1

CBER Assessment of Porcine Circovirus in Vaccines

Philip R. Krause, M.D.

FDA/CBER/OVRR

7 May 2010

2

Introduction• Historical Perspective

– SV40 and polio vaccines– EAV in egg-derived vaccines– TSE issues and vaccines– Use of novel cell substrates for vaccines

• CBER Laboratory Assessment of Porcine Circovirus in vaccines

3

SV40 and polio vaccines• In 1960, Sweet & Hilleman discovered SV40• Formalin inactivation of IPV did not completely inactivate

SV40• SV40-free vaccine was produced as quickly as possible

– Licensure of OPV was delayed until SV40 free preparation could be made

– No recall of IPV, but no release of additional IPV lots until SV40 was removed

• Consequences:– Some IPV recipients seroconverted to SV40– OPV recipients (including those in clinical trials who were

exposed to virus) did not seroconvert to SV40– Concerns re: SV40 and human cancers– overall, evidence does

not support a role

4

EAV in egg-produced vaccines• A more sensitive PCR test (PERT) for the enzyme,

reverse transcriptase (RT), present in retroviruses was developed.

• In 1996, this test showed that previously undetectable quantities of RT were present in some avian cell-produced vaccines

• Additional studies showed that EAV (endogenous avian retrovirus) is a defective particle and does not induce productive infections in culture

• Long safety record of hens’ egg produced vaccines, together with absence of evidence of potential harm to humans were also important considerations

5

TSE issues and vaccines• In early 2000s, concern was raised that bovine derived

materials sourced from countries with BSE or at risk of BSE could pose a theoretical risk to vaccinees

• Sourcing of bovine derived materials used for vaccine production was changed to countries not categorized as having BSE or at risk of BSE

• For products with potential exposures to bovine derived materials from countries with or at risk of BSE, risk assessments were performed, based on maximum possible exposures, dilution factors, potential infectious doses, etc.

• Many virus seeds were re-derived to remove all doubt

6

Cell substrates• Numerous discussions & advisory

committee presentations on introduction of new cell substrates for vaccine production

• Discussions addressed issues posed by tumorigenic phenotype, theoretical infectivity and oncogenicity risks of cell DNA or other cell components

• Issues addressed by risk assessment

7

Previous FDA evaluation of PCV• CREON Pancreatic enzyme preparation

– Advisory committee discussion 2008– Discussion was broad-ranging, covering all

potential porcine viruses– though PPV and PCV were recognized as the viruses most likely to survive the production process

– Potential for exposure to porcine agents is mentioned in the package insert

8

CBER’s Scientific Approach to Evaluating PCV in Vaccines

• Major question: Is PCV in rotavirus vaccines likely to represent infectious virus?– Molecular studies

• Can PCV nucleic acid be detected in vaccines?• How much PCV nucleic acid is in vaccines?• Are PCV nucleic acids in vaccines particle associated?• Are PCV nucleic acids present as full-length genomes

– In vaccine– In particles purified from vaccines

• How does sequence of PCV1 from vaccines compare with sequence of known infectious PCV1?

– Cell culture studies• Do vaccine-inoculated previously uninfected cells show evidence of

virus infection?

9

PCR assays used to detect PCV1

• Four conventional PCR assays were used in initial studies

• We used sequencing & Southern hybridization to confirm results

10

Rotarix contains PCV1 DNA• Rotarix was consistently

positive for PCV1 DNA• Rotateq was negative for

PCV1 DNA (to limits of this assay’s sensitivity)

• All assay controls (negatives, extraction controls, positives) yielded expected results

• These results are consistent with findings reported by GSK, Dr. Delwart

1

SERUM

1 2 3 4 1 2 3 1 2

POSITIVE NEGATIVE

PrimersExtraction # 1 2 3

1223311223

ROTATEQROTATEQ

3

RO

TA

RIX

RO

TA

TE

Q

RO

TA

RIX

SP

IKE

11

Long PCR & Sequencing• Long PCR

– We detected and cloned a 1638 nt PCR product (PCV1 genome size: 1759 nt) directly from final Rotarix product

• Sequencing of PCV1 from Rotarix– Entire genome of virus from Rotarix final container available

