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Real time PCR: A rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines
Biologicals 40 (2012) 92e95
Contents lists available at SciVerse ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Real time PCR: A rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines M. Prabhu a,1, M.S. Siva Sankar a,1, V. Bhanuprakash a, *, G. Venkatesan a, D.P. Bora a, R. Yogisharadhya a, V. Balamurugan b a b
Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital (Distt.), Mukteswar 263 138, Uttarakhand, India Project Directorate on Animal Disease Monitoring and Surveillance, H A Farm, Hebbal, Bangalore 560 024, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2011 Received in revised form 16 August 2011 Accepted 12 September 2011
In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (3.324) and (3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID50 method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines. Ó 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Camelpox and buffalopox vaccines Potency C18L gene SYBR Green and TaqMan real time PCRs
1. Introduction Camelpox is one of the common contagious skin diseases of camelids [1] caused by camelpox virus (CMLV). The disease is characterized by mild local skin lesions with rare systemic infections and considerable loss in terms of morbidity, mortality, productivity and reduced draught capacity of the animal [2]. It is confined to camel rearing belts of developing countries and economically important. Both inactivated and live attenuated vaccines are available for the control of the disease in few countries. Likewise, buffalopox is a contagious viral disease that affects buffaloes (Bubalus bubalis) and rarely cows with a morbidity of 80% causing economic loss to the farmers. It is an emerging/reemerging zoonotic disease and affects humans also. In India, the incidence has been in increasing trend [3e6]. Control of the disease contributes to increased buffalo productivity and international trade. Vaccination is the method of choice for controlling the disease in enzootic countries like India. Both CMLV and BPXV are classified in the Orthopoxvirus (OPV) genus of the subfamily Chordopoxvirinae in Poxviridae family [7,8]. Therefore, the Division of * Corresponding author. Tel.: þ91 5942 286346; fax: þ91 5942 286347. E-mail address: [email protected] (V. Bhanuprakash). 1 Equal contribution.
Virology has developed live camelpox and buffalopox vaccines, which proved effective in camels and buffaloes, respectively (unpublished data). Assigning the number of live virus particles per dose is one of the important quality control aspects of a live vaccine as they determine the efficacy of vaccine and sero-conversion. Generally, the potency of a live attenuated viral vaccine is evaluated using plaque forming unit (PFU), median cell culture infective dose assays (CCID50) [9] and egg infective dose (EID50) assays based on virusinduced cytopathology [10]. But the drawbacks associated with these gold standard assays impede them from long-term implementation for potency estimation of live vaccines. These assays are subjective leading to inconsistent results depending on laboratory, personnel and time factors [11-13]. Furthermore, they are labor intensive, time consuming and have restricted throughput. Moreover, these traditional assays are not preferred in assigning the titres to the individual components of bivalent or multivalent vaccines. Conventional methods require highly specific neutralizing antibody against each component of multivalent or combined vaccine, which however is not full proof method as one hundred percent neutralization is highly unlikely. In order to circumvent these problems, real time polymerase chain reaction (rt-PCR) has been applied over recent years for estimating number of live virus particles in the monovalent [14],
1045-1056/$36.00 Ó 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2011.09.012
M. Prabhu et al. / Biologicals 40 (2012) 92e95
