G Model
ARTICLE IN PRESS
JVAC-16434; No. of Pages 8
Vaccine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Review
Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction Gourapura J. Renukaradhya a,∗ , Xiang-Jin Meng b , Jay G. Calvert c , Michael Roof d , Kelly M. Lager e,∗∗ a Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, United States b Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States c Zoetis, Kalamazoo, MI, United States d Boehringer Ingelheim Vetmedica, Inc., Ames, IA, United States e Virology Swine Research Unit, National Animal Disease Center, U.S. Department of Agriculture, Ames, IA, United States
a r t i c l e
i n f o
Article history: Received 10 February 2015 Received in revised form 18 April 2015 Accepted 30 April 2015 Available online xxx Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Inactivated/killed PRRSV Immunity Cross-protection Delivery system
a b s t r a c t Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in major pork producing countries causing reproductive failure in sows and respiratory disease in young pigs. Prevention of PRRS in sows is critical to avoid passing
∗ Corresponding author at: The Ohio State University, FAHRP, OARDC, 1680 Madison Avenue, Wooster 44691, United States. Tel.: +1 330 263 3748; fax: +1 330 263 3677. ∗ ∗ Corresponding author at: Virology Swine Research Unit, National Animal Disease Center, U.S. Department of Agriculture, Ames, IA, United States. Tel.: +1 515 337 7371; fax: +1 515 337 7149. E-mail addresses: [email protected] (G.J. Renukaradhya), [email protected] (K.M. Lager).
PRRS virus (PRRSV) to susceptible pigs through vertical and horizontal transmission resulting in endemic disease and chronic economic loss. PRRSV is an Arterivirus that was discovered almost simultaneously in 1991 on the European and North American continent which lead to the classification of type 1 and 2 PRRSV, respectively. Since the discovery of PRRSV there has been a tremendous growth in the PRRS literature. Genetic studies have revealed this virus to have one of the highest known mutation rates for an RNA virus which promotes extensive antigenic and genetic variation (reported evolutionary rate of 4.7–9.8 × 10−2 /site/year [1]). Currently, PRRSV consists of at least 9 distinct genetic lineages within type 2 PRRSV, and 3 subtypes within type 1 PRRSV [2,3]. This constant change in the virus is a driving force in the cyclical PRRS epidemics observed in the field, and a major obstacle for developing a broadly-protective vaccine against genetically diverse field
http://dx.doi.org/10.1016/j.vaccine.2015.04.102 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Renukaradhya GJ, et al. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.04.102
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JVAC-16434; No. of Pages 8
2
Route/no. of doses
Pigs age
PRRSV chal. and route
Adjuvant
Viremia
Promise (type 2) Promise (type 2) Promise (type 1) Progressis (type 1) Progressis (type 1) Progressis (type 1)
IM/2 IM/6 IM/2 IM/2 IM/2 IM/5
8 wks Sows 10 wks ∼6 wks 6–8 wks Gilts Sows
− − − + IN homologous − −
+ + + + + +
− − ↔ – − −
