 2; and on days 0, 3, 6, 9, 12, 15, and 18 postvac-cination during experiment 3. Peripheral blood mononuclear cells(PBMC) were prepared by diluting 20 ml of blood in 10 ml of phosphate-buffered saline (PBS) (Invitrogen), layering it over 20 ml of Ficoll (Ficoll-Paque Plus; GE Healthcare, Uppsala, Sweden), and centrifuging it at1,455 � g for 30 min at room temperature (RT), without braking, in arotating bucket centrifuge (Beckman Coulter, High Wycombe, UnitedKingdom). PBMC were aspirated from the plasma-Ficoll interface andwashed three times in PBS by centrifugation at 930 � g for 5 min at 4°C.PBMC were resuspended in RPMI 1640 medium supplemented with 10%FBS, 100 IU/ml penicillin, and 100 �g/ml streptomycin (cRPMI), and celldensity was determined by analyzing 50 �l of cell suspension using avolumetric flow cytometer (Miltenyi Biotec) and gating on events withtypical forward scatter (FSC) and side scatter (SSC) for PBMC. Cells werecryopreserved for later analyses. PBMC were adjusted to a density of 1 �107 to 2 � 107 cells/ml, resuspended in cold (4°C) 10% dimethyl sulfoxide(DMSO) (Sigma, Poole, United Kingdom) in FBS, and transferred toprecooled (4°C) labeled cryotubes. These tubes were immediately trans-ferred to a precooled (4°C) Cryo 1°C freezing container (Nalgene; FisherScientific, Loughborough, United Kingdom) containing 250 ml of 100%isopropyl alcohol (4°C), which was placed in a �80°C freezer for a mini-mum of 4 h to a maximum of 24 h. Cryotubes were then transferred to aliquid nitrogen storage container.

Stimulation of peripheral blood mononuclear cells. To resuscitatecryopreserved cells, cryovials were rapidly thawed in a 37°C water bathand cells were transferred to tubes containing 10 ml of prewarmed (37°C)cRPMI. Cells were washed by centrifugation, 930 � g for 5 min at RT, andresuspended in fresh warm cRPMI. Cell densities were calculated usingflow cytometry as described above and adjusted to 1 � 107 cells/ml, and100 �l was transferred to wells of a 96-well round-bottom microtiter plate(Costar; Fisher Scientific). PBMC were stimulated with 100 �l of CSFVAlfort 187 strain at an MOI of 1. Mock-virus-infected PK15 supernatantand concanavalin A (ConA; Sigma, 10 �g/ml) were used as negative andpositive controls, respectively. Cells were incubated at 37°C for 14 to 16 h,then brefeldin A (GolgiPlug; BD Biosciences) was added (0.2 �l/well), andcells were further incubated for 6 h before performing flow cytometricanalysis. To detect cytotoxic degranulation, PBMC were incubated with

TABLE 2 Monoclonal antibodies for flow cytometric analysis of porcine T cell responses

MAb Source Clone Concn (mg/ml) Isotype Labeling Working dilution

CD45 AbD Serotec K252-1E4 0.1 IgG1 FITC NeatCD107a AbD Serotec 4E9/11 0.05 IgG1 Alexa Fluor 647 NeatCD8� BD Pharmingen 76-2-11 0.2 IgG2a PE 1/10CD4 BD Pharmingen 74-12-4 0.2 IgG2b PerCP-Cy5.5 1/10CD3 Southern Biotech BB23-8E6 0.5 IgG1 Unconjugated 1/2a

�-TcR1-N4 VMRD PGBL22A 1.0 IgG1 Unconjugated 1/2a

CD25 AbD Serotec K231.3B2 Not determined IgG1 Unconjugated 1/5CD27 In-house b30c7 Not determined IgG1 Unconjugateda 1/4CD44 eBioscience IM7 0.2 IgG2b eFluor450 1/16CD62L AbD Serotec CC32 1.0 IgG1 Unconjugated 1/100IFN-� AbD Serotec CC302 0.05 IgG1 FITC or Alexa Fluor 647 1/10TNF-� BioLegend MAb11 0.5 IgG1 Pacific Blue 1/10IL-2 Invitrogen A150D 3F1 1.0 IgG2a Unconjugated 1/4a

Mouse IgG1 BD Pharmingen A85-1 0.2 Rat IgG1 APC 1/20IgG1 isotype control AbD Serotec NAc 0.05 IgG1 FITC or Alexa Fluor 647 Neat, 1/10b

a MAb dilution following Zenon labeling.b Isotype control used neat to control CD107a staining and 1/10 for IFN-� staining.c NA, not applicable.