bivalent or multivalent vaccines in combination with virus propagation [9,15,16]. This assay is used to achieve accurate quantification of DNA or RNA template. The quantitative PCR (QPCR) based potency estimation has been successfully employed for measles vaccine [15], rotavirus vaccine (Rota TeqÒ) [17], multivalent measles, mumps and rubella vaccine [9,15], goatpox vaccine [16] and adenovirus based vaccine and vectors for gene therapy [14]. In the current study, the camelpox and buffalopox vaccines were evaluated for their potencies in terms of number of live viral particles based on the C18L gene specific SYBR GreenÒ [18] and TaqMan (19) rt-PCR assays respectively, which are being applied in our laboratory for rapid diagnosis. To the best of authors’ knowledge, this appears to be the first report on estimation of live camelpox and buffalopox virus particles in camelpox and buffalopox vaccines, respectively. 2. Materials and methods 2.1. Vaccine and cell lines The camelpox (CMLV-I, P-49) and buffalopox (BPXV-Vij, P-49) seed vaccine viruses used for the preparation of respective vaccine batches were propagated in Vero cells and maintained at the Division of Virology. The camelpox vaccine batch (A- 01/150409) with a mean titre of 106.53 0.09 TCID50/ml and buffalopox vaccine batch (A-01/060109) with a mean titre of 106.62 0.32 TCID50/ml were used as reference batches. Vero cells (African green monkey kidney) obtained from ATCC (CCL81) were used for growth and titration of camelpox and buffalopox vaccine viruses. The cells were propagated in Eagle’s minimum essential medium (EMEM) (Sigma, USA) containing 10% bovine calf serum (BCS) and for maintenance of cells and titration experiments, EMEM with 2% BCS was used. Different batches of live attenuated camelpox and buffalopox vaccines were produced and used for the potency estimation by QPCR assays. 2.2. Real time PCRs For camelpox, the SYBR GreenÒ based rt-PCR assay was carried out using published oligonucleotide primers and protocols [18]. This semi-quantitative assay had shown the melting temperature of the specific amplicon at 77.6 C with an efficiency of 102% and slope of 3.254 [18]. Similarly for buffalopox, the TaqMan PCR was employed using published oligonucleotide primers and probe targeting the C18L gene of BPXV [19]. The assay had an efficiency of 98% with high specificity and sensitivity. High accuracy and efficiency of the rt-PCR assays encouraged us to explore their potentials over conventional end point dilution assay in potency estimation of these live vaccines. In SYBR Green method, the reaction was carried out in a 50 ml volume using 10 pmol of primers and QuantiTect SYBR Green PCR Master Mix (Qiagen Inc., Valencia, CA) in an Mx3000P machine (Stratagene Inc., La Jolla, CA) [18]. Similarly, TaqMan rt-PCR was carried out in a 50 ml reaction volume containing 2 ml extracted DNA, 10 pmol of each primer, 5 pmol of TaqMan probe, 5 ml of the 10 buffer, 10 mmol/l dNTPs, and 0.25e0.5 IU of Taq DNA polymerase (M/s Invitrogen, Carlsbad, CA, USA). The PCR cycling conditions were initial denaturation at 95 C for 4 min and 40 cycles of denaturation at 95 C for 30s, annealing and extension at 61 C for 1 min[19]. 2.3. Estimation of critical time of harvest The camelpox reference vaccine batch (A- 01/150409) was reconstituted in EMEM and serially diluted (10-folds) ranging from
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neat to 104. 100 ml of each dilution was inoculated onto six-well plates having a confluent Vero cell monolayer with a cell control and the plates were maintained in an incubator at 37 C under 2.5% CO2. Each well of the plate was harvested at an interval of 6 h for 42 h using 300 ml of sterile phosphate buffered saline (PBS) (pH, 7.4) and freeze-thawed three times before employing rt-PCR. Total genomic DNA (gDNA) was extracted from all the harvests using AuPrep GEN DNA extraction kit (Life Technologies India (PVT) Ltd, New Delhi, India) as per manufacturer’s protocol and rt-PCR was carried out to determine the Ct (cycle threshold) values. To establish the standard curve, the Ct values were plotted against the log10 dilutions of virus titre and linear regression analysis was performed. The slopes of standard curves (SC) at different time interval harvests were compared and an SC of particular time interval harvest with maximum slope was selected as critical time for harvest for test batches. The Ct values for the test batches of camelpox vaccine were ascertained and the titre of each batch was determined by plotting Ct values on the standard curve. The critical time of harvest for buffalopox vaccine was carried out using the reference batch (A-01/060109) as described for camelpox except that each well of the plate was harvested at 6 h interval for 48 h using 500 ml of sterile PBS (pH, 7.4) and freezethawed three times before use. The TaqMan PCR was carried out with the DNA isolated using a commercial kit (AuPrep GEN DNA extraction kit, Life Technologies India (PVT) Ltd, India) from each hr interval. The standard curve and titre of each buffalopox vaccine batches were established in way similar to camelpox vaccine. 