Lelystad–1 × 108 TCID50 Suvaxyn (type 1)
BPL-killed IM/2 IM/2
6 wks Gilts
+ IN homologous + IN homologous
w/o emulsion +
Progressis (type 1) Lelystad -0.5 × 106
IM/2 Killed IN/2
20 wks 4 wks
+ IN homologous −
Killed vac (China) Lelystad–1 × 108 Lelystad–1 × 108 Lelystad–1 × 108 07V063–1 × 108 VR2332 2.5 × 106
IM/2 BEI-killed IM/2 BEI-killed IM/2 BEI killed IM/2 BEI-killed IM/2 UV-killed nanoparticle -IN/1 UV-killed nanoparticle -IN/1 UV-killed nanoparticle -IN/2
4 wks 6 wks 6 wks 6 wks Gilts 6 wks
+ IN homologous + IN homologous + IN Homologous + IN homologous + IN homologous + IN heterologous (MN184)* + IN homologous
+ CpG–10,100, 1000 g + IFA Alhydrogel Suvaxyn o/w Suvaxyn o/w None
1 log10 ↓ 2 log10 ↓ on day 9 ↔ −
6 wks
+ IN heterologous (MN184)*
M.tb WCL
BEI-killed IM/2
3 wks
+ IM homologous
Montanide
↔ 2 log10 ↓ 2 log10 ↓ 2 log10 ↓ Early clearance 2 log10 ↓ Early clearance 2 log10 ↓ early clearance 4 log10 ↓ RNA & Neg - virus isolation 2 log10 ↓
VR2332 2.5 × 106 VR2332 2.5 × 10
6
FL12/GP5 DM virus 1 × 108
6 wks
None
Viral load in lungs
VN titer (log2 )
IFN␥ ELISPOT
Cell proliferation
Citations
300 spots 2006 [14]
− ++ – – −
2003 [83] 2004 [19] 2004 [16] 2005 [18] 2005 [18]
− −
3 >4 – − Improved reproductive performance >3 >5
− 2007 [17]
2006 [23]
↔ −
>5 −
− +++
2007 [15] 2007 [84]
− − − − − −
>4 >3 >2 >4 >3 >3
+ − − − − −
2007 [85] 2009 [10] 2009 [10] 2009 [10] 2012 [24] 2012 [20]
−
>3
−
2013 [21]
6 log10 ↓ RNA & Neg - virus isolation −
>4 in serum & ∼5 in lungs
− Preweaning mortality↓ >100 spots IFN␥ in culture sup ↑ − − − − − IFN␥ in culture sup ↑ IFN␥ in culture sup ↑ >1000 spots & IFN␥+ cells↑
++++
2014 [22,40]
4
−
−
2014 [86]
− – – − −
Notes: IN—intranasal; IM—intramuscular; ID—intradermal; wks—weeks; virus titers in TCID50 —tissue culture infective dose 50: Homologous—genetically identical PRRSV strain; Heterologous – genetically partially different PRRSV strain; ‘−’—negative/none/nil; ‘+’—positive; + weak; ++ mild; +++ high; ++++ very high. Commercial vaccines: Promise (Bayer); Progressis (Merial); Suvaxyn (FortDodge). * PRRSV strains VR2332 and MN184 are 10–16% genetically different in ORF 2 to 5 sequences [87].
ARTICLE IN PRESS
Vaccine and dose
G.J. Renukaradhya et al. / Vaccine xxx (2015) xxx–xxx
Please cite this article in press as: Renukaradhya GJ, et al. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.04.102
Table 1 Summary of inactivated PRRSV vaccine studies (2003–2014).
G Model JVAC-16434; No. of Pages 8
ARTICLE IN PRESS 2014 [42] − − − − MPLA 10 g
−
Quil A 500 g
Transdermal/2 GP45M-DC-SIGN
8 wks
BEI, IM/2
3 wks
+ IN homologous + IM homologous − DNA-Eu GP3-GP5 500 g
FL12/GP5DM
4 wks
GP3, GP4 and GP5 each 100 g IM/2 IM/2 Plant derived PRRSV proteins
4 wks IM/2 PRV-GP5M 1 × 106 TCID50
4 wks IM/4 PRRSV ORF5 & ORF7
4 wks 4 wks orally/2 IM/4 PRRSV N-CT-B PRRSV ORF5 & ORF7
3 wks IM&ID/3 GST-ORF5 300 g
3 wks
Suvaxyn o/w
None
IFN␥
CT-B IL-2
None
None
+ IT homologous + IT homologous − + IN homologous + IN homologous + IN homologous − IM&ID/3 DNA-ORF5 100 g
3 wks
PRRSV chal. and route Pigs age Route/no. of doses Vaccine and dose
Table 2 Summary of subunit PRRSV vaccine studies (1998–2014).
