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Optimization of the Protocols for Double Vaccination Against Marek's Diseaseby Using Commercially Available Vaccines: Evaluation of Protection, VaccineReplication, and Activation of T CellsAuthor(s): Isabel M. Gimeno, Aneg L. Cortes, Richard L. Witter and Arun R. PandiriSource: Avian Diseases, 56(2):295-305. 2012.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9930-091311-Reg.1URL: http://www.bioone.org/doi/full/10.1637/9930-091311-Reg.1

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Optimization of the Protocols for Double Vaccination Against Marek’s Disease byUsing Commercially Available Vaccines: Evaluation of Protection, Vaccine

Replication, and Activation of T Cells

Isabel M. Gimeno,AD Aneg L. Cortes,A Richard L. Witter,B and Arun R. PandiriAC

ADepartment of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607BAvian Disease and Oncology Laboratory, United States Department of Agriculture, Agricultural Research Service, East Lansing, MI 48823

Received 13 September 2011; Accepted 10 December 2011; Published ahead of print 12 December 2011

SUMMARY. Revaccination against Marek’s disease is a widespread practice in some countries. The rationale of this practice isunknown, and there is no consensus in the protocols. Recently, we have demonstrated that administration of the first vaccine at18 days of embryonation followed by a more protective second vaccine at hatch (18ED/1d) reproduced systematically the benefitsof revaccination under laboratory conditions. Here, we have used the same model to optimize the revaccination protocols by usingcurrently available vaccines and to determine whether two features associated with Marek’s disease vaccine-induced protection(activation of T cells and replication of vaccine virus) are involved in the revaccination protocols. Protection conferred by threerevaccination protocols (turkey herpesvirus [HVT] 18ED/HVT+SB-1 1d, HVT 18ED/CVI988 1d, and HVT+SB-1 18ED/CVI988 1d) was evaluated. Revaccination protocols also were compared with single vaccination protocols (HVT 18ED, HVT+SB-118ED, HVT+SB-1 1d, CVI988 18ED, and CVI988 1d). Our results demonstrated that it is possible to improve efficacy of thecurrently available vaccines by using them in revaccination programs. Administration of HVT 18ED/CVI988 1d and HVT+SB-118ED/CVI988 1d were the two protocols that conferred the highest protection against a very early challenge (2 days of age) with veryvirulent plus Marek’s disease virus strain 648A. In a separate experiment, we evaluated vaccine replication and activation of T cells insingle and revaccination protocols. Our results demonstrated that replication of the second vaccine, although decreased compared withsingle vaccination, could be detected at 3 days (HVT, CVI988) or at 6 days (SB-1). Administration of the first vaccine (HVT) at 18EDresulted in a high percentage of activated T cells. Administration of a second vaccine (either HVT-SB-1 or CVI988) at 1d resulted inincreased intensity of MHC-II stain in activated T cells.

RESUMEN. Optimizacion de los protocolos de vacunacion doble contra la enfermedad de Marek mediante el uso de vacunascomercialmente disponibles: Evaluacion de la proteccion, replicacion de la vacuna y activacion de celulas T.

La revacunacion contra la enfermedad de Marek es una practica muy extendida en algunos paıses. La justificacion de esta practicaes desconocida, y no hay un consenso en los protocolos. Recientemente, se demostro que la administracion de la primera vacuna alos 18 dıas de desarrollo embrionario (DE) seguido por la aplicacion de una segunda vacuna mas protectora a la eclosion (18DE/1d), reproduce de manera sistematica los beneficios de la revacunacion bajo condiciones de laboratorio. En este trabajo, se utilizo elmismo modelo para optimizar los protocolos de la revacunacion con las vacunas actualmente disponibles y para determinar si dosaspectos asociados con proteccion inducida por la vacuna contra la enfermedad de Marek (la activacion de las celulas T y lareplicacion de virus vacunal) estan involucrados en los protocolos de la revacunacion. La proteccion conferida por tres protocolos dela revacunacion (virus herpes de pavo [HVT] a los18DE/HVT + SB-1 al dıa 1; HVT 18DE/CVI988 al dıa 1; y HVT + SB-118DE/CVI988 al dıa 1) se evaluaron. Los protocolos de revacunacion tambien se compararon con los protocolos de vacunacionindividuales (HVT 18ED, HVT + SB-1 18ED, HVT + SB-1 dıa 1, CVI988 18ED, y CVI988 dıa 1). Los resultados observadosdemostraron que es posible mejorar la eficacia de las vacunas actualmente disponibles mediante su aplicacion dentro de programasde revacunacion. La administracion de HVT 18ED/CVI988 dıa 1 y HVT + SB-1 18ED/CVI988 dıa 1, fueron los dos protocolosque le confirieron la maxima proteccion contra a un desafıo muy temprano (dos dıas de edad) con una cepa muy virulenta del virusde la enfermedad de Marek, la cepa 648A. En un experimento por separado, se evaluaron la replicacion del virus vacunal y laactivacion de celulas T en los protocolos individuales y de revacunacion. Los resultados demuestran que la replicacion de la segundavacuna, aunque disminuida en comparacion con una sola vacunacion, se pudo detectar a los tres dıas (HVT, CVI988) o a los seisdıas (SB-1). La administracion de la primera vacuna (HVT) a los 18 dıas de desarrollo embrionario, resulto en un alto porcentaje decelulas T activadas. La administracion de una segunda vacuna (ya sea HVT-SB-1 o CVI988) al primer dıa de edad resulto en unaumento de la intensidad de la deteccion de moleculas MHC-II en las celulas T activadas.