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virus/mock supernatants for 6 h before the addition of CD107a-AlexaFluor 647 or IgG1 isotype control-Alexa Fluor 647 MAbs (both from AbDSerotec; 10 �l/well) (Table 2), brefeldin A (as above), and monensin(Golgi Stop; BD Bioscience; 0.2 �l/well) and then cultured for a further14 h.

Multiparameter cytofluorometric analysis of peripheral bloodmononuclear cells. Cells were harvested and washed in Dulbecco’s PBSwithout Mg2� and Ca2� (Invitrogen). To assess viability, cells werestained with Near InfraRed Fixable Live/Dead viability dye (Invitrogen)for 30 min at 4°C and then washed twice with Dulbecco’s PBS withoutMg2� and Ca2� supplemented with 2% FBS and 0.09% sodium azide(FACS buffer). Cells were stained with the MAbs specific for surfacemarkers for 10 min at RT and then washed twice with FACS buffer. MAbsused to discriminate T and NK cell populations were CD8�-phycoeryth-rin (PE) (76-2-11; BD Pharmingen), CD4-peridinin chlorophyll protein(PerCP)-Cy5.5 (74-12-4; BD Pharmingen), and CD3 (BB238E6; South-ern Biotech, Cambridge Bioscience, Cambridge, United Kingdom). CD3MAb was labeled using the Zenon Alexa Fluor 647 IgG1 labeling kit,according to the manufacturer’s instructions (Invitrogen). To directlyassess the contribution of � T cells to CSFV-specific IFN-� responses,CSFV/mock-virus-stimulated PBMC from three representative C-strain-vaccinated/Brescia-challenged pigs from each of experiments 1 (day 5postchallenge) and 2 (day 21 postchallenge) were stained with CD8�-PEMAb and a MAb against �-TcR1-N4 (PGBL22A; VMRD, Pullman, WA,USA) conjugated to the Alexa Fluor 647 Zenon IgG1 labeling kit.

Since results showed that CSFV-specific IFN-�� cells were locatedsolely in CD3� �-TcR1� CD4� CD8�low and CD3� �-TcR1� CD4�

CD8�high T cell populations (see Fig. 2 and 4 and also Fig. S1 in thesupplemental material), subsequent stainings were conducted with theomission of CD3 MAb and used only CD4 and CD8� MAbs to discrimi-nate these two T cell populations. The following MAbs were used to assesssurface expression of activation/memory markers: CD25 (K231.3B2; AbDSerotec), CD27 (b30c7 [31]), CD44-eFluor450 (IM7; eBioscience, Hat-field, United Kingdom), and CD62L (CC32; AbD Serotec). Since cryo-preservation of human PBMC had been reported to induce a decrease inCD62L expression (32), an experiment was conducted to compare expres-sion of CD62L, CD44, CD25, and CD27 on antigen-specific IFN-�� CD8T cells stimulated from fresh versus cryopreserved/thawed PBMC. Twopigs previously infected with porcine reproductive and respiratory syn-drome virus (PRRSV) were utilized, and PBMC were stimulated with asynthetic PRRSV peptide previously shown to be recognized by CD8 Tcells from these animals (H. Mokhtar and S. P. Graham, unpublisheddata). Staining with CD25, CD27, and CD62L MAbs was visualized byincubation of cells with allophycocyanin (APC)-conjugated rat anti-mouse IgG1 (BD Biosciences) for 10 min, and then cells were washedtwice with FACS buffer.

Surface-stained cells were fixed and permeabilized using CytoFix/Cy-toPerm solution (BD Bioscience) for 20 min at 4°C. After two washes inBD Perm/Wash buffer (BD Biosciences), PBMC were incubated with cy-tokine-specific MAbs at RT for 10 min in the dark. MAbs used wereIFN-�–FITC or -Alexa Fluor 647 (CC302; AbD Serotec), TNF-�–PacificBlue (MAb11; Biolegend, Cambridge Bioscience), and IL-2 (A150D 3F1;Invitrogen), labeled using the Zenon Alexa Fluor 647 mouse IgG2a label-ing kit (Invitrogen). IgG1-FITC or -Alexa Fluor 647 isotype control MAbswere used to control staining with IFN-� MAbs. Unstained cells were usedas controls for IL-2 and TNF-�. The cells were given two final washes inBD Perm/Wash buffer and resuspended in FACS buffer prior to flowcytometric analysis. Further details of all the antibodies used in this studyare reported in Table 2. All the washes used centrifugation at 930 � g for2 min, and cells were analyzed on a MACSQuant (Miltenyi Biotec) or aCyAn ADP (Beckman Coulter) flow cytometer. Cells were analyzed byexclusion of doublets followed by gating on viable cells (Live/Dead FixableDead Cell stain negative) in the lymphocyte population, and defined lym-phocyte subpopulations were then gated upon and their expression ofcytokines was assessed. Gates were set using the corresponding isotype/