Protective immune responses induced by most inactivated viral vaccines are mediated by an enhanced virus neutralization (VN) antibody response [6]. This is the case for two of the four Arteriviruses, equine arteritis virus (EAV) [7] and lactate dehydrogenase-elevating virus (LDV) [8], where VN activity induced by KV has been shown to be protective. Although a number of commercial and experimental PRRSV KV have been evaluated, the induction of PRRSV protective immunity may not be satisfactory implying a potential difference in the pathophysiology of PRRSV compared to EAV and LDV. Little is known about protective immunity for the fourth Arterivirus, Simian hemorrhagic fever virus SHFV. Following experimental infection with PRRSV, swine clear the virus indicating a successful adaptive immune response. However, the time required to eliminate the virus can range from days to months. This variation appears dependent on the age of the animal at time of infection, and may depend on the strain of virus, dose of challenge, comorbidities, and overall level of physiologic stress. The clearance of PRRSV from the lungs and serum coincides with the appearance of VN antibodies in serum and BAL fluid [9,10] indicating there may be a role of VN in viral clearance. Passive transfer studies have demonstrated VN antibody were responsible for viral clearance from the lungs and reducing transplacental transmission of PRRSV [11–13] suggesting VN antibody can play a significant role in the protective immune response. Given the apparent role that PRRSV VN can play in clearing virus from a pig, and presumably preventing infection, it seems reasonable that PRRSV KV vaccines should be able to induce VN antibodies that are protective resulting in efficacious vaccines. The minimal PRRSV VN titer in recipient pigs that provides 100% protection against viremia, virus replication in the lungs,
Adjuvant
2. Commercial PRRSV KV
Notes: IN—intranasal; IM—intramuscular; ID—intradermal; wks—weeks; TCID50 —tissue culture infective dose 50: Homologous—genetically identical PRRSV strain; Heterologous—genetically partially different PRRSV strain; ‘−’—negative/none/nil; ‘+’—positive; + weak; ++ mild; +++ high; ++++ very high.
2014 [43] − − 4
2014 [41] +++ − >4
1.5 log10 ↓ RNA copies −
0.5 log10 ↓ RNA copies 1.0 log10 ↓ RNA copies −
2014 [89] − − − −
−
2007 [85] ++ − >6 − Early clearance
2004 [88] − − 2-fold↓ −
−
2004 [83] 2004 [88] − − − − − 2-fold↓ − −
− −
↔
ARTICLE IN PRESS
JVAC-16434; No. of Pages 8
Vaccine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Review
Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction Gourapura J. Renukaradhya a,∗ , Xiang-Jin Meng b , Jay G. Calvert c , Michael Roof d , Kelly M. Lager e,∗∗ a Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, United States b Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States c Zoetis, Kalamazoo, MI, United States d Boehringer Ingelheim Vetmedica, Inc., Ames, IA, United States e Virology Swine Research Unit, National Animal Disease Center, U.S. Department of Agriculture, Ames, IA, United States
a r t i c l e
i n f o
Article history: Received 10 February 2015 Received in revised form 18 April 2015 Accepted 30 April 2015 Available online xxx Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Inactivated/killed PRRSV Immunity Cross-protection Delivery system
a b s t r a c t Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in major pork producing countries causing reproductive failure in sows and respiratory disease in young pigs. Prevention of PRRS in sows is critical to avoid passing
∗ Corresponding author at: The Ohio State University, FAHRP, OARDC, 1680 Madison Avenue, Wooster 44691, United States. Tel.: +1 330 263 3748; fax: +1 330 263 3677. ∗ ∗ Corresponding author at: Virology Swine Research Unit, National Animal Disease Center, U.S. Department of Agriculture, Ames, IA, United States. Tel.: +1 515 337 7371; fax: +1 515 337 7149. E-mail addresses: [email protected] (G.J. Renukaradhya), [email protected] (K.M. Lager).