Key words: revaccination, Marek’s disease, control, vaccine

Abbreviations: 1d 5 1 day of age; acTP 5 acute transient paralysis; CEF 5 chicken embryo fibroblast; Ct 5 cycle threshold;ED 5 days of embryonation; FP 5 feather pulp; HB 5 Horsfall-Bauer; HVT 5 turkey herpesvirus; MD 5 Marek’s disease;MDV 5 Marek’s disease virus; MHC 5 major histocompatibility complex; PBS 5 phosphate-buffered saline; s.c. 5 subcutaneous;vv+ 5 very virulent plus; wpc 5 weeks postchallenge

Marek’s disease (MD) is a lymphoproliferative disease of chickens(Gallus gallus domesticus) that, in the absence of control measures, is

capable of causing devastating losses in commercial poultry flocks.MD has been successfully controlled by vaccination since 1969 (7).However, vaccine efficacy has decreased concomitantly with theincrease in virulence of MD virus (MDV; 46). Of particular concernis the poor protection that bivalent vaccines (HVT+SB-1), themainstay for MD control in United States for the past 25 yr, confersagainst the emerging very virulent plus (vv+) MDVs (46).DCorrepsonding author. E-mail: isabel_gimeno@ncsu.edu

CPresent address: Experimental Pathology Laboratories, Inc., ResearchTriangle Park, NC 27709.

AVIAN DISEASES 56:295–305, 2012

295

Attenuated serotype 1 CVI988 strain is the only licensed vaccine ableto protect against vv+ MDV (50). However, it is likely that theefficacy of CVI988 will decrease in the future, as demonstrated inEurope (27,45). Over the past 30 yr, the scientific community hasstruggled to develop a vaccine that could exceed the efficacy of strainCVI988 (33,35,44,47,50). Several recombinant vaccines have beendeveloped but none of them are as efficacious as CVI988 strain(1,10,12,22,28,31,32,33,39). There are a few experimental vaccinesthat have shown to be more effective than CVI988 against earlychallenge with vv+MDV but either they induce severe lymphoid organatrophy (30,47) or they maintain residual oncogenicity (11,51) insusceptible chickens lacking maternal antibodies against MD. Thesenegative features prevent these vaccines from being licensed undercurrent regulations that require safety tests for MD vaccinesconducted in chickens lacking maternal antibodies against MDV.

Revaccination, the practice of administering MD vaccine a secondtime, has been sporadically used in commercial poultry flocks formany years (3,4,6,43,54). However, this practice became widespreadin several European and Asian countries in the late 1990s (forreview, see 16). The rationale for this practice is unknown, and thereis no consensus in the protocols. A variety of vaccine combinations,vaccine schedules, and vaccine doses have been reported previously(16). Several attempts have been made to reproduce the benefit ofMD revaccination under laboratory conditions, but all of them havebeen unsuccessful (3,4,6,14,29,36,43,54). Recently, Wu et al. (53)evaluated the positive effect of revaccination in an outbreak of MD,but they were not able to experimentally reproduce the benefits ofrevaccination. The lack of a model to study this phenomenon in thelaboratory has hindered the research community from undertakingfurther studies to better understand the mechanisms behindrevaccination against MD and to optimize protocols.

We have recently standardized a model to study revaccinationagainst MD in the laboratory (21). In our previous study, wedemonstrated that heterologous revaccination (administration of twodifferent vaccines, with the second vaccine being more protectivethan the first vaccine) but not homologous revaccination (admin-istration of same vaccine twice) could reproduce experimentally thebenefits of MD revaccination. Our previous work also showed thatadministration of the first vaccine at 18 days of embryonation(18ED) followed by a more protective second vaccine at hatchreproduced systematically the benefits of revaccination (21).

Using this model, we are planning to optimize revaccinationprotocols and to elucidate some of the mechanisms behind thebenefits of revaccination. The specific objectives of this work are tooptimize the use of currently available vaccines in revaccination

protocols and to determine whether two features associated withMD vaccine-induced protection (activation of T cells and replicationof vaccine virus) are involved in the revaccination protocols.

MATERIALS AND METHODS

Chickens. Commercially available specific-pathogen-free SPAFASchickens (Charles River Laboratories Inc., North Franklin, CT) were used.

Viruses. Oncogenic serotype 1 MDV, vv+ strain 648A at passage 10in duck embryo fibroblasts (46), was used. Vaccine strains CVI988 atpassage 43 in chicken embryo fibroblasts (CEFs; 37), FC-126 at passage10 in CEFs (46), and SB-1 at passage 13 in CEFs were used (38).

Experimental design. Two animal experiments, in two replicates each,were conducted. In experiment 1, various revaccination protocols wereevaluated, and load of challenge MDV DNA load was measured by real-time PCR. In Expt. 2, activation of T cells was evaluated by flowcytometry, and replication of vaccine viruses was studied by real-time PCR.No statistically significant differences were found between replicates, andthe data are pooled for brevity. For both experiments, the inoculation dosewas confirmed by plaque assay titration at the time of inoculation. Allchickens were handled according to the guidelines of the North CarolinaState University Animal care and use committee. In Expt. 1, all chickenswere sampled at different time points. In Expt. 2, chickens sampled at eachtime point were selected randomly based on the wing band numbers.