unstained controls, and values were corrected by subtraction of the per-cent positive events in the biological negative control (mock virus super-natant stimulated). The number of singlet, live lymphocytes acquired foranalysis ranged from 200,000 to 400,000, which translated to 20,000 to80,000 CD8 T cells being analyzed. To determine the absolute numbers ofIFN-�-producing cells, the PBMC density in peripheral blood was as-sessed by CD45 staining as described above except with the exclusion ofgranulocytes, which were distinguished by their forward and side scattercharacteristics. This value was then used to convert the percentage ofIFN-�-secreting cells within the viable PBMC gate to a cell density/�l ofblood.

Data analysis and statistics. Graphical and statistical analysis was per-formed using GraphPad Prism 5.04 (GraphPad Software Inc., La Jolla,CA). Data were represented as means with standard errors of means(SEM) quoted to indicate the uncertainty around the estimate of thegroup mean. The differences between mean values were analyzed by atwo-tailed unpaired t test or a one-way analysis of variance (ANOVA)followed by Bonferroni’s posttest or Dunnett’s test, and a P value of 0.05was considered statistically significant.

RESULTSCD8 T cells are the primary source of virus-specific IFN-� afterC-strain vaccination and challenge with virulent CSFV. Therapid onset of protection conferred by the C-strain vaccine haspreviously been correlated with the induction of a broadly cross-reactive T cell IFN-� response (7). To characterize this early re-sponse, an in vivo experiment was conducted where pigs werevaccinated with the C-strain vaccine and challenged after 5 dayswith the virulent Brescia strain. Vaccinated pigs were solidly pro-tected against CSF. In contrast, unvaccinated challenge controlpigs developed clinical signs of the disease, viremia, and leukope-nia (Fig. 1). PBMC from vaccinated and challenged control pigswere collected longitudinally during the experiment, at days �2,0, 3, and 5 postchallenge, and cryopreserved. CSFV-specific IFN-�release from different lymphocyte populations was assessed usingflow cytometry in order to identify the cellular source (Fig. 2A). Asshown in Fig. 2B, CD8 T cells, as defined by the CD3� CD4�

CD8�high phenotype (33), were identified as the initial source ofCSFV-specific IFN-� after vaccination with the C-strain virus.Neither CD4� CD8�� nor CD4� CD8�low T cells, which in swinehave been proposed to represent naive and activated/memorypopulations, respectively (33), showed a significant increase inCSFV-specific IFN-�� cells over the time period analyzed. CD3�

CD8�� NK cells and CD8�low/� T cells, which are mostly � Tcells (33), did not show significant IFN-� expression after virusstimulation at any of the time points examined. To exclude thepossibility that CD8�� � T cells falling within the CD3� CD4�

CD8�high gate were contributing to the IFN-�� cells detected,assays were performed with a pan-�-T cell MAb and utilizingPBMC from 3 representative vaccinated and challenged pigs (seeFig. S1 in the supplemental material). Both challenged and un-challenged vaccinated animals, but not the challenge-only pigs,displayed significant virus-specific CD8 T cell IFN-� responsesfrom 8 days after vaccination (day 3 postchallenge) (P 0.05).Interestingly, CD8 T cells from vaccinated but unchallenged ani-mals showed a higher frequency of IFN-�� CD8 T cells than didvaccinated and challenged animals at 8 days postvaccination (P 0.05; Fig. 2C). The frequency of virus-specific CD8 T cells in-creased most markedly in the vaccinated and challenged pigs onday 5 postchallenge (10 days after vaccination), with the greatestnumber of cells being observed in those vaccinated intranasally(P 0.05).

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In order to further characterize this early T cell response, theability of the IFN-�-expressing CD8 T cells to degranulate andrelease other antiviral cytokines upon stimulation with CSFV wasinvestigated. Translocation of lysosome-associated membraneprotein 1 (LAMP1/CD107a) to the cell membrane has been con-firmed as a marker of cytotoxic degranulation by CD8 T and NKcells (34). While CD107a mobilization has been reported as amarker for porcine NK cell degranulation (35), this is the firstreport of its application to assess porcine CD8 T cell degranula-tion. PBMC isolated 5 days postchallenge were stimulated withCSFV. A proportion of IFN-�-expressing CD8 T cells from allvaccinated animals expressed CD107a on their surface, suggestiveof cytotoxic activity against CSFV-infected antigen-presentingcells (Fig. 3A). Assessment of the coexpression of IFN-� andTNF-� by CD8 T cells after virus stimulation showed individualvariability between animals (data not shown), but on average, themajority of CSFV-specific CD8 T cells expressed IFN-� and notTNF-� (Fig. 3B). Comparison of TNF-� and CD107a expressionon IFN-�� CD8 T cells between experimental groups showed asignificantly greater number of TNF-�� IFN-�� cells in all vacci-nated groups than in the challenge control group (P 0.05), butonly the intranasally vaccinated and challenged animals showed agreater number of TNF-�� IFN-�� CD8 T cells (P 0.05). Thisgroup was also the only one which expressed a greater number ofboth CD107a�- and CD107a�-expressing IFN-�� CD8 T cells.There were no significant differences in the proportions ofTNF-�� IFN-�� or CD107a� IFN-�� CD8 T cells betweenmock- and CSFV-stimulated conditions (data not shown).