2.4. Median tissue culture infective dose assay (TCID50) The potencies of live camelpox and buffalopox vaccine batches by conventional end point dilution method (TCID50) were determined using a standard protocol [20]. The rt-PCR results obtained for each vaccine batch of camelpox and buffalopox were compared with that of respective virus titres estimated by conventional end point dilution (TCID50) and correlation was drawn between these methods by student’s t-test. 3. Results and discussion The potency of live vaccines is determined in vivo either in the target host/laboratory animals or in vitro titration of the virus in suitable cell culture system/embryonated eggs [21]. The gold standard assays are besotted with certain drawbacks as mentioned earlier [11e13]. Real time PCRs are additional tools to overcome the drawbacks of conventional assays. Hence, these strategies have successfully been employed earlier for estimation of potency in a number of human and animal vaccines [15e17,22]. Further, template nucleic acid concentration (both infectious and noninfectious virus) of an unknown sample can be quantified using the standard nucleic acid of known concentration. To do so, cells are initially to be infected with the vaccine virus and presumed that, if the live virus is first propagated in cell culture, the amount of gDNA produced corresponds to amount of viable particles in the vaccine preparation. The rt-PCR methods can estimate the titre of the vaccine much before the appearance of CPE and this is particularly useful in delayed CPE producing or non-cytopathic viruses. In case of CMLV, the CPE starts appearing 24 h post infection (hpi) and completes before 72 hpi. Similarly, in case of BPXV the CPE appears at 24 h post infection (hpi) progressed to 80% by 36e48 h and completes by 72 hpi. In this study, rt-PCR assays were applied to determine the exact titre of CMLV and BPXV in a given vaccine batches as early as 36 and 30 hpi, respectively. The rt-PCR methods reduced the time to read the titre of vaccine and subjective errors associated with cell
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M. Prabhu et al. / Biologicals 40 (2012) 92e95
Ct values
A
Estimation of critical time for harvest 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18
6 hrs p.i. (Slope -1.225) 12 hrs p.i. (Slope -1.553) 18 hrs p.i. (Slope -1.711) 24 hrs p.i. (Slope -1.658) 30 hrs p.i. (Slope -1.822) 36 hrs p.i. (Slope -3.324) 42 hrs p.i. (Slope -2.164)
y = -3.324x + 25.05 R² = 0.9925
0
1
2
3
4
5
6
7
Virus titer (Log 10)
B
Estimation of critical time for harvest 40 6 hr harvest
35
12 hr harvest
Ct values
30
18 hr harvest 24 hr harvest
25
30 hr harvest (Slope - 3.321)
20
36 hr harvest
y = -3.321x + 35.59 R2 = 0.9981
15 10 5 0 0
1
2
3
4
5
6
7
Virus titer (Log10) Fig. 1. Standard curves established for different time interval harvest of camelpox (A) and buffalopox (B) vaccines using rt-PCR assays.
culture based assays. The vaccine batches were selected in such a way that the titres/Ct values of the test batches might fall within the wide range of these reference vaccines dilutions. This would enable minimum number of dilutions of test vaccine batches. Further, the critical time point harvests for reference vaccines were determined and they correspond to the minimum time required for the appearance of sufficient viral particles in all the dilutions of the virus infected cells. The slope of the curve was maximum at 36 (R ¼ 3.324) and 30 hpi (3.321) harvest than that of other intervals, respectively for camelpox and buffalopox vaccines (Fig. 1A and B) and they were considered as the critical
time points to harvest the reference as well as test vaccine batches so as to get maximum respective live viral particles. The slopes obtained by 30 and 36 hpi were almost comparable with minor differences for buffalopox. A total of eight (n ¼ 8) batches of live attenuated camelpox vaccine and seven (n ¼ 7) batches of buffalopox vaccines including their respective reference batches were selected to estimate their titres by respective rt-PCRs. The titres obtained were satisfactory and within the acceptable range of accuracy (Tables 1A and B) as reported earlier for goatpox vaccine [16] which showed a high degree of correlation (0.96) with no significant difference between
Table 1A Estimation of virus infectivity titres of camelpox vaccines by SYBR QPCR and end point dilution (TCID50) assays. Vaccine Batches A-01/091209 B-01/091209 C-01/091209 D-02/161209 E-02/161209 F-02/161209 G-03/301209 H-04/060110
Ct values of QPCR against different dilutions of camelpox vaccine virus 1
Neat
10
23.03 24.25 23.40 20.11 19.25 20.49 19.73 19.42
23.78 26.44 24.90 21.33 21.41 21.61 21.14 21.49
10
2
25.25 28.93 26.61 22.07 22.47 23.41 23.95 22.42
10
3
26.63 29.46 29.19 26.00 28.79 26.20 26.12 25.54
