U.S. patent application number 12/137938 was filed with the patent office on 2009-01-22 for pcv2 immunogenic compositions and methods of producing such compositions.
This patent application is currently assigned to BOEHRINGER INGELHEIM VETMEDICA, INC.. Invention is credited to Marc Eichmeyer, Greg Nitzel, Merrill Schaeffer.
Application Number | 20090022750 12/137938 |
Document ID | / |
Family ID | 36615566 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090022750 |
Kind Code |
A1 |
Eichmeyer; Marc ; et
al. |
January 22, 2009 |
PCV2 IMMUNOGENIC COMPOSITIONS AND METHODS OF PRODUCING SUCH
COMPOSITIONS
Abstract
An improved method for recovering the protein expressed by open
reading frame 2 from porcine circovirus type 2 is provided. The
method generally involves the steps of transfecting recombinant
virus containing open reading frame 2 coding sequences into cells
contained in growth media, causing the virus to express open
reading frame 2, and recovering the expressed protein in the
supernate. This recovery should take place beginning approximately
5 days after infection of the cells in order to permit sufficient
quantities of recombinant protein to be expressed and secreted from
the cell into the growth media. Such methods avoid costly and
time-consuming extraction procedures required to separate and
recover the recombinant protein from within the cells.
Inventors: |
Eichmeyer; Marc; (Bondurant,
IA) ; Nitzel; Greg; (Mattawan, MI) ;
Schaeffer; Merrill; (Saint Joseph, MO) |
Correspondence
Address: |
MICHAEL P. MORRIS;BOEHRINGER INGELHEIM USA CORPORATION
900 RIDGEBURY RD, P O BOX 368
RIDGEFIELD
CT
06877-0368
US
|
Assignee: |
BOEHRINGER INGELHEIM VETMEDICA,
INC.
Saint Joseph
MO
|
Family ID: |
36615566 |
Appl. No.: |
12/137938 |
Filed: |
June 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11319975 |
Dec 29, 2005 |
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12137938 |
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11034797 |
Jan 13, 2005 |
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11319975 |
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60640510 |
Dec 30, 2004 |
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Current U.S.
Class: |
424/186.1 |
Current CPC
Class: |
A61K 2039/552 20130101;
A61K 2039/5258 20130101; C12N 2770/10034 20130101; A61K 2039/55505
20130101; C12N 2710/14163 20130101; A61P 37/04 20180101; C12N
2750/10034 20130101; C07K 14/005 20130101; A61K 2039/55555
20130101; A61P 31/00 20180101; A61P 37/00 20180101; A61P 31/20
20180101; A61K 45/06 20130101; C12N 2750/10051 20130101; A61K 39/12
20130101; A61P 31/12 20180101; C12N 2750/10022 20130101; C12N
2710/14143 20130101; A61K 2039/545 20130101; A61K 33/00
20130101 |
Class at
Publication: |
424/186.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61P 37/04 20060101 A61P037/04 |
Claims
1. A kit comprising a container and an instruction manual: a.
wherein said container comprises at least one dose of an
immunogenic composition of PCV2 ORF2 protein, wherein one dose
comprises at least 2 .mu.g PCV2 ORF2 protein; and b. wherein said
instruction manual includes the information for the intramuscular
application of at least one dose of the immunogenic composition
into piglets to lessen the severity of clinical symptoms associated
with PCV2 infection.
2. The kit of claim 1, wherein said immunogenic composition of PCV2
ORF2 protein comprises: i) a polypeptide comprising a sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; ii) any polypeptide
that is at least 80% homologous to the polypeptide of i); iii) any
immunogenic portion of the polypeptides of i) and/or ii); iv) the
immunogenic portion of iii), comprising at least 10 contiguous
amino acids included in a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID No: 9, SEQ ID No:
10, and SEQ ID No: 11, v) a polypeptide that is encoded by a DNA
comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 4; vi) any
polypeptide that is encoded by a polynucleotide that is at least
80% homologous to the polynucleotide of v); vii) any immunogenic
portion of the polypeptides encoded by the polynucleotide of v)
and/or vi); viii) the immunogenic portion of vii), wherein
polynucleotide coding for said immunogenic portion comprises at
least 30 contiguous nucleotides included in the sequences of SEQ ID
No: 3, or SEQ ID No: 4.
3. The kit of claim 1, wherein said immunogenic composition further
comprises an inactivated viral vector.
4. The kit of claim 3, wherein said inactivated viral vector is a
recombinant baculovirus coding for the PCV2 ORF2 protein.
5. The kit of claim 1, wherein said composition further comprises
an additional component selected from the group consisting of cell
culture supernate, BEI, sodium thiosulfate, carriers, adjuvants,
media, viral inactivators, diluents, isotonic agents,
immunomodulatory agents, antibiotics, and combinations thereof.
6. The kit of claim 5, wherein said adjuvant is selected from the
group consisting of acrylic acid, methacrylic acid, and any polymer
thereof.
7. The kit of claim 5, wherein said adjuvant comprises