CSFV C-strain-induced CD8 T cell responses are sustainedand boosted upon later rechallenge infection. To further inves-tigate the possible role for T cells in providing protection against

CSFV, another experiment was conducted whereby C-strain-vac-cinated animals were challenged with the Brescia strain after 5days and then rechallenged with a 10-fold-higher dose after 28days. All animals were protected against both challenge infectionswith no reduction in leukocyte counts or clinical signs of CSFbeing observed (data not shown). PBMC were collected longitu-dinally during the experiment, and the CSFV-specific IFN-� re-sponse was again investigated ex vivo using flow cytometry (Fig.4). Significant virus-specific CD8 (CD3� CD4� CD8�high) (Fig.4A) and “memory” CD4 (CD3� CD4� CD8�low) (Fig. 4B) T cellresponses were detected in vaccinated and challenged animals, butnot control animals, from days 7 and 14 postchallenge, respec-tively. No virus-specific IFN-� expression was detected in “naïve”CD4 T cell (CD3� CD4� CD8�high), CD8�low/� T cell, or NK cellpopulations (data not shown). As described above, the contribu-tion of � T cells was directly assessed on a subset of pigs using aT-cell receptor (TcR) -chain MAb, and no CSFV-specific in-crease in IFN-�� � T cells was detected (see Fig. S1 in the sup-plemental material). The CD8 T cell response showed an initialpeak on day 14 postchallenge, contracted to a degree, and thenpeaked again 7 days after the second challenge infection (day 35)(Fig. 4A). The percentage of cells remaining in the contractedCSFV-specific CD8 T cell population following this second peakwas similar to that observed after the first, at �0.4%. Individualdifferences between animals were observed in the kinetics of theresponse, with pigs which showed the greatest peak in response tothe first challenge infection displaying less evidence of a recall inresponse to the second challenge (data not shown). The kinetics ofthe memory CD4 T cell IFN-� response was largely similar to theCD8 response except that the frequency of cells was lower and wasaccompanied with an increase, albeit to lower levels, in the per-

FIG 1 Outcome to challenge with virulent CSFV following vaccination with live attenuated C-strain CSFV. Five days after intranasal (i.n.) or intramuscular(i.m.) vaccination, animals were challenged by intranasal inoculation with the CSFV Brescia strain. Two additional groups were vaccinated but not challengedor challenged only. (A) Clinical signs of CSF were scored twice daily. (B) Viral RNA copy numbers in peripheral blood were determined by real-time RT-PCR.(C) Leukocyte counts assessed by volumetric flow cytometry. Results are expressed as the mean data for each group, and error bars represent SEM.

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FIG 2 CD8 T cells are the primary source of CSFV-specific IFN-� following vaccination and challenge. At days �2, 0, 3, and 5 postchallenge, PBMC werestimulated in vitro with CSFV or mock virus supernatant and IFN-� expression was assessed by flow cytometry. (A) Gating strategy used to interrogate responsesin singlet, live lymphocytes and representative data from pig 51, vaccine (i.n.)/challenge group, day 5 postchallenge, are displayed. T cell populations were definedas CD3� CD8�high CD4�, CD3� CD8�low CD4�, CD3� CD8�� CD4�, and CD3� CD8�low/� CD4�, and NK cells were defined as CD3� CD8�low CD4�.Finally, intracytoplasmic IFN-� expression was assessed in each population and compared to staining with an isotype control MAb. (B) Representative data ofthe expression of IFN-� versus CD8� on live, singlet CD3� CD4� T cells after CSFV stimulation (pig 51, vaccine [i.n.]/challenge group, day 5 postchallenge). (C)Mean numbers of IFN-�-producing cells/�l blood in response to CSFV stimulation for each lymphocyte population following correction for the IFN-� expression inresponse to mock virus supernatant. The mean data from three randomly selected pigs/group/time point are presented, and error bars represent SEM.