Virus titre 10
4
29.28 33.16 30.14 29.53 30.39 29.48 28.60 28.86
QPCR 6.77 5.87 6.35 7.30 7.10 7.17 7.26 7.38
TCID50 0.87 0.58 0. 73 0.55 0.39 0.53 0.49 0.52
6.53 6.23 6.53 7.00 6.67 6.77 6.50 6.50
0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09
M. Prabhu et al. / Biologicals 40 (2012) 92e95
95
Table 1B Estimation of virus infectivity titres of buffalopox vaccines by TaqMan PCR and end point dilution (TCID50) assays. Vaccine Batches
A-01/060109 B-01/091209 C-02/060110 D-03/311209 E-04/161209 F-05/311209 G-06/091209
Ct values for different dilutions of buffalopox vaccine virus
Virus titre
Neat
101
102
103
104
QPCR
13.45 14.56 14.34 15.33 13.93 13.35 13.51
17.45 18.79 18.18 18.70 17.03 17.39 18.09
22.13 21.82 21.76 22.55 20.93 22.03 22.14
24.50 24.36 26.01 26.67 25.84 24.36 25.20
28.23 27.18 28.73 29.81 28.49 28.10 29.07
6.74 6.68 6.41 6.32 6.56 6.72 6.57
these assays. A similar degree of correlation was obtained in this study between rt-PCRs and end point dilution assay with no significant difference (p 0.05) for live attenuated camelpox and buffalopox vaccines. These assays are advantageous over end-point dilution methods. QPCR based estimation overcomes the variation between laboratories due to discrepancy in conventional assay procedures as the potency of an unknown vaccine batch was estimated relative to a reference batch [11]. However, the virus in the test vaccine batches should have the same replication rate as that of the reference batch for accuracy. As the difference in the sample/ vaccine composition might affect the replication rate of the virus [15], vaccine batches of similar composition of both camelpox and buffalopox (reference and test) were used in this study. In conclusion, the potency of live attenuated camelpox and buffalopox vaccines were evaluated, respectively using C18L gene based SYBR Green and TaqMan rt-PCRs. The critical time point for harvest of camelpox and buffalopox, respectively were 36 and 30 hpi to estimate the maximal respective virus load. The titres obtained in rtPCRs and the conventional end point dilution methods (TCID50) were well comparable with each other without significant differences. Therefore, rt-PCRs are additional tools for quantification of live camelpox and buffalopox vaccines. Nevertheless, these novel rt-PCRs based potency estimation warrant validation using large number of vaccine batches to attain better repeatability and reproducibility prior contemplating for widespread application and effective vaccination leading to prevention and control of the disease. Acknowledgment The authors thank the Director, Indian Veterinary Research Institute (IVRI) for providing necessary facilities to carry out this work, the staff of Pox Virus Laboratory for providing technical assistance. Further, first two authors acknowledge IVRI for financial support in terms of fellowships to carry out their degree programs.
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
References [19] [1] Bhanuprakash V, Prabhu M, Venkatesan G, Balamurugan V, Hosamani M, Pathak KML, et al. Camelpox: epidemiology, diagnosis and control measures. Exp Rev Anti-Infect Ther 2010;8(10):1187e201. [2] Jezek Z, Kriz B, Rothbaur V. Camelpox and its risk to the human population. J Hyg Epi Microbio Immunol 1983;27:29e42. [3] Singh RK, Hosamani M, Balamurugan V, Bhanuprakash V, Rasool TJ, Yadav MP. Buffalopox: an emerging and re-emerging zoonosis. Anim Health Res Rev 2007;8(1):105e14. [4] Yadav S, Hosamani M, Balamurugan V, Bhanuprakash V, Singh RK. Partial genetic characterization of viruses isolated from pox-like infection in cattle
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TCID50 0.15 0.09 0.21 0.21 0.12 0.10 0.22
6.62 6.54 6.54 6.30 6.42 6.64 6.52
0.32 0.04 0.12 0.18 0.25 0.20 0.16
and buffaloes: evidence of buffalo pox virus circulation in Indian cows. Arch Virol 2010;155(2):255e61. Bhanuprakash V, Venkatesan G, Balamurugan V, Hosamani M, Yogisharadhya R, Gandhale P, et al. Zoonotic infections of buffalopox in India. Zoonoses Public Health 2010;57(7e8):e149e155. Venkatesan G, Balamurugan V, Prabhu M, Yogisharadhya R, Bora DP, Gandhale PN, et al. Emerging and re-emerging zoonotic buffalopox infection: a severe outbreak in Kolhapur (Maharashtra), India. Vet Ital 2010;46(4): 439e48. Van Regenmortel MHV, Fauquet CM, Bishop DHL. Virus taxonomy: seventh report of the international committee on taxonomy of viruses. San Diego: Academic Press; 2000. ICTV. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus Taxonomy: 8th Report of the international Committee on Taxonomy of viruses. Academic Press; 2005. p. 123. Schalk JA, de Vries CG, Jongen PM. Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay. Biologicals 2005;33:71e9. Gaush CR, Smith TF. Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl Microbiol 1968;16(4):588e94. Forsey T, Heath AB, Minor PD. A collaborative study to assess the proficiency of laboratory estimates of potency of live measles vaccines. Biologicals 1992; 20:233e4. Forsey T, Heath AB, Minor Pd. A European collaborative study to assess the proficiency of laboratory estimates of potency of live measles, mumps and rubella tri-valent vaccines. Biologicals 1993;21:239e49. Fukuda A, Sengun F, Sarpay HE, Konobe T, Saito S, Umino Y, et al. Parameters for plaque formation in the potency assay of Japanese measles vaccines. J Virol Methods 1996;61:1e6. Wang F, Puddy AC, Mathis BC, Montalvo AG, Louis AA, McMackin JL. Using QPCR to assign infectious potencies to adenovirus based vaccines and vectors for gene therapy: toward a universal method for the facile quantitation of virus and vector potency. Vaccine 2005;23:4500e8. Schalk JAC, van den Elzen C, Ovelgonne H, Bass C, Jongen PMJM. Estimation of the number of infectious measles viruses in live vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179e87. Kallesh DJ, Hosamani M, Balamurugan V, Bhanuprakash V, Yadav V, Singh RK. A Quantitative PCR: a quality control assay for estimation of viable virus content in live attenuated goatpox vaccine. Indian J Exp Biol 2009;47(11): 911e5. Ranheim T, Mathis PK, Joelsson DB, Smith ME, Campbell KM, Lucas G, et al. Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (Rota TeqR). J Virol Methods 2006;131: 193e201. Balamurugan V, Bhanuprakash V, Hosamani M, Jayappa KD, Venkatesan G, Chauhan B, et al. A polymerase chain reaction strategy for the diagnosis of camelpox. J Vet Diagn Invest 2009;21(2):231e7. Singh RK, Balamurugan V, Hosamani M, Kallesh DJ, Bhanuprakash V. Sequence analysis of C18L gene of buffalopox virus: PCR strategy for specific detection and differentiation of buffalopox from orthopoxviruses. J Virol Methods 2008; 154(1e2):146e53. Reed LJ, Muench H. A simple method of estimating fifty percent end point. Am J Hyg 1938;27:493. Terpstra C, Kroese AH. Potency control of modified live viral vaccines for veterinary use. Vaccine 1996;14:570e5. Sastry L, Johnson T, Hobson MJ, Smucker B, Cometta K. Titering lentiviral vectors: Comparison of DNA, RNA and marker expression methods. Gene Ther 2002;17:1155.
Contents lists available at SciVerse ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Real time PCR: A rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines M. Prabhu a,1, M.S. Siva Sankar a,1, V. Bhanuprakash a, *, G. Venkatesan a, D.P. Bora a, R. Yogisharadhya a, V. Balamurugan b a b
Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital (Distt.), Mukteswar 263 138, Uttarakhand, India Project Directorate on Animal Disease Monitoring and Surveillance, H A Farm, Hebbal, Bangalore 560 024, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2011 Received in revised form 16 August 2011 Accepted 12 September 2011
In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (3.324) and (3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID50 method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines. Ó 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Camelpox and buffalopox vaccines Potency C18L gene SYBR Green and TaqMan real time PCRs
1. Introduction Camelpox is one of the common contagious skin diseases of camelids [1] caused by camelpox virus (CMLV). The disease is characterized by mild local skin lesions with rare systemic infections and considerable loss in terms of morbidity, mortality, productivity and reduced draught capacity of the animal [2]. It is confined to camel rearing belts of developing countries and economically important. Both inactivated and live attenuated vaccines are available for the control of the disease in few countries. Likewise, buffalopox is a contagious viral disease that affects buffaloes (Bubalus bubalis) and rarely cows with a morbidity of 80% causing economic loss to the farmers. It is an emerging/reemerging zoonotic disease and affects humans also. In India, the incidence has been in increasing trend [3e6]. Control of the disease contributes to increased buffalo productivity and international trade. Vaccination is the method of choice for controlling the disease in enzootic countries like India. Both CMLV and BPXV are classified in the Orthopoxvirus (OPV) genus of the subfamily Chordopoxvirinae in Poxviridae family [7,8]. Therefore, the Division of * Corresponding author. Tel.: þ91 5942 286346; fax: þ91 5942 286347. E-mail address: [email protected] (V. Bhanuprakash). 1 Equal contribution.