PRRS virus (PRRSV) to susceptible pigs through vertical and horizontal transmission resulting in endemic disease and chronic economic loss. PRRSV is an Arterivirus that was discovered almost simultaneously in 1991 on the European and North American continent which lead to the classification of type 1 and 2 PRRSV, respectively. Since the discovery of PRRSV there has been a tremendous growth in the PRRS literature. Genetic studies have revealed this virus to have one of the highest known mutation rates for an RNA virus which promotes extensive antigenic and genetic variation (reported evolutionary rate of 4.7–9.8 × 10−2 /site/year [1]). Currently, PRRSV consists of at least 9 distinct genetic lineages within type 2 PRRSV, and 3 subtypes within type 1 PRRSV [2,3]. This constant change in the virus is a driving force in the cyclical PRRS epidemics observed in the field, and a major obstacle for developing a broadly-protective vaccine against genetically diverse field
http://dx.doi.org/10.1016/j.vaccine.2015.04.102 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Renukaradhya GJ, et al. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.04.102
G Model
JVAC-16434; No. of Pages 8
2
Route/no. of doses
Pigs age
PRRSV chal. and route
Adjuvant
Viremia
Promise (type 2) Promise (type 2) Promise (type 1) Progressis (type 1) Progressis (type 1) Progressis (type 1)
IM/2 IM/6 IM/2 IM/2 IM/2 IM/5
8 wks Sows 10 wks ∼6 wks 6–8 wks Gilts Sows
− − − + IN homologous − −
+ + + + + +
− − ↔ – − −
Lelystad–1 × 108 TCID50 Suvaxyn (type 1)
BPL-killed IM/2 IM/2
6 wks Gilts
+ IN homologous + IN homologous
w/o emulsion +
Progressis (type 1) Lelystad -0.5 × 106
IM/2 Killed IN/2
20 wks 4 wks
+ IN homologous −
Killed vac (China) Lelystad–1 × 108 Lelystad–1 × 108 Lelystad–1 × 108 07V063–1 × 108 VR2332 2.5 × 106
IM/2 BEI-killed IM/2 BEI-killed IM/2 BEI killed IM/2 BEI-killed IM/2 UV-killed nanoparticle -IN/1 UV-killed nanoparticle -IN/1 UV-killed nanoparticle -IN/2
4 wks 6 wks 6 wks 6 wks Gilts 6 wks
+ IN homologous + IN homologous + IN Homologous + IN homologous + IN homologous + IN heterologous (MN184)* + IN homologous
+ CpG–10,100, 1000 g + IFA Alhydrogel Suvaxyn o/w Suvaxyn o/w None
1 log10 ↓ 2 log10 ↓ on day 9 ↔ −
6 wks
+ IN heterologous (MN184)*
M.tb WCL
BEI-killed IM/2
3 wks
+ IM homologous
Montanide
↔ 2 log10 ↓ 2 log10 ↓ 2 log10 ↓ Early clearance 2 log10 ↓ Early clearance 2 log10 ↓ early clearance 4 log10 ↓ RNA & Neg - virus isolation 2 log10 ↓
VR2332 2.5 × 106 VR2332 2.5 × 10
6
FL12/GP5 DM virus 1 × 108
6 wks
None
Viral load in lungs
VN titer (log2 )
IFN␥ ELISPOT
Cell proliferation
Citations
300 spots 2006 [14]
− ++ – – −
2003 [83] 2004 [19] 2004 [16] 2005 [18] 2005 [18]
− −
3 >4 – − Improved reproductive performance >3 >5
− 2007 [17]
2006 [23]
↔ −
>5 −
− +++
2007 [15] 2007 [84]
− − − − − −
>4 >3 >2 >4 >3 >3
+ − − − − −
2007 [85] 2009 [10] 2009 [10] 2009 [10] 2012 [24] 2012 [20]
−
>3
−
2013 [21]
6 log10 ↓ RNA & Neg - virus isolation −
>4 in serum & ∼5 in lungs
− Preweaning mortality↓ >100 spots IFN␥ in culture sup ↑ − − − − − IFN␥ in culture sup ↑ IFN␥ in culture sup ↑ >1000 spots & IFN␥+ cells↑
++++
2014 [22,40]
4
−
−
2014 [86]
− – – − −
Notes: IN—intranasal; IM—intramuscular; ID—intradermal; wks—weeks; virus titers in TCID50 —tissue culture infective dose 50: Homologous—genetically identical PRRSV strain; Heterologous – genetically partially different PRRSV strain; ‘−’—negative/none/nil; ‘+’—positive; + weak; ++ mild; +++ high; ++++ very high. Commercial vaccines: Promise (Bayer); Progressis (Merial); Suvaxyn (FortDodge). * PRRSV strains VR2332 and MN184 are 10–16% genetically different in ORF 2 to 5 sequences [87].