Expt. 1. Chickens were distributed in 10 groups (Table 1). Lots 1, 2, 3,6, 7, and 8 were vaccinated in ovo at 18ED with the vaccine indicated inTable 1. Inoculation was done as described previously (40,41). In brief,the external surface of the broad end of the egg was swabbed with 70%ethyl alcohol, and a 1-mm hole was punched in the eggshell. The viralinoculum in a 0.1-ml volume was injected by inserting the entire length ofa 3.1-cm-long, 22-gauge needle through the hole. This method ofinoculation resulted in deposition of the inoculum in amniotic fluid inmost eggs (40). After hatch, chickens from lots 4–8 were vaccinatedsubcutaneously (s.c.) with the vaccine indicated in Table 1. Chickens werekept in biosecurity level II Horsfall-Bauer (HB) isolation units for thelength of the experiment (9 wk). Lots 1–9 were challenged subcutaneouslywith vv+ strain 648A at 2 days of age. Lot 10 served as unvaccinated andunchallenged control group. Samples of feather pulp (FP) were taken at14 and 28 days postchallenge to evaluate load of viral DNA.

Expt. 2. Chickens were distributed in nine groups (Table 2). Lots 1,4, 5, and 6 were vaccinated in ovo at 18ED with the correspondingvaccine. After hatch, chickens of lots 2–6 were vaccinated subcutane-ously with the vaccine indicated in Table 2. Lot 7 served as unvaccinatedcontrol group. Chickens were kept in BSL-II HB isolation units for thelength of the experiment (6 days). At days 3 and 6 postinoculation, 14chickens per group (seven per replicate) were euthanized, and spleenswere collected to make cell suspensions for further analysis by flowcytometry. FP samples were collected at 3 and 6 days postinoculation toevaluate load of vaccine DNA by real-time PCR.

Table 1. Expt. 1 design.A

LotNo.

chickens

First vaccination Second vaccination Challenge

Vaccine Dose (PFU) Route Age (days) VaccineDose(PFU) Route

Age(days) Strain

Dose(PFU) Route

Age(days)

1 30 HVT 2000 i.o. 18ED NA NA NA NA 648A 500 s.c. 22 30 HVT+SB-1 1000 each i.o. 18ED NA NA NA NA 648A 500 s.c. 23 30 CVI988 2000 i.o. 18ED NA NA NA NA 648A 500 s.c. 24 30 HVT+SB-1 1000 each s.c. 1 NA NA NA NA 648A 500 s.c. 25 30 CVI988 2000 s.c. 1 NA NA NA NA 648A 500 s.c. 26 30 HVT 2000 i.o. 18ED HVT+SB-1 2000 s.c. 1 648A 500 s.c. 27 30 HVT 2000 i.o. 18ED CVI988 2000 s.c. 1 648A 500 s.c. 28 30 HVT+SB-1 1000 each i.o. 18ED CVI988 2000 s.c. 1 648A 500 s.c. 29 30 NA NA NA NA NA NA NA NA 648A 500 s.c. 2

10 30 NA NA NA NA NA NA NA NA NA NA NA NAAPFU 5 plaque-forming units; i.o. 5 in ovo vaccination; NA 5 not applicable.

296 I. M. Gimeno et al.

Real-time PCR. DNA was extracted from feather pulp usingPuregeneTM DNA isolation kit (Gentra Systems, Inc., Minneapolis,MN), and real-time PCR assay was performed as described previously(17,42). Primers specific for pp38 gene of serotype 1 MDV (all serotype1 minus CVI988 and specific for CVI988; 42); polymerase gene (DNA-Pol Stp 2) of serotype 2 MDV (25); a 62-bp fragment that lies betweenopen reading frames HVT072 and HVT073 of the turkey herpesvirus(HVT) genome; and chicken GAPDH gene were used. Sequences forthe respective forward and reverse primers and for the probes arepresented in Table 3. All amplifications were done using theMX3005PH PCR system (Stratagene, La Jolla, CA).

Amplifications of MDV serotypes 2 and 3 genes and chickenGAPDH gene were done in a 25-ml PCR reaction containing 50 ng ofDNA and 0.2 mM each primer and probe. SYBR Green-based MasterMix (BRILLIANTH SYBR Green QPCR) and Probe-based Master Mix(Brilliant QPCR Master Mix) that contain the appropriate buffers,nucleotides, and Taq polymerase (Biocrest-Stratagene, Cedar Creek,TX) were used. Reactions were cycled 55 times at 95 C denaturation for15 sec and a 60 C combined annealing and extension for 60 sec.Fluorescence was acquired at the end of the annealing and extensionphase.

Amplification of pp38-serotype 1 genes (both non–CVI988-specificand CVI988-specific) required the use of HotStart-IT SYBR GreenqPCR Master Mix (Biocrest-Stratagene) and different cycle parameters.After an initial template melting step at 95 C for 3 min, the DNA wasamplified during 40 cycles of 95 C for 1 min, 63 C for 30 sec, and 72 Cfor 20 sec. A final elongation step at 72 C for 5 min completed the PCRreaction. The non-CVI988 PCR used the forward non–CVI988-specificand reverse primers (Table 3). The PCR conditions were the same as forthe CVI988-specific amplification, except that the annealing tempera-ture was 60 C. The melting curves for SYBR Green cycles were obtainedat the end of amplification by cooling the sample at 20 C/sec to 60 Cand then increasing the temperature to 95 C at 0.1 C/sec. The specificity

and sensitivity of these primer sets is published separately (42). TheCVI988-specific primers were able to amplify 2 3 103 copies of aCVI988 plasmid. The non–CVI988-specific primers were not assensitive and detected 2 3 104 copies of the non-CVI988 plasmid(42). No false positive was detected with the CVI988-specific primerswhen using MD tumors from chickens that had not being vaccinatedwith CVI988 (42). No false positive was detected with the non–CVI988-specific primers when using CEFs infected with CVI988 (42).