(combination of CBER and data from Dr. Delwart)– Some variability in sequence, but no nucleotide changes not already

reported in Genbank for wild-type virus– No changes in ORFs

0 100

200

300

40050

0

600

700

800900

1000

11

00

1200

1300

1400

1500

1600

PCV1

1638 nt

12

Quantitative PCR• Used three PCV1 primer pairs and two PCV2 primer

pairs

• Studied DNA extracted from vaccine samples– Total DNA extracted from vaccines (“Direct prep”)– Particle-associated DNA was assessed by “Capsid prep”, which

included nuclease treatment and ultracentrifugation• Quantified PCV DNA by TaqMan qPCR

0 100

200

300

40050

0

600

700

800900

1000

11

00

1200

1300

1400

1500

1600

PCV1

70 nt

0 100

200

300

40050

0

600

700

800900

1000

11

00

1200

1300

1 400

1500

1600

PCV1

529 nt

0 100

200

300

400

500

600

700

800900

1000

11

00

1200

1300

1400

1500

1600

PCV1

1638 nt

0 10 0

200

300

400500

600

700

800900

1000

1100

1200

1300

140 0

1500

1600

PCV2

87 nt

0 10 0

200

300

400500

600

700

800900

1000

1100

1200

1300

1400

1500

1600

PCV2

357 nt

13

PCV quantitation: Rotarix• Both Rotarix samples were

positive for PCV1• Both Rotarix samples were

negative for PCV2• Not all virus DNA is full-length;

longer PCRs yielded lower quantities of PCV1 DNA

• At each PCR fragment size, most PCV1 DNA was particle-associated

• Each vaccine dose contained around 100,000 copies of near full-length, particle-associated PCV1 DNA

GSK 2 GSK 3

PC

V c

opie

s/m

L

70 529

163

8

35787

≤100

106

107

105

104

103

102

101

70 529

163

8

35787

LOQ

Virus: PCV1 PCV2 Prep: Capsid Direct

14

Further studies on Rotateq

• Using a more sensitive Taqman qPCR, PCV1 and PCV2 DNA fragments were detected in Rotateq final container

• Particle-associated PCV2 DNA fragments were detected in Rotateq bulks

• Further studies are ongoing

15

GSK IPV-containing vaccines

• CBER’s final container PCRs for PCV1 and PCV2 are inconclusive due to PCR inhibition in spiking experiments – Follow up experiments pending

• Further studies will be performed when viral harvest samples are available– PCR– Infectivity

16

Infectivity studies: Considerations

• Inactivation of rotavirus could also inactivate PCV

• Theoretical concern that bovine serum might contain neutralizing antibodies to PCV and thus reduce sensitivity of studies

• Need to assure that no exogenous PCV is added to culture

• Different cell types have different susceptibility to virus

• If cells become confluent, virus will not grow even in susceptible cell types

• Virus does not cause cytopathic effect

S phase

17

Infectivity studies: Considerations

• Inactivation of rotavirus could also inactivate PCV

• Theoretical concern that bovine serum might contain neutralizing antibodies to PCV and thus reduce sensitivity of studies

• Need to assure that no exogenous PCV is added to culture

• Different cell types have different susceptibility to virus

• If cells become confluent, virus will not grow even in susceptible cell types

• Virus does not cause cytopathic effect

CBER approach• Heat inactivation under conditions where

PCV is resistant• Use Ig-depleted bovine serum for cultures

• Use recombinant trypsin and pre-tested reagents

• Use swine testis (ST) cells• Inoculate sub-confluent cells & maintain

cells in growth phase by sub-passage @ day 3

• Evaluate cultures by qPCR to detect virus DNA

S phase

18

Infectivity study: Rotarix• Mock-infected Swine testis (ST) cells

yielded negative results• Cells inoculated with Rotarix (GSK1

and GSK 2) showed increasing PCV1 DNA quantities, measured by 529 nt qPCR

• Particle-associated PCV1 DNA was also produced

• (Not shown) Particle-associated PCV1 DNA was also detected in cell culture supernatants at day 3 and day 6

• (Not shown) Inoculation of cell lysates from day 6 of vaccine cultures onto fresh ST cells revealed 2-4 log increases in PCV1 DNA quantity after 3 additional days in culture