ARTICLE IN PRESS
Vaccine and dose
G.J. Renukaradhya et al. / Vaccine xxx (2015) xxx–xxx
Please cite this article in press as: Renukaradhya GJ, et al. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.04.102
Table 1 Summary of inactivated PRRSV vaccine studies (2003–2014).
G Model JVAC-16434; No. of Pages 8
ARTICLE IN PRESS 2014 [42] − − − − MPLA 10 g
−
Quil A 500 g
Transdermal/2 GP45M-DC-SIGN
8 wks
BEI, IM/2
3 wks
+ IN homologous + IM homologous − DNA-Eu GP3-GP5 500 g
FL12/GP5DM
4 wks
GP3, GP4 and GP5 each 100 g IM/2 IM/2 Plant derived PRRSV proteins
4 wks IM/2 PRV-GP5M 1 × 106 TCID50
4 wks IM/4 PRRSV ORF5 & ORF7
4 wks 4 wks orally/2 IM/4 PRRSV N-CT-B PRRSV ORF5 & ORF7
3 wks IM&ID/3 GST-ORF5 300 g
3 wks
Suvaxyn o/w
None
IFN␥
CT-B IL-2
None
None
+ IT homologous + IT homologous − + IN homologous + IN homologous + IN homologous − IM&ID/3 DNA-ORF5 100 g
3 wks
PRRSV chal. and route Pigs age Route/no. of doses Vaccine and dose
Table 2 Summary of subunit PRRSV vaccine studies (1998–2014).
Protective immune responses induced by most inactivated viral vaccines are mediated by an enhanced virus neutralization (VN) antibody response [6]. This is the case for two of the four Arteriviruses, equine arteritis virus (EAV) [7] and lactate dehydrogenase-elevating virus (LDV) [8], where VN activity induced by KV has been shown to be protective. Although a number of commercial and experimental PRRSV KV have been evaluated, the induction of PRRSV protective immunity may not be satisfactory implying a potential difference in the pathophysiology of PRRSV compared to EAV and LDV. Little is known about protective immunity for the fourth Arterivirus, Simian hemorrhagic fever virus SHFV. Following experimental infection with PRRSV, swine clear the virus indicating a successful adaptive immune response. However, the time required to eliminate the virus can range from days to months. This variation appears dependent on the age of the animal at time of infection, and may depend on the strain of virus, dose of challenge, comorbidities, and overall level of physiologic stress. The clearance of PRRSV from the lungs and serum coincides with the appearance of VN antibodies in serum and BAL fluid [9,10] indicating there may be a role of VN in viral clearance. Passive transfer studies have demonstrated VN antibody were responsible for viral clearance from the lungs and reducing transplacental transmission of PRRSV [11–13] suggesting VN antibody can play a significant role in the protective immune response. Given the apparent role that PRRSV VN can play in clearing virus from a pig, and presumably preventing infection, it seems reasonable that PRRSV KV vaccines should be able to induce VN antibodies that are protective resulting in efficacious vaccines. The minimal PRRSV VN titer in recipient pigs that provides 100% protection against viremia, virus replication in the lungs,
Adjuvant
2. Commercial PRRSV KV
Notes: IN—intranasal; IM—intramuscular; ID—intradermal; wks—weeks; TCID50 —tissue culture infective dose 50: Homologous—genetically identical PRRSV strain; Heterologous—genetically partially different PRRSV strain; ‘−’—negative/none/nil; ‘+’—positive; + weak; ++ mild; +++ high; ++++ very high.
2014 [43] − − 4
2014 [41] +++ − >4
1.5 log10 ↓ RNA copies −
0.5 log10 ↓ RNA copies 1.0 log10 ↓ RNA copies −
2014 [89] − − − −
−
2007 [85] ++ − >6 − Early clearance
2004 [88] − − 2-fold↓ −
−
2004 [83] 2004 [88] − − − − − 2-fold↓ − −
− −
↔