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centage of IFN-�-expressing CD4 T cells in response to the mockvirus supernatant, an effect not observed in the control animalsdespite these animals having been inoculated with this superna-tant in place of the vaccine/challenge viruses. Subtraction of theresponse to mock virus supernatant from the CSFV-stimulatedresponses and conversion of this percentage to the number ofIFN-�-expressing cells per �l of blood clearly revealed that theCD8 T cell population was the major contributor to the CSFV-specific T cell response (Fig. 4C). The comparison of fluorescenceintensity measurements revealed no differences between IFN-�expression levels of CD8 (Fig. 4A) and memory CD4 T cells, andthe levels did not differ significantly over time (data not shown).

Following challenge, CSFV C-strain-induced CD8 T cell re-sponses are polyfunctional and show a putative mixed effector/effector memory phenotype. Phenotypic and functional analysiswas performed on PBMC previously cryopreserved at the peaksfollowing primary and secondary challenge infections, days 14and 35 post-primary challenge, respectively, and PBMC werecompared to cells collected from pigs at the peak of response post-C-strain vaccination (day 9) (28). Due to the limited number ofcryopreserved cells, for some parameters only 5 randomly chosenvaccinated and challenged pigs out of 8 were analyzed. On day 9postvaccination, and days 14 and 35 postchallenge, the majority ofIFN-�-expressing CD8 T cells expressed high levels of the celladhesion and indicative effector memory marker CD44 (Fig. 5A)

and lacked expression of the peripheral lymph node homing re-ceptor, L-selectin/CD62L (Fig. 5B). Evaluation of the expressionof CD44 and CD62L at the other time points postchallenge re-vealed that CD44high CD62L� was the dominant phenotypethroughout (data not shown). In contrast, CSFV-specific T cellsfrom both groups and selected time points displayed variable lev-els of both CD25 and CD27 (Fig. 5C and D). There were no sig-nificant differences in expression of these markers by IFN-�� CD8T cells between mock- or CSFV-stimulated conditions (data notshown). To assess the potential effect of previous cryopreservationon porcine CD8 T cell memory/activation marker expression,which might have influenced these data, a separate experimentwas conducted to directly compare expression of CD62L, CD44,CD25, and CD27 by antigen-specific IFN-�� CD8 T cells stimu-lated from fresh versus cryopreserved/thawed PBMC (see Fig. S2in the supplemental material). Following stimulation with amodel antigenic peptide, the phenotype of specific IFN-�� CD8 Tcells did not differ between fresh and previously cryopreservedPBMC, with a CD62L� CD44high CD25� CD27� phenotype be-ing discerned in both cases. However, when considering the entireCD8 T cell population, there was a small but measurable decreasein the proportion of cells expressing CD62L, CD25, and CD27following cryopreservation.

The polyfunctionality of CSFV-specific CD8 T cells at thepeaks of response postvaccination and vaccination/challenge was

FIG 3 Functional and phenotypic characterization of “early” CSFV-specific CD8 T cells. Five days postchallenge, PBMC were stimulated in vitro with CSFV ormock virus supernatant and the expression of CD107a, IFN-�, and TNF-� by singlet, live CD8�high CD4� T cells was assessed by flow cytometry. The gatingstrategy was identical to the one illustrated in Fig. 2A except with the exclusion of the CD3 MAb. Representative dot plots show quadrant gates defining expressionof IFN-� and CD107a (A) and IFN-� and TNF-� (B) by the live, singlet CD8�high CD4� T cell population. Data represent the means of 6 replicates (2replicates/pig/group) following subtraction of responses to mock virus supernatant, and error bars represent SEM. Values were compared using a two-tailedunpaired t test, and significance is indicated by *** (P 0.001), ** (P 0.01), and * (P 0.05). Data for CD107a� IFN-�� and TNF-�� IFN-�� cells are notpresented since the frequency of these cells did not differ significantly between CSFV- and mock-virus supernatant-stimulated cultures.