The parameter cycle threshold (Ct) was calculated for each PCRreaction by establishing a fixed threshold. Ct is defined as the fractionalcycle number at which the fluorescence passes the fixed threshold.Relative quantification of the load of MDV DNA was evaluated by thecomparative Ct method as reported previously (17). Three Ct ratioswere established for each sample (Ct ratio GAPDH-pp38 5 CtGAPDH/Ct pp38, Ct ratio GAPDH-DNA-Pol serotype 2 5 CtGAPDH/Ct DNA Pol serotype 2, and Ct ratio GAPDH-HVT 5 CtGAPDH/Ct HVT). The higher the Ct ratio, the higher the load of viralgenome.

Flow cytometry analysis. The level of activated T cells was evaluatedby studying the expression of major histocompatibility complex (MHC)class II in CD3+ cells by flow cytometry as reported previously (19). Inbrief, spleens were collected aseptically and gently forced through sterilegauze. Tissue debris was separated by decantation, and cells were washedin phosphate-buffered saline (PBS) twice. Cell suspensions were stainedfor direct immunofluorescence in a two-color staining as describedpreviously (23). Cells were then washed and resuspended in cold flowcytometry media consisting of PBS supplemented with 2% fetal bovineserum and 0.1% sodium azide (NaN3). Finally, cells (1 3 106) wereincubated with the appropriate dilution of primary monoclonalantibody for 20 min on ice. The excess primary antibody was removedby three successive washes with ice-cold flow cytometry media. Tenthousand cells were analyzed by flow cytometry using an FACSort cellsorter (Becton Dickinson Immunocytometry Division, San Jose, CA).

Table 2. Expt. 2 design.A

Lot No. chickens

First vaccination Second vaccination

Vaccine Dose (PFU) Route Age (days) Vaccine Dose (PFU) Route Age (days)

1 28 HVT 2000 i.o. 18ED NA NA NA NA2 28 HVT+SB-1 1000 each s.c. 1 NA NA NA NA3 28 CVI988 2000 s.c. 1 NA NA NA NA4 28 HVT 2000 i.o. 18ED HVT+SB-1 1000 each s.c. 15 28 HVT 2000 i.o. 18ED CVI988 2000 s.c. 16 28 HVT+SB-1 1000 each i.o. 18ED CVI988 2000 s.c. 17 28 NA NA NA NA NA NA NA NA

APFU 5 plaque-forming units; i.o. 5 in ovo vaccination; NA 5 not applicable.

Table 3. Oligonucleotides used for real-time PCR.

Target gene Sequence Purpose

GAPDH 59-GGAGTCAACGGATTTGGCC-39 Forward59-TTTGCCAGAGAGGACGGC-39 Reverse

pp38 serotype 1 minus CVI988A 59-GAGGGAGAGTGGCTGTCAAA-39 Forward59-TCCGCATATGTTCCTCCTTC-39 Reverse

pp38 CVI988 specificB 59-GAGGGAGAGTGGCTGTCAAG-39 Forward59-TCCGCATATGTTCCTCCTTC-39 Reverse

DNA-Pol serotype 2 59-TCGTCAAGATGTTCATTCCCTG-39 Forward59 GAAAGGTTTTCCGCTCCCATA-39 Reverse59-FAM-CGCCCGTAATGCACCCGTGACT-39-TAMRA TaqMan probe

Noncoding region serotype 3 59-CGGGCCTTACGTTTCACCT-39 Forward59-GCGCCGAAAAGCTAGAAAAG-39 Reverse59-FAM-CCCGGGTCGCCTCATCTGGA-39-TAMRA TaqMan probe

AThis primer set detects every serotype 1 MDV strain except the vaccine strain CVI988. Underlined nucleotide indicates intentional mismatch(42).

BThis primer set is specific for gene pp38 of strain CVI988, and it does not amplify the pp38 genes of other serotype 1 strains. Underlinednucleotide indicates intentional mismatch (42).

Revaccination against Marek’s disease 297

Antibodies. All the antibodies used in this study were purchased fromSouthern Biotechnology Associates (Birmingham, AL) either as afluorescein isothiocyanate-conjugated or phycoerythrin-conjugated an-tibodies. Chicken antigens CD3 and CD45 were detected using CT3(5) and CD45 (34) monoclonal antibodies, respectively. MHC antigenclass II was detected using monoclonal antibody CIa (15).

Data analysis. Chickens were classified into three categories:negative, latency, and tumors, based on the load of challenge MDVDNA in FP. A chicken was considered negative when the FP sample wasamplified for the GAPDH but not for the pp38 gene of serotype 1MDV, latency when the Ct ratio GAPDH/pp38 was ,1.17, and tumorwhen the Ct ratio GAPDH/pp38 was $1.17. The threshold linebetween latency and tumor by using this primer set differs from thatreported previously using primer sets that recognized all serotype 1MDV strains gB gene (9,17,18). The threshold line for this protocol hasbeen calculated in a previous study (42).

Data were analyzed with Statistica software (StatSoft, Tulsa, OK).Comparisons among groups were conducted by an ANOVA. In allcases, Scheffe test was used as a post hoc analysis. The level of statisticalsignificance considered was P , 0.05.