• We have not formally evaluated the sensitivity of this assay

≤101

107

108

106

105

104

103

102

0 1 2 3 4 5 6 7

PC

V1

copi

es

Time (Days)

Sample: GSK 2 GSK 3 MockPrep: Capsid prep Direct extraction

19

CBER and Manufacturer’s resultsVaccine CBER Manufacturer

Rotarix (GSK)

PCV1 DNA in product PCV1 DNA in product, bulks, seeds, cells

Particle-associated near full length PCV1 DNA in product

Infectious PCV1 in cell culture Infectious PCV1 in cell culture

IPV containing vaccines (GSK)

Pending PCV1 DNA in harvest, seeds, cells, but not in bulks or final container

Rotateq (Merck)

Particle-associated PCV2 DNA in harvest; PCV1 and PCV2 DNA in final container

PCV1 and PCV2 DNA in harvest, PCV DNA in final container

20

Implications• Tests of increasing sensitivity can detect very

low quantities of virus DNA and of virus• We do not know if it is possible to completely

eliminate PCV from porcine trypsin• Porcine and other animal-derived reagents

used in vaccine manufacture could potentially contain other viruses, known and as-yet unknown

• It is impossible to absolutely assure that vaccines do not contain adventitious viruses

21

Conclusions• PCV1 DNA, particles, infectious virus are

present in Rotarix• PCV1 and PCV2 DNA is present at low levels

in Rotateq– Further evaluation is ongoing

• PCV1 DNA is present in poliovirus harvests– Not detected by manufacturer in final bulks or final

container– Quantities are low and purification/inactivation

procedures are in place

22

• Laboratory work– Christine Uhlenhaut– Shasta McClenahan– Shuang Tang

• Reagents– Gordon Allan– Steve Krakowka– Andrew Cheung– XJ Meng

• FDA Scientific Advice– Jerry Weir– Robin Levis– Keith Peden– Arifa Khan– Konstantin Chumakov– Erik Henchal

Acknowledgements

23

DISCUSSION POINTS

1. Based on the information presented today on the detection of porcine circovirus (PCV) or PCV DNA in US licensed rotavirus vaccines:

a) Please discuss the available scientific evidence and identify factors to be considered in assessing the potential risk of the use of US licensed rotavirus vaccines.

b) Please discuss any additional scientific studies and information that you would recommend FDA consider in its deliberations.

24

DISCUSSION POINTS

2. Given the available data about porcine circoviruses, including the lack of known infectivity and pathogenic effects in humans, and that porcine circoviruses or PCV DNA may be present in both US licensed rotavirus vaccines, please discuss factors to be considered in determining whether, or in what circumstances, the benefits of using rotavirus vaccines outweigh the theoretical risk from PCV.

25

DISCUSSION POINTS

3. Please discuss the application of emerging technologies, and the implications for their use in the detection of known and unknown adventitious agents in vaccines currently licensed, as well as those under development.

26

Backup slides

27

PCV quantitation: Rotateq• Weak signals were

observed for both PCV1 and PCV2 DNA fragments in Rotateq final container

• This assay is more sensitive than that performed initially

• Virus DNA was not demonstrated to be particle-associated

• Near full-length PCV1 or 357 nt PCV2 sequences were not detected

70 529

163

8

35787

Merck 1 Merck 2 Merck 3

70 529

163

8

35787 70 529

163

8

35787

PC

V c

opie

s/m

L

≤100

106

107

105

104

103

102

101

LOQ

Virus: PCV1 PCV2 Prep: Capsid Direct

28

PCV quantitation: Rotateq bulks• Bulks are more concentrated

than final container vaccines, so PCV concentration is expected to be higher

• In spiking experiments, inhibition was detected in the direct prep, but not in the capsid preps.

• Rotateq bulks were negative for particle-associated PCV1

• Using the shortest (87 nt) PCR, some PCV2 DNA was particle-associated

Bulk 1Bulk 2Bulk 3Bulk 4Bulk 5

PC

V c

opie

s/m

L

529

35787

≤100

106

107

105

104

103

102

101

529

35787

529

35787

529

35787

529

35787

LOQ

s

Virus: PCV1 PCV2 Prep: Capsid Direct