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again investigated by their ability to degranulate and coexpresscytokines upon virus stimulation. As had been observed in theearlier experiment, only a proportion (�50%) of IFN-�� CD8 Tcells from the vaccinated-only group had mobilized CD107a, in-dicative of cytotoxic degranulation (Fig. 5E). In contrast, the ma-jority of cells from vaccinated and challenged animals, on both day14 and day 35, showed evidence of degranulation. Coexpression ofTNF-� and IL-2 by CSFV-specific IFN-�� CD8 T cells was as-sessed using the gating strategy illustrated in Fig. 6A. Interestingly,significant variability was observed in the cytokine profile betweenindividual animals, groups, and time points (Fig. 6B). Nine dayspostvaccination, the majority of CSFV-specific CD8 T cells se-creted only IFN-�, with a significant minority (19.4 to 42% ofCSFV-specific IFN-�� CD8 T cells) coexpressing TNF-�. Only 2of the 4 animals were found to present a small proportion of CD8T cells expressing all three cytokines (2.9% and 5.1% of CSFV-

specific IFN-�� CD8 T cells). Analysis of the vaccinated and chal-lenged animals showed that on day 14 postchallenge, cells coex-pressing IFN-� and TNF-� were the largest population in 2/5animals (43.6% and 49.9% of CSFV-specific IFN-�� CD8 T cells),with the single IFN-� expressers being the major population in theother animals. In 4 of the 5 animals, there was a significant popu-lation of cells expressing all three cytokines, which ranged from9.1% to 29.6% of CSFV-specific IFN-�� CD8 T cells. On day 35postchallenge (7 days post-rechallenge), there was an increase inthe relative proportion of single IFN-� expressers in three ani-mals, while in the other two the greatest increase was observed inthe triple expressers (pig 8) and IFN-� and TNF-� dual expressers(pig 10). There were no significant differences in the proportionsof TNF-�� IFN-��, IL-2� IFN-��, or CD107a� IFN-�� CD8 Tcells between mock- and CSFV-stimulated conditions (data notshown). Assessment of the relative IFN-� expression levels, by

FIG 4 Vaccine-induced CD8 T cells display a sustained response and are boosted by rechallenge with virulent CSFV. PBMC from eight pigs vaccinated withC-strain CSFV and challenged with CSFV Brescia strain after 5 days and a further 28 days (vaccinated/challenged) and from unvaccinated/unchallenged pigs(control) were isolated on the indicated days and stimulated in vitro with CSFV or a mock virus supernatant. (A and B) IFN-� expression by CD8 (CD3� CD4�

CD8�high) (A) and CD4 (CD3� CD4� CD8�low) (B) T cells was assessed by flow cytometry using the gating strategy illustrated in Fig. 2A. (C) The absolutenumbers of CSFV-specific CD8 and CD4 T cells were calculated from mock-virus stimulation-corrected percent IFN-�� cells and absolute PBMC count data.The data represent the means of 16 and 4 replicates for the vaccinated/challenged and control groups (2 replicates/pig/group), respectively, � SEM. Mean valueswere compared using a two-tailed unpaired t test, and significance is indicated by ** (P 0.01) and * (P 0.05).

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FIG 5 Phenotypic and functional characterization of CSFV-specific CD8 T cells. PBMC from pigs vaccinated with C-strain (animal experiment 3) or C-strain-vaccinated pigs challenged with CSFV Brescia (animal experiment 2) were stimulated with CSFV and mock virus supernatant 9 days after vaccination or 14 daysafter challenge (day 14) and 7 days after rechallenge (day 35), respectively. The expression of CD44 (A), CD62L (B), CD25 (C), CD27 (D), and CD107a (E) onIFN-�� singlet, live CD8�high CD4� T cells was assessed by flow cytometry. Data represent the mean mock-virus stimulation-corrected percent IFN-�� CD8 Tcells for 8 or 16 replicate cultures (2 replicates for four vaccine-only pigs and eight vaccinated and challenged pigs, respectively), and error bars represent SEM.Values were compared using a two-tailed unpaired t test, and significance is indicated by *** (P 0.001), ** (P 0.01), and * (P 0.05).

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FIG 6 Characterization of polyfunctional cytokine expression by CSFV-specific CD8 T cells. PBMC from pigs vaccinated with C-strain CSFV (animal experiment 3; n �4) or C-strain-vaccinated pigs challenged with CSFV Brescia (animal experiment 2; n � 5) were stimulated with CSFV and mock virus supernatant 9 days aftervaccination or 14 days after challenge (day 14) and 7 days after rechallenge (day 35), respectively. The expression of TNF-� and IL-2 by CSFV-specific IFN-�� singlet,live CD8�high CD4� T cells was determined by flow cytometry. (A) Representative dot plots. (B) Mock-corrected mean proportions of IFN-�-secreting CD8 T cellsexpressing either of the two other cytokines, for duplicate cultures/animal. (C) Mean fluorescence intensity (MFI) of IFN-� staining of each population is presented. Datarepresent the mean mock-virus stimulation-corrected percent IFN-�� CD8 T cells for 8 or 10 replicate cultures (two replicates for four vaccinated pigs and eightvaccinated and challenged pigs, respectively), and error bars represent SEM. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s multiplecomparison test against CD8 T cells secreting only IFN-�. Significance is indicated by *** (P 0.001), ** (P 0.01), and * (P 0.05).