RESULTS

Comparison of various revaccination protocols. Here, we usedthree revaccination protocols that were compared with singlevaccination protocols by using the same vaccines. In addition, weused the strain CVI988 administered in ovo at 18ED (CVI98818ED) as the most protective single vaccine currently availableagainst early challenge with a vv+ MDV. Results are presented inTable 4 and Fig. 1.

The first revaccination protocol (HVT 18ED/HVT+SB-1 1d)was compared with single vaccination protocols (HVT 18ED,HVT+SB-1 1d). None of them were able to protect against thedevelopment of MD in most chickens (91.7%–100% developedMD lesions). However, mean death age due to MD was higher inchicken vaccinated with HVT 18ED/HVT+SB-1 1d (41 days) thanin chickens vaccinated with HVT 18ED (36 days) or withHVT+SB-1 1d (33 days). Chickens vaccinated with HVT+SB-11d died of acute transient paralysis (acTP) at higher incidence (40%)

than chickens vaccinated with HVT 18ED (20%) or with HVT18ED/HVT+SB-1 (20%). The mean death age due to acTP washigher in chickens vaccinated with HVT 18ED/HVT+SB-1 1d(15 days) than in chickens vaccinated with HVT 18ED (13 days) orwith HVT+SB-1 1d (13 days).

The second revaccination protocol (HVT 18ED/CVI988 1d) wascompared with single vaccination protocols (HVT 18ED andCVI988 1d). The best protection was achieved in the group ofchickens receiving HVT 18ED/CVI988 1d (66.7% developed MDvs. 90%–100% in the single vaccinated groups). The mean death agedue to MD was higher in chickens vaccinated with HVT 18ED(36 days) than in chickens vaccinated with CVI988 1d (31 days) orHVT 18ED/CVI9881d (32 days). Administration of CVI988 1ddid not protect against the development of acTP (66.7%).Moreover, it increased the percentage of chickens that developedacTP (20% in chickens vaccinated with HVT 18ED and 50% inchickens vaccinated with HVT 18ED/CVI988 1d). Administrationof CVI988 also decreased the mean death age due to acTP (13 daysin chickens vaccinated with HVT 18ED vs. 11 days in chickensvaccinated with CVI988 1d or HVT 18ED/CVI9881d).

The third revaccination protocol (HVT+SB-1 18ED/CVI988 1d)was compared with single vaccination protocols (HVT+SB-1 18EDand CVI988 1d). Protection against the development of MD washigher when HVT+SB-1 18ED/CVI988 1d was administered(69.2% developed MD vs. 88%–90% in the single vaccinatedgroups). Chickens vaccinated with HVT+SB-1 18ED andHVT+SB-1 18ED/CVI988 1d had higher mean death age due toMD (40 and 41 days, respectively) than chickens receiving CVI9881d (31 days). Administration of HVT+SB-1 18ED was able toreduce the development of acTP. However, administration ofCVI988 1d increased the number of chickens that developed acTP(5.9% of the chickens vaccinated with HVT+SB-1 18ED vs. 23.5%of chickens vaccinated with HVT+SB-1 18ED/CVI988 1d). Themean death age due to acTP, however, was the same for chickensreceiving HVT+SB-1 18ED and HVT+SB-1 18ED/CVI988 1d(14 days).

Administration of CVI988 18ED was able to reduce thepercentage of chickens that developed MD lesions but did not

Table 4. Protection conferred by various single and double vaccination protocols after challenge at 2 days with vv+ strain 648A.

Lot ComparisonA Treatment No. chickens % Death acTPMean death age

due to acTP

% Chickenssurviving acTP that

developed MDMean death age

due to MDB

1 1 HVT 18ED 30 20.0a*{ 13a*{ 100.0a* 36a4 1 HVT+SB-1 1d 30 40.0b*{ 13a*{ 100.0a* 33a6 1 HVT 18ED/ HVT+SB-1 1d 30 20.0a*{ 15b*{ 91.7a* 41b*1 2 HVT 18ED 30 20.0a*{ 13a*{ 100.0a* 36a5 2 CVI988 1d 30 66.7b{ 11b 90a* 31b7 2 HVT 18ED/CVI988 1d 30 50.0b{ 11b 66.7b 32b2 3 HVT+SB-1 18ED 30 5.9a*{ 14a*{ 88.0a 40a5 3 CVI988 1d 30 66.7b{ 11b 90.0a* 31b8 3 HVT+SB-1 18ED/CVI988 1d 30 23.5c*{ 14a*{ 69.2b 41a*3 * CVI988 18ED 30 63.0 10 70.0 369 { Unvaccinated/challenged 30 100 10 NAC NA

10 Unvaccinated/unchallenged 30 0.0 NA 0.0 NAAComparisons among treatments within groups of vaccines were done by ANOVA (P , 0.05). There were three groups of vaccines evaluated

(group 1: HVT18ED, HVT+SB-1 1d, and HVT 18ED/HVT+SB-1 1d; group 2: HVT 18ED, CVI988 1d, and HVT 18ED/CVI988 1d; andgroup 3: HVT+SB-1 18ED, CVI988 1d, HVT+SB-1 18ED/CVI988 1d). Same lowercase letters indicate lack of statistically significant differencesamong treatments within each group of vaccine comparison. In addition, each treatment group was compared with CVI988 18 ED (statisticallysignificant differences are marked with an asterisk) and with the group of unvaccinated but challenged chickens (statistically significant differencesare marked with a dagger).