Virology has developed live camelpox and buffalopox vaccines, which proved effective in camels and buffaloes, respectively (unpublished data). Assigning the number of live virus particles per dose is one of the important quality control aspects of a live vaccine as they determine the efficacy of vaccine and sero-conversion. Generally, the potency of a live attenuated viral vaccine is evaluated using plaque forming unit (PFU), median cell culture infective dose assays (CCID50) [9] and egg infective dose (EID50) assays based on virusinduced cytopathology [10]. But the drawbacks associated with these gold standard assays impede them from long-term implementation for potency estimation of live vaccines. These assays are subjective leading to inconsistent results depending on laboratory, personnel and time factors [11-13]. Furthermore, they are labor intensive, time consuming and have restricted throughput. Moreover, these traditional assays are not preferred in assigning the titres to the individual components of bivalent or multivalent vaccines. Conventional methods require highly specific neutralizing antibody against each component of multivalent or combined vaccine, which however is not full proof method as one hundred percent neutralization is highly unlikely. In order to circumvent these problems, real time polymerase chain reaction (rt-PCR) has been applied over recent years for estimating number of live virus particles in the monovalent [14],
1045-1056/$36.00 Ó 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2011.09.012
M. Prabhu et al. / Biologicals 40 (2012) 92e95
bivalent or multivalent vaccines in combination with virus propagation [9,15,16]. This assay is used to achieve accurate quantification of DNA or RNA template. The quantitative PCR (QPCR) based potency estimation has been successfully employed for measles vaccine [15], rotavirus vaccine (Rota TeqÒ) [17], multivalent measles, mumps and rubella vaccine [9,15], goatpox vaccine [16] and adenovirus based vaccine and vectors for gene therapy [14]. In the current study, the camelpox and buffalopox vaccines were evaluated for their potencies in terms of number of live viral particles based on the C18L gene specific SYBR GreenÒ [18] and TaqMan (19) rt-PCR assays respectively, which are being applied in our laboratory for rapid diagnosis. To the best of authors’ knowledge, this appears to be the first report on estimation of live camelpox and buffalopox virus particles in camelpox and buffalopox vaccines, respectively. 2. Materials and methods 2.1. Vaccine and cell lines The camelpox (CMLV-I, P-49) and buffalopox (BPXV-Vij, P-49) seed vaccine viruses used for the preparation of respective vaccine batches were propagated in Vero cells and maintained at the Division of Virology. The camelpox vaccine batch (A- 01/150409) with a mean titre of 106.53 0.09 TCID50/ml and buffalopox vaccine batch (A-01/060109) with a mean titre of 106.62 0.32 TCID50/ml were used as reference batches. Vero cells (African green monkey kidney) obtained from ATCC (CCL81) were used for growth and titration of camelpox and buffalopox vaccine viruses. The cells were propagated in Eagle’s minimum essential medium (EMEM) (Sigma, USA) containing 10% bovine calf serum (BCS) and for maintenance of cells and titration experiments, EMEM with 2% BCS was used. Different batches of live attenuated camelpox and buffalopox vaccines were produced and used for the potency estimation by QPCR assays. 2.2. Real time PCRs For camelpox, the SYBR GreenÒ based rt-PCR assay was carried out using published oligonucleotide primers and protocols [18]. This semi-quantitative assay had shown the melting temperature of the specific amplicon at 77.6 C with an efficiency of 102% and slope of 3.254 [18]. Similarly for buffalopox, the TaqMan PCR was employed using published oligonucleotide primers and probe targeting the C18L gene of BPXV [19]. The assay had an efficiency of 98% with high specificity and sensitivity. High accuracy and efficiency of the rt-PCR assays encouraged us to explore their potentials over conventional end point dilution assay in potency estimation of these live vaccines. In SYBR Green method, the reaction was carried out in a 50 ml volume using 10 pmol of primers and QuantiTect SYBR Green PCR Master Mix (Qiagen Inc., Valencia, CA) in an Mx3000P machine (Stratagene Inc., La Jolla, CA) [18]. Similarly, TaqMan rt-PCR was carried out in a 50 ml reaction volume containing 2 ml extracted DNA, 10 pmol of each primer, 5 pmol of TaqMan probe, 5 ml of the 10 buffer, 10 mmol/l dNTPs, and 0.25e0.5 IU of Taq DNA polymerase (M/s Invitrogen, Carlsbad, CA, USA). The PCR cycling conditions were initial denaturation at 95 C for 4 min and 40 cycles of denaturation at 95 C for 30s, annealing and extension at 61 C for 1 min[19]. 2.3. Estimation of critical time of harvest The camelpox reference vaccine batch (A- 01/150409) was reconstituted in EMEM and serially diluted (10-folds) ranging from