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comparing fluorescence intensity measurements, revealed that onday 9 postvaccination and days 14 and 35 postchallenge, the triple-cytokine-expressing CD8 T cells produced significantly largeramounts of IFN-� than did the single and dual expressers(Fig. 6C).

DISCUSSION

For the first time, our results provide detailed phenotypic andfunctional data on the T cell response induced by vaccination witha live attenuated C-strain vaccine and boosted upon subsequentchallenge infections. We demonstrate that CD8 T cells (CD3�

CD8�high CD4�) are the initial source of CSFV-specific IFN-�after vaccination with the C-strain vaccine and can be detected inthe blood as early as 8 days postvaccination. IFN-�-producingCD8 T cells were detected in all vaccinated groups, independentlyof the route of vaccine administration (intranasal [i.n.] or intra-muscular [i.m.]); in contrast, infection of unvaccinated animalsdid not result in a detectable T cell IFN-� response against thevirus and animals developed clinical signs of the disease, leucope-nia, and viremia. Our results are in accordance with previous pub-lications, where it has been reported that a CSFV-specific T cellIFN-� response can be identified in the blood as early as 6 or 8 daysafter C-strain vaccination (5–7), and confirm the correlation be-tween the T cell IFN-� response and protection against CSF. Theinability to detect a CSFV-specific IFN-� response in unvacci-nated challenge control pigs is in accordance with earlier studiesconducted by our group utilizing moderately virulent CSFVstrains (6, 7) but contrasts with a previous report where IFN-�-producing CD4 and CD8 T cells were detected in unvaccinatedpigs challenged with a virulent strain of CSFV (11). While CD8 Tcells were detected as the first source of IFN-� produced in re-sponse to CSFV, they were also shown to be the major producers,with 400 to 1,200 CSFV-specific CD8 T cells/�l being detected inblood. In comparison, virus-specific CD4 T cells were present at 2-to 6-fold-lower numbers, which is in general accordance with ourearlier studies (7). Moreover, rechallenge of animals resulted in asignificant boosting of specific CD8 T cell numbers while CSFV-responding CD4 T cells remained relatively constant in number.Interestingly, vaccinated/challenged pigs showed an increase inthe percentage of IFN-�-expressing memory CD4 T cells in re-sponse to culture with the mock virus supernatant. We mightspeculate that this is due to reactivity against an (as yet) unidenti-fied antigen present in the PK15 lysate. The presence of CSFV mayhave adjuvanted the response to this antigen so that it was ob-served only following vaccination and challenge and not seen incontrol animals inoculated with the mock virus supernatant.

We next addressed the memory/activation phenotype of theCSFV-specific CD8 T cells, since it has become apparent in recentyears that the efficacy of T-cell-based vaccines is highly dependentupon the quality of the memory population induced. CSFV-spe-cific IFN-�� CD8 T cells expressed high levels of the cell adhesionglycoprotein CD44 and lacked expression of the lymph nodehoming receptor CD62L. Based upon current models of CD8 Tcell differentiation, this would classify these cells as either effectors(TEFF) or TEM cells, which traffic via nonlymphoid tissues, ratherthan TCM cells which express CD62L (17). CD27, a costimulatoryreceptor that provides a critical survival signal to activated T cells(36), is required for CD8 memory T cell formation and respon-siveness. CD27 expression is lost on human CD8 TEFF cells, andexpression increases upon differentiation to memory cells with

low levels expressed on TEM cells and higher levels on TCM cells(37, 38). A recent study of CD27 expression by porcine CD4 T cellshas shown that antigen-specific IFN-�-expressing CD4� CD8��

T cells may be either CD27� or CD27� (39). Compared to CD27�

CD4� CD8�� T cells, CD27� CD4� CD8�� T cells respond topolyclonal activation with enhanced cytokine production and re-duced proliferation. They also lack or show reduced expression oflymph node homing receptors, and it has therefore been proposedthat CD27� and CD27� porcine CD4 T cells may represent TCM