BMean death age of chickens that survived acTP but died of MD.CNA 5 not applicable.

298 I. M. Gimeno et al.

protect against the development of acTP (63%). Moreover, themean death age due to acTP was as low as that of the unvaccinatedand challenged group (10 days).

Challenge MDV DNA load in FP. Load of challenge MDVDNA in the FP was evaluated at 2 and 4 wk postchallenge (wpc) byreal-time PCR. Results are presented in Fig. 2. Chickens weredivided into three categories based on the load of viral DNA:

negative, latency, and tumors, as described in Materials andMethods. Remarkable differences were found among groups in thefrequency of chickens within the tumor category at 2 wpc (Fig. 2A).Every group receiving double vaccination had significantly fewertumors than those in the single vaccinated groups. The groupvaccinated with HVT+SB-1 18ED/CVI988 1d had the lowestincidence of chickens within the tumor category at 2 wpc (23.5%).

Fig. 1. Survival curves of chickens receiving various single or double vaccination protocols. (A) Survival curve of first vaccine combination (HVT18ED, HVT+SB-1 1d, HVT 18ED/HVT+SB-1 1d), CVI988 18ED, and unvaccinated but challenged with strain 648A10. (B) Survival curve ofsecond vaccine combination (HVT 18ED, CVI988 1d, HVT 18ED/CVI988 1d), CVI988 18ED, and unvaccinated but challenged with strain648A10. (C) Survival curve of the third vaccine combination (HVT+SB-1 18ED, CVI988 1d, HVT+SB-1 18ED/CVI988 1d), CVI988 18ED, andunvaccinated but challenged with strain 648A10.

Revaccination against Marek’s disease 299

All revaccination protocols resulted in lower frequency of chickens inthe tumor category (23.5–47.5) than those groups vaccinated withCVI988 (the best vaccine currently available) when administered as asingle vaccine (either at 18ED [53%] or at 1 day [80%]). Theincidence of chickens in the tumor category was very high in allgroups at 4 wpc (74%–100%), and no statistically significantdifferences were observed among groups (Fig. 2B).

Activation of T cells. Activation of T cells was evaluated in Expt.2, and results are presented in Figs. 2 and 3. Activation wasmeasured as frequency of MHC-II+-CD3+ cells (Fig. 3) and as theintensity of MHC-II stain in those cells (Fig. 4). At 3 days of age,frequency of activated T cells was significantly higher in all in ovo

inoculated groups (single or double vaccination) than in groupsvaccinated at 1 day (Fig. 3A,C). Differences, however, were notdetected among studied groups at 6 days of age (Fig. 3B,D).

Administration of vaccines more protective than HVT(HVT+SB-1 or CVI988), either as single vaccines or in revaccina-tion protocols, tended to have higher intensity of MHC-II stain inthe activated T cells (Fig. 4). Differences were only significantbetween chickens inoculated with CVI988 and chickens receivingonly HVT 18ED (Fig. 4C,D).

Vaccine DNA load in FP. Load of vaccine DNA in the FP wasevaluated by real-time PCR at 3 and 6 days of age. The load of HVT(Fig. 5A), SB-1 (Fig. 5B), and CVI988 (Fig. 5C) when administered

Fig. 2. Effect of revaccination on the viral DNA (challenge virus) load at various time points. Viral DNA load was measured by real-time PCR,and results were calculated based on the Ct ratio (see Materials and Methods for details). Chickens were classified based on the Ct ratio into threecategories: negative, latency, and tumors as explained in Materials and Methods. Expt. 1 was conducted in two replicates, and results are presentedtogether. (A) Load of MDV DNA in FP at 2 wpc. (B) Load of MDV DNA in FP at 4 wpc. Comparisons among groups for each combinations ofvaccines (combination 1: HVT 18ED, HVT+SB-1 1d, HVT 18ED/HVT+SB-1 1d; combination 2: HVT 18ED, CVI988 1d, HVT 18ED/CVI9881d; and combination 3: HVT+SB-1 18ED, CVI988 1d, HVT+SB-1 18ED/CVI988 1d) were conducted by ANOVA. Same letters indicate lack ofstatistically significant differences among groups (P , 0.05).

300 I. M. Gimeno et al.

alone and in combinations. HVT DNA load was not affected by theadministration of other serotypes. However, SB-1 and CVI988 DNAload was reduced by the previous administration of HVT.Nonetheless, replication of both vaccines, the first vaccine adminis-tered at 18ED and the second vaccine administered at 1 day, could bedetected at 3 days (HVT, CVI988) and at 6 days (SB-1) of age by real-time PCR.

DISCUSSION

In a separate study, we standardized a method to study thebenefits of revaccination against MD in the laboratory (21). In ourprevious work, we demonstrated that in ovo vaccination at 18EDfollowed by revaccination at 1 day of age (18ED/1d) was the mostadvantageous revaccination protocol. Here, we have used the same

model to optimize the use of currently available vaccines inrevaccination protocols and to determine whether two featuresassociated with MD vaccine-induced protection (activation of T cellsand replication of vaccine virus) are involved in the revaccinationprotocols. Our results demonstrated that it is possible to improveefficacy of the currently available vaccines by using them as part ofrevaccination programs. HVT 18ED/CVI988 1d and HVT+SB-118ED/CVI988 1d were the two protocols that conferred the highestprotection against a very early challenge (2 days of age) with vv+MDV strain 648A (Table 4). Administration of the first vaccine at18ED resulted in a high percentage of activated T cells at 3 days postvaccination (Fig. 3). Administration of a second vaccine at 1 day(more protective than the first administered vaccine) resulted inincreased intensity of MHC-II stain in activated T cells (Fig. 4).Replication of both vaccines, the first vaccine administered at 18ED