93
neat to 104. 100 ml of each dilution was inoculated onto six-well plates having a confluent Vero cell monolayer with a cell control and the plates were maintained in an incubator at 37 C under 2.5% CO2. Each well of the plate was harvested at an interval of 6 h for 42 h using 300 ml of sterile phosphate buffered saline (PBS) (pH, 7.4) and freeze-thawed three times before employing rt-PCR. Total genomic DNA (gDNA) was extracted from all the harvests using AuPrep GEN DNA extraction kit (Life Technologies India (PVT) Ltd, New Delhi, India) as per manufacturer’s protocol and rt-PCR was carried out to determine the Ct (cycle threshold) values. To establish the standard curve, the Ct values were plotted against the log10 dilutions of virus titre and linear regression analysis was performed. The slopes of standard curves (SC) at different time interval harvests were compared and an SC of particular time interval harvest with maximum slope was selected as critical time for harvest for test batches. The Ct values for the test batches of camelpox vaccine were ascertained and the titre of each batch was determined by plotting Ct values on the standard curve. The critical time of harvest for buffalopox vaccine was carried out using the reference batch (A-01/060109) as described for camelpox except that each well of the plate was harvested at 6 h interval for 48 h using 500 ml of sterile PBS (pH, 7.4) and freezethawed three times before use. The TaqMan PCR was carried out with the DNA isolated using a commercial kit (AuPrep GEN DNA extraction kit, Life Technologies India (PVT) Ltd, India) from each hr interval. The standard curve and titre of each buffalopox vaccine batches were established in way similar to camelpox vaccine. 2.4. Median tissue culture infective dose assay (TCID50) The potencies of live camelpox and buffalopox vaccine batches by conventional end point dilution method (TCID50) were determined using a standard protocol [20]. The rt-PCR results obtained for each vaccine batch of camelpox and buffalopox were compared with that of respective virus titres estimated by conventional end point dilution (TCID50) and correlation was drawn between these methods by student’s t-test. 3. Results and discussion The potency of live vaccines is determined in vivo either in the target host/laboratory animals or in vitro titration of the virus in suitable cell culture system/embryonated eggs [21]. The gold standard assays are besotted with certain drawbacks as mentioned earlier [11e13]. Real time PCRs are additional tools to overcome the drawbacks of conventional assays. Hence, these strategies have successfully been employed earlier for estimation of potency in a number of human and animal vaccines [15e17,22]. Further, template nucleic acid concentration (both infectious and noninfectious virus) of an unknown sample can be quantified using the standard nucleic acid of known concentration. To do so, cells are initially to be infected with the vaccine virus and presumed that, if the live virus is first propagated in cell culture, the amount of gDNA produced corresponds to amount of viable particles in the vaccine preparation. The rt-PCR methods can estimate the titre of the vaccine much before the appearance of CPE and this is particularly useful in delayed CPE producing or non-cytopathic viruses. In case of CMLV, the CPE starts appearing 24 h post infection (hpi) and completes before 72 hpi. Similarly, in case of BPXV the CPE appears at 24 h post infection (hpi) progressed to 80% by 36e48 h and completes by 72 hpi. In this study, rt-PCR assays were applied to determine the exact titre of CMLV and BPXV in a given vaccine batches as early as 36 and 30 hpi, respectively. The rt-PCR methods reduced the time to read the titre of vaccine and subjective errors associated with cell
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Ct values
A
Estimation of critical time for harvest 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18
6 hrs p.i. (Slope -1.225) 12 hrs p.i. (Slope -1.553) 18 hrs p.i. (Slope -1.711) 24 hrs p.i. (Slope -1.658) 30 hrs p.i. (Slope -1.822) 36 hrs p.i. (Slope -3.324) 42 hrs p.i. (Slope -2.164)
y = -3.324x + 25.05 R² = 0.9925
0
1
2
3
4
5
6
7
Virus titer (Log 10)
B
Estimation of critical time for harvest 40 6 hr harvest
35
12 hr harvest
Ct values
30
18 hr harvest 24 hr harvest
25
30 hr harvest (Slope - 3.321)
20
36 hr harvest
y = -3.321x + 35.59 R2 = 0.9981
15 10 5 0 0
1
2
3
4
5
6
7
Virus titer (Log10) Fig. 1. Standard curves established for different time interval harvest of camelpox (A) and buffalopox (B) vaccines using rt-PCR assays.