and TEM, respectively. While a comparable analysis of CD27 ex-pression by porcine CD8 T cells is still to be conducted, a longitu-dinal study of CD27 expression on porcine CD8 T cells from birthto 6 months of age suggested that early effector cells expressed lowlevels of CD27, which was absent on late effector or memory cells(40). Thus, we may only speculate at this time that the variablelevel of CD27 expression on CSFV-specific CD8 T cells reflects amixture of effector and memory phenotypes. Variable expressionof CD25, the alpha-chain of the IL-2 receptor that is expressed onthe surface of activated lymphocytes (11, 41, 42), was also seen.Heterogeneity in expression of CD25 on CD8 T cells has beendescribed in an acute murine virus infection model, with lowCD25 expression associated with generation of long-lived mem-ory and high expression associated with terminal effector differ-entiation (43). This expression pattern may therefore also imply amixed CSFV-specific TEFF and TEM population and explain thepreviously described lack of association between enhanced CSFV-specific IFN-� production and upregulation of CD25 on CD8 Tcells (11). Since this analysis was conducted following in vitrorestimulation of previously cryopreserved PBMC, it remains to bedetermined whether these phenotypes accurately represent thoseof CSFV-specific CD8 T cells in vivo. We have recently identified anumber of CD8 T cell epitopes on CSFV (G. Franzoni and S. P.Graham, unpublished data), and together with the recent devel-opment of porcine major histocompatibility complex (MHC)class I tetramers (44), tools could be developed to confirm thephenotype of CSFV-specific CD8 T cells by direct ex vivo exami-nation. We finally assessed the polyfunctionality of the CSFV-specific CD8 T cell population since this may be central to theirprotective capacity. Based on current models, CD8 TEM cells havebeen shown to have high ex vivo cytotoxic potential and rapidlyproduce cytokines upon reexposure to antigens, whereas CD8TCM cells possess a greater proliferative capacity (15). Interest-ingly, we found that early postchallenge or in the absence of chal-lenge only a proportion of CSFV-specific IFN-� CD8 T cells mo-bilized CD107a, compared to later time points postchallenge,where the majority expressed CD107a on their surface. CD107a isexpressed on the cell surface following activation-induced degran-ulation, and it has been described as a marker for cytotoxic CD8 Tcell activity whose expression has been associated with loss of per-forin following antigen stimulation (34). The data presented heresuggest that CSFV-specific IFN-�-expressing cells may also havecytotoxic capacity and consolidate our previous results where weshowed that IFN-� expression was restricted to CD8 T cells ex-pressing intracellular perforin (7) and other earlier studies thatdemonstrated CSFV-specific cytotoxic activity by T cell culturesisolated from vaccinated pigs (8–10). While IFN-�-secreting TEM

cells have been implicated in protection against a variety of intra-cellular pathogens, simultaneous production of IFN-�, TNF-�,and IL-2 detected at the single-cell level by multiparameter flowcytometry has been correlated with enhanced vaccine-induced

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protection (45–51). Moreover, data suggest that such polyfunc-tional CD8 T memory cells are critical to long-term protectionagainst other viruses belonging to the family Flaviviridae (20, 22).In the present study, we observed that the majority of CD8 T cellsproduced only IFN-� or IFN-� and TNF-�, with only a smallpercentage coexpressing IFN-�, TNF-�, and IL-2. As seen withCD107a mobilization, there appeared to be a trend toward anincrease in functionality following challenge, although interani-mal variability was observed. Since all animals were solidly pro-tected against the challenge infections, we were unable to correlatepolyfunctionality with enhanced protection. Nevertheless, triple-cytokine-expressing T cells showed the highest “quality” of re-sponse followed by the cells coexpressing TNF-�, similar to thatobserved in other systems (48, 52, 53).

Overall, our data support the hypothesis that CD8 TEFF/TEM

cells play a key role in the protection afforded by C-strain CSFV inthe early period preceding the appearance of neutralizing antibod-ies and are also readily recalled by later challenge. This informa-tion and the novel flow cytometric panels developed and em-ployed for the first time in the pig to characterize these responseswill hopefully aid the development and evaluation of next-gener-ation vaccines against CSFV and more broadly against other por-cine pathogens.

ACKNOWLEDGMENTS

We thank Nicole Piontkowski, Riemser Arzneimittel AG, Germany, forsupplying the C-strain CSFV vaccine; Alexandra Meindl-Böhmer, Uni-versity of Veterinary Medicine, Hannover, Germany, for provision of theCSFV Brescia strain; Adam Whelan and Bernardo Villarreal-Ramos,AHVLA TB Department, for discussions on cross-reactive T cell markerMAbs; Christine Jansen, Utrecht University, the Netherlands, for adviceon CD107a mobilization assays; Bentley Crudgington and HelenMokhtar, AHVLA Virology Department, for technical assistance duringthe first animal experiment; Derek Clifford and the AHVLA Animal Ser-vices Unit for the care of animals and provision of samples; and FalkoSteinbach, AHVLA Virology Department, for critical review of the man-uscript.

This work was supported by project SE0796 from the Department forthe Environment, Food and Rural Affairs of the United Kingdom. N.V.K.was supported by a DBT CREST Award from the Ministry of Science &Technology, Government of India.

The authors declare that they have no commercial or other associationthat may pose a conflict of interest.

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