Technical Opinion no. 1591/2008

Proceedings:  01200.000528/2008-01
Applicant:  Intervet do Brasil Veterinária Ltda.
CNPJ:   07.954.091/0001-43
Address: Avenida Alfredo Egídio de Souza Aranha, 100, 6º Andar, Bloco B, 04726-170 São Paulo, SP. Telefones: (11) 2165 6632, (11) 2165 6673, Fax: (11) 2165 6611.
Matter: Requests technical opinion on biosafety of a product derived from genetically modified organisms – vaccine  against Porcine Circovirosis – Porcilis Cirvumvent PCV - for commercial use.
Previous extract: 1282/2008. Published in the Federal Official Gazette no. 42,  of March 07, 2008.
Meeting: 116th Regular Meeting held on  September 18, 2008.
Decision: GRANTED.

SYNOPSIS:
CTNBio, following examination of the request for Technical Opinion on biosafety of a product derived from genetically modified organism – vaccine  against Porcine Circovirosis – Porcilis Circumvent PCV – for importing and marketing the product, was for the APPROVAL of the  request  on the terms of this Technical Opinion. Mr. Leonardo Costa,  Chairman of  the Internal Biosafety Commission of the  company Internet do Brasil Veterinária Ltda., holder of CQB 248/08, requests  CTNBio  the commercial release of product  Porcilis Circumvent PCV, targeted to active immunization of healthy swine aged three weeks, to  be used in prevention of  viremia or elimination of PCV2 virus in feces. The product shall be imported ready and finished, and  the phases of production, purification and packaging shall be conducted  in the United States (Delaware, USA)  by the company Intervet Inc. Its use is targeted  to application in four week old swine. An analysis of the documents related to commercial release of the circovirus vaccine shows that the proceedings  for importing and marketing the finished product in the Brazilian territory  is well documented. The  result  of the voting in the plenary CTNBio meeting  was  twenty (20) votes favorable  to approval of the request for commercial release of the inactivated  vaccine against Porcine Circovirosis – Porcilis Circumvent PCV, on the terms of this Technical Opinion. In the context of the jurisdiction granted by Law no. 11,105,  as regulated by Decree-Law no. 5,591/2005, the Commission held that the experimental protocols and remaining biosafety measures proposed comply with CTNBio rules and applicable legislation, which intend  to secure biosafety to the  environment, agriculture, and human and animal health.
1. General Information:
Post-weaning Multi-systemic Wasting Syndrome (PMWS) associated  to  type 2 PCV infection (PCV2) is  a severe emerging disease scattered all over the world. This disease affects mainly piglets from 5 to  18 weeks. Clinic signs  include loss of weight, emaciation, tachypnea, dyspnea, jaundice and/or mucosa paleness, lymphadenopathy (swollen lymph nodes) and diarrhea.
Economic losses caused by circovirosis may be significant and are mainly due to progressive thinning of infected animals, reduction in weight gain and reduced food  conversion. Mixed infections (co-infection) of PCV 2 jointly with other  microorganisms causing respiratory, enteric and reproductive infections are common. The  weakened immunologic  system of  pigs  infected  by PCV 2, which may cause reduced  immunity, is another area currently under studies.
Compared  with other viral infections  affecting swine farming, both the PCV 2 identification and the probable clinical signs of porcine circovirosis  syndrome have been described in the recent past. However, serologic  and mainly etiologic studies  show that  PCV 2 infection is  largely disseminated in world swine herds, especially in countries where production features high technical level.
In Brazil, the first  PCV 2 identification report occurred in 2000. Later, new diagnostic descriptions were made. Currently there are at least five research teams dedicated to  the study of  PCV 2 in this country. Special  emphasis shall be given to the pioneering  team, still  active in the  field, of researchers linked to  the Swine and Bird National Research Center (EMBRAPA, Concórdia, SC). Besides, research teams  in the states of Rio Grande do Sul, São Paulo, Rio de Janeiro and  Minas Gerais have spent  time studying porcine circovirosis. Preliminary results  from these studies have  shown that porcine circovirosis is highly spread in  Brazilian herds. Studies in Brazil have shown a reproduction of a disease  associated  to PCV 2 when administered jointly with Porcine Parvovirus, as published by Fernandes  et.al. (Experimental co-infection of porcine circovirus type 2 (PCV 2) isolated  in Brazil and porcine parvovirus (PPV) in SPF pigs (Arquivo Brasileiro de Medicina Veterinária e Zootecnia, Belo Horizonte, MG, v.58, no.1, p.1-8,2006.