Fig. 3. Frequency of activated T splenocytes (CD3+-MHC-II+) after single or double vaccination. Results are expressed as percentage of the age-matched unvaccinated control chickens. Expt. 2 was conducted in two replicates, and results are presented together. There were seven chickenssampled per treatment and per time point in each replicate (14 chickens total). (A) Frequency of activated T cells in HVT 18ED, HVT+SB-1 1d,and HVT 18ED/HVT+SB-1 1d vaccinated chickens at 3 days of age. (B) Frequency of activated T cells in HVT 18ED, HVT+SB-1 1d, and HVT18ED/HVT+SB-1 1d vaccinated chickens at 6 days of age. (C) Frequency of activated T cells in HVT 18ED, CVI988 1d, and HVT 18ED/CVI9881d vaccinated chickens at 3 days of age. (D) Frequency of activated T cells in HVT 18ED, CVI988 1d, and HVT 18ED/CVI988 1d vaccinatedchickens at 6 days of age. Comparisons among groups for each combination of vaccines were conducted by ANOVA. Same letters indicate lack ofstatistically significant differences among groups (P , 0.05). Asterisks indicate that results are statistically significant with the age-matchedunvaccinated controls. Bars indicate 95% confidence interval.

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and the second vaccine administered at 1 day, could be detected at3 days (HVT, CVI988) or at 6 days (SB-1) of age by real time PCR(Fig. 5). Replication of HVT was not affected by administrationwith other serotypes. However, replication of SB-1 and CVI988 wasreduced when HVT was administered previously.

Our results demonstrated that administration of a second vaccineagainst MD had a benefit on the protection against the developmentof the disease. However, several factors could influence the positiveeffect of the second vaccine (e.g., interval of vaccination and time ofchallenge, efficacy of the vaccine strains used, and the presence orabsence of maternal antibodies against MDV). When the intervalbetween the administration of the second vaccine and challenge wasmore than a week (21), combinations of HVT 18ED/HVT+SB-1 1dwere very efficacious. However, the same vaccine protocol did notprotect against MD when the challenge with a vv+ MDV occurred1 day after the second vaccine either in previous work (21) or in the

present study. In contrast, combinations that include HVT 18ED/CVI988 1d or HVT+SB-1 18ED/CVI988 1d were able to protectbetter against an early challenge with a vv+ MDV. In this study, wehave used a very stringent challenge system: high doses of a vv+ MDVat 2 days. Under these conditions, we have demonstrated thatrevaccination protocols using HVT 18ED/CVI988 1d and HVT+SB-1 18ED/CVI988 1d were the most efficacious methods of control.Further studies need to be conducted to confirm these results incommercial chicken lines with maternal antibodies against MDV.

Vaccination and maternal antibodies protects against thedevelopment of acTP (20,49). Chickens used in this study did nothave maternal antibodies against MDV. All chickens werevaccinated, but challenge occurred before the immune responseelicited by vaccines could stop the onset of acTP. Great differenceswere observed in the ability of various vaccines to protect againstacTP under these conditions (Table 4). In ovo vaccination with

Fig. 4. Intensity of MHC-II stain in activated T splenocytes (CD3+-MHC-II+) after single or double vaccination. Results are expressed aspercentage of the age-matched unvaccinated control chickens. Expt. 2 was conducted in two replicates, and results are presented together. There were7 chickens sampled per treatment and per time point in each replicate (14 chickens total). (A) Intensity of MHC-II stain in activated T splenocytesin HVT 18ED, HVT+SB-1 1d, and HVT 18ED/HVT+SB-1 1d vaccinated chickens at 3 days of age. (B) Intensity of MHC-II stain in activated Tsplenocytes in HVT 18ED, HVT+SB-1 1d, and HVT 18ED/HVT+SB-1 1d vaccinated chickens at 6 days of age. (C) Intensity of MHC-II stain inactivated T splenocytes in HVT 18ED, CVI988 1d, and HVT 18ED/CVI988 1d vaccinated chickens at 3 days of age. (D) Intensity of MHC-IIstain in activated T splenocytes in HVT 18ED, CVI988 1d, and HVT 18ED/CVI988 1d vaccinated chickens at 6 days of age. Comparisons amonggroups for each combination of vaccines were conducted by ANOVA. Same letters indicate lack of significant differences among groups (P , 0.05).Asterisks indicate that results are statistically significant with the age-matched unvaccinated controls. Bars indicate 95% confidence interval.

302 I. M. Gimeno et al.

HVT or HVT+SB-1 reduced greatly the percentage of chickens thatdied from acTP. However, CVI988 confer poor protection againstacTP regardless of the administration route (in ovo at 18ED orsubcutaneously at 1 day). In addition, CVI988 seem to have anegative effect on the protection against acTP conferred by othervaccines. Administration of HVT 18ED by itself protects 80% ofthe chickens against development of acTP, but it only protects 50%of the chickens when HVT 18ED/CVI988 1d was administered.Similarly, administration of HVT+SB-1 18ED protected 94.1% ofthe chickens from acTP, but only 76.5% of the chickens wereprotected when HVT+SB-1 18ED/CVI988 1d was administered.These findings suggest that there are major differences in thepathogenesis of CVI988 and that of HVT or HVT+SB-1 that willdeserve further studies.