culture based assays. The vaccine batches were selected in such a way that the titres/Ct values of the test batches might fall within the wide range of these reference vaccines dilutions. This would enable minimum number of dilutions of test vaccine batches. Further, the critical time point harvests for reference vaccines were determined and they correspond to the minimum time required for the appearance of sufficient viral particles in all the dilutions of the virus infected cells. The slope of the curve was maximum at 36 (R ¼ 3.324) and 30 hpi (3.321) harvest than that of other intervals, respectively for camelpox and buffalopox vaccines (Fig. 1A and B) and they were considered as the critical
time points to harvest the reference as well as test vaccine batches so as to get maximum respective live viral particles. The slopes obtained by 30 and 36 hpi were almost comparable with minor differences for buffalopox. A total of eight (n ¼ 8) batches of live attenuated camelpox vaccine and seven (n ¼ 7) batches of buffalopox vaccines including their respective reference batches were selected to estimate their titres by respective rt-PCRs. The titres obtained were satisfactory and within the acceptable range of accuracy (Tables 1A and B) as reported earlier for goatpox vaccine [16] which showed a high degree of correlation (0.96) with no significant difference between
Table 1A Estimation of virus infectivity titres of camelpox vaccines by SYBR QPCR and end point dilution (TCID50) assays. Vaccine Batches A-01/091209 B-01/091209 C-01/091209 D-02/161209 E-02/161209 F-02/161209 G-03/301209 H-04/060110
Ct values of QPCR against different dilutions of camelpox vaccine virus 1
Neat
10
23.03 24.25 23.40 20.11 19.25 20.49 19.73 19.42
23.78 26.44 24.90 21.33 21.41 21.61 21.14 21.49
10
2
25.25 28.93 26.61 22.07 22.47 23.41 23.95 22.42
10
3
26.63 29.46 29.19 26.00 28.79 26.20 26.12 25.54
Virus titre 10
4
29.28 33.16 30.14 29.53 30.39 29.48 28.60 28.86
QPCR 6.77 5.87 6.35 7.30 7.10 7.17 7.26 7.38
TCID50 0.87 0.58 0. 73 0.55 0.39 0.53 0.49 0.52
6.53 6.23 6.53 7.00 6.67 6.77 6.50 6.50
0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09
M. Prabhu et al. / Biologicals 40 (2012) 92e95
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Table 1B Estimation of virus infectivity titres of buffalopox vaccines by TaqMan PCR and end point dilution (TCID50) assays. Vaccine Batches
A-01/060109 B-01/091209 C-02/060110 D-03/311209 E-04/161209 F-05/311209 G-06/091209
Ct values for different dilutions of buffalopox vaccine virus
Virus titre
Neat
101
102
103
104
QPCR
13.45 14.56 14.34 15.33 13.93 13.35 13.51
17.45 18.79 18.18 18.70 17.03 17.39 18.09
22.13 21.82 21.76 22.55 20.93 22.03 22.14
24.50 24.36 26.01 26.67 25.84 24.36 25.20
28.23 27.18 28.73 29.81 28.49 28.10 29.07
6.74 6.68 6.41 6.32 6.56 6.72 6.57
these assays. A similar degree of correlation was obtained in this study between rt-PCRs and end point dilution assay with no significant difference (p 0.05) for live attenuated camelpox and buffalopox vaccines. These assays are advantageous over end-point dilution methods. QPCR based estimation overcomes the variation between laboratories due to discrepancy in conventional assay procedures as the potency of an unknown vaccine batch was estimated relative to a reference batch [11]. However, the virus in the test vaccine batches should have the same replication rate as that of the reference batch for accuracy. As the difference in the sample/ vaccine composition might affect the replication rate of the virus [15], vaccine batches of similar composition of both camelpox and buffalopox (reference and test) were used in this study. In conclusion, the potency of live attenuated camelpox and buffalopox vaccines were evaluated, respectively using C18L gene based SYBR Green and TaqMan rt-PCRs. The critical time point for harvest of camelpox and buffalopox, respectively were 36 and 30 hpi to estimate the maximal respective virus load. The titres obtained in rtPCRs and the conventional end point dilution methods (TCID50) were well comparable with each other without significant differences. Therefore, rt-PCRs are additional tools for quantification of live camelpox and buffalopox vaccines. Nevertheless, these novel rt-PCRs based potency estimation warrant validation using large number of vaccine batches to attain better repeatability and reproducibility prior contemplating for widespread application and effective vaccination leading to prevention and control of the disease. Acknowledgment The authors thank the Director, Indian Veterinary Research Institute (IVRI) for providing necessary facilities to carry out this work, the staff of Pox Virus Laboratory for providing technical assistance. Further, first two authors acknowledge IVRI for financial support in terms of fellowships to carry out their degree programs.
[5]
[6]
[7]
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[17]
[18]
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