 Undoubtedly, porcine circovirosis  is currently a major concern  in animal health, afflicting  hog farmers  and professionals in the area. The reduction caused  by the disease in productivity entails significant economic losses  in  this important sector of Brazilian hog farming and reduces the competitiveness of Brazilian pork in the international market due to  increased production costs.
The method of manufacturing and  quality controls are described in their different phases: growth of cell culture with virus, incubation, collection  of the antigen, inactivation of viral cultures with  BEI and neutralization of BEI with sodium thiosulphite, addition of thimerosal, standardization of  the antigen and storage. Finally, preparation of the finished product is made  by adding adjuvant, preservative and excipient, followed by labeling, packaging and shipping.
GMO description:
The PCV 2 gene used is the ORF 2 (viral capsid), a gene preserved among samples of PCV 2 and a target for neutralizing antibodies. The gen originated from a virus isolated in pig lung tissues of  a clinical  case of PMWS. The ORF 2  gene was  inserted in the pFASTBac Hta plasmid (Invitrogen) and subcloned  in pAcAS3. The nuclear polyhedrosis virus of insect Autographa californica baculovirus was used as structure biologic agent to produce the pre-mother-seed. Plasmid pACAS3 was  cloned  from the DNA of a baculovirus and used to transfect Sf9 cells, and recombinant DNA was extracted and purified, and a precipitation with ethanol took place in this phase (Vox Sang 91:292-300, 2006).
This DNA was used  to transfect  certified Sf21 cells. In August 2004, the mother-seed virus was approved in the USA, from which the working viruses are prepared in no more than  five steps. Sf21 cells were granted approval in 2005, using a bovine fetal serum irradiated with gamma ray with certified adequateness (-40ºC with 30 to 40 kGy (Vox Sang 84:36-44,2003), to start cultivating the mother cell. Regarding production, cells were cultivated in a medium without JRH Excell420 serum. He procedures were employed with a view to minimize the likelihood of contamination with  spongiform encephalopathies transmissible in the mother-seed sample.
(1) Biosafety aspects
This is a vaccine produced from an inactivated  viral subunit.  Sterility and mycoplasma tests, and test of inactivation control with BEI (Clin. Microbiol 3:209-10, 1976) were described in addition to specific measures related to preventing the transmission of animal spongiform encephalopaties and  determination of antigen content.  Tests were performed in 1,154 animals aged three weeks. Besides, the product has already been used  in million of pigs in Canada and USA in 2007, and the ORF2 carrier baculovirus was referred in the literature as efficient (Vet. Res. Commun. 4: 487-96, 2007; VBaccine 26:3443-51, 2008;Vaccine 26:2488-99, 2008).
Therefore, being  an inactivated  GMO derivative, multiplied in a serumless medium, there is no risk of introducing an exotic microorganism into the country. There is no risk of  using  this recombinant virus as immunogenic to animal health, either swine or other species, to  public health, through infection of  human beings and  to  the environment.
Vaccine safety tests were performed in high doses  of the vaccine  in  newly born susceptible pigs. Application of the vaccine failed to reveal any toxic  reactions and, according to the studies submitted, it  was deemed as safe  for pigs over three weeks of age.
Environmental safety:
As the vaccine is an inactivated (dead) organism, the risk of leakage to the environment is very low, and may be held acceptable, requiring little care regarding this aspect. The likelihood of an inactivated vaccine to establish itself in the environment is even remoter, requiring the concurrent materialization of rare events, thus lowering the probability of occurrence.
2. Final CTNBio opinion:
CTNBio is favorable to the commercial release  of the product named vaccine against Porcine Circovirosis – Porcilis Circumvent PCV, considering that:
(1) There are no reports on problems associated to the capsid protein of PCV 2 (isolated from insect and Escherichia coli recombinants);
(2) the vaccine with Porcilis Circumvent PCV is produced in the facilities of Intervet Inc., in the United States of America;
(3) The nuclear polyhedrosis virus of  Baculovirus  Autographa californica of insects used  to produce the vaccine is inactivated  at the completion of the procedure, has been innocuous to pigs, mice and guinea pigs  both in laboratory and field conditions;
(4) There is no expected condition for  the inactivated virus to establish in the environment;
(5) There are no reports  of human diseases  associated to this porcine Circovirus, though antibodies against  such virus have been found in serum;
(6) The risks to public health, animal health and the environment  are low.
Twenty (20) CTNBio members voted  favorably to the commercial release of the product.

Walter Colli
President of CTNBio