In previous studies (8,17), we demonstrated that early diagnosis ofMD can be done by measuring load of challenge MDV DNA ineither blood (17) or FP (8). In this study, we demonstrated thatgroups receiving double vaccination had lower MDV DNA levels inFP at 2 wk than groups that received only one vaccine, regardless ofthe vaccine strain and the administration route or chicken age(Fig. 2). Because of the severity of the challenge, most chickensdeveloped MD, and high load of MDV DNA was detected at 4 wkof age. However, the lower percentage of chickens with high load ofMDV DNA (compatible with tumor levels) at 2 wk in therevaccinated groups could be associated with reduced incidence ofMD and extended long mean death age due to MD observed inthose groups. It would be necessary to repeat this experiment incommercial chickens bearing maternal antibodies against MDV that

Fig. 5. Effect of revaccination on the vaccine DNA load in FP at various time points. Viral DNA load was measured by real-time PCR andresults were calculated based on the Ct ratio (see Materials and Methods for details). (A) Load of HVT DNA in FP at 3 and 6 days of age when usingvarious single or double vaccination protocols. (B) Load of SB-1 DNA in FP at 3 and 6 days of age when using various single or double vaccinationprotocols. (C) Load of CVI988 DNA in FP at 3 and 6 days of age when using various single or double vaccination protocols. Comparisons amonggroups were conducted by ANOVA. Same letters indicate lack of statistically significant differences among groups (P , 0.05). Bars indicate 95%confidence interval.

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are challenged by contact at day of age to further evaluate protectionconferred by revaccination protocols.

The mechanisms behind the benefits associated with revaccinationare poorly understood, mainly because the lack of a proper model toreproduce them in the laboratory. Using a model that we developedpreviously (21), we have evaluated two features that have beenassociated with MD vaccine–induced protection: activation of T cells(19,26) and vaccine replication (2,19). Our results demonstrated that inovo vaccination with HVT resulted in an early increase of activated Tcells (Fig. 3). The administration of a second vaccine (more protectivethan the first vaccine) resulted in increased intensity of MHC-II stain inactivated T cells (Fig. 4). In ovo administration of HVT followed by abetter vaccine at day of age (HVT+SB-1 or CVI988) resulted in anincreased frequency of activated T cells (statistically significant) andslight increase in the intensity of MCH-II stain in activated T cells(although not statistically significant). The earlier activation of theimmune system might help in delaying the onset of infection with wild-type virus and give additional time for the second vaccine to elicit astrong immune response. Wu et al. (53) showed that revaccinationinduced expansion of T cells for a longer period than single vaccination.However, their results could not be correlated with higher protectionbecause they could not reproduce the benefits of revaccination in thelaboratory. Our results demonstrated that MD revaccination using theschedule 18ED/1d not only expands T cell subpopulations but alsoelicits an earlier and stronger activation of T cells. Additional studies onthe immune mechanisms behind the beneficial effects of revaccinationare warranted.

Vaccination against MDV does not prevent superinfection withother MDVs. Equally, it has been demonstrated recently thatinfection with an oncogenic strain does not prevent superinfectionwith another oncogenic strain (13). Most studies evaluatingsuperinfection involve infection of vaccinated chickens withoncogenic viruses (all challenge studies on vaccinated chickens) orinfection and superinfection with oncogenic viruses (13,24,48).However, the pathogenesis of superinfection of vaccinated chickenswith vaccine strains is poorly understood. Recently, it wasdemonstrated that the time interval between exposures, weak initialexposures, or both are major factors for the success of superinfectionwith oncogenic MDV strains (13). This also might be applicable tosuperinfection with vaccine viruses in the context of revaccination.Our study demonstrated that replication of the second vaccine ispossible, although it is decreased compared with single administra-tion (Fig. 5). Administration of HVT18ED followed by adminis-tration of HVT+SB-1 or CVI988 at 1 day resulted in replication ofall involved vaccines (HVT, SB-1, and CVI988). Such replicationcould be detected by real-time PCR as early as 3 days of age (HVT,CVI988) or 6 days of age (SB-1). In this study, we have used a shorttime interval between vaccines (3 days) and heterologous revaccina-tion with a second vaccine more protective than the first vaccine. Wuet al. (53) found that homologous revaccination with a time intervalof 7 days between vaccines resulted in two waves of productioninfection, suggesting that second administration of the same vaccinemight result in another wave of replication. In our study, however,double vaccination of HVT (at 18ED and at 1 day in combinationwith SB-1) did not result in higher load of HVT MDV at 6 days ofage. Discrepancies could be due to differences in the chicken strainor in the time interval between vaccines (3 days in our study vs.7 days in Wu et al. (53)). Further evaluation on the pathogenesis ofMD vaccines when using in revaccination protocols is warranted.

This study has demonstrated that it is possible to increase theprotection conferred by currently available vaccines by using them inrevaccination protocols using specific-pathogen-free chickens. Ad-

ministration of the first vaccine in ovo and the second betterprotective vaccine at 1 day of age resulted in early and strongactivation of T cells that can be related to improved protection. Thisstudy also demonstrated that superinfection of vaccinated chickenswith a different vaccine strain is possible. A better understanding ofthe mechanisms behind the benefits of MD revaccination will aid inthe optimization of this practice in the field.

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ACKNOWLEDGMENT

We thank the U.S. Poultry and Egg Association for funding this study(grant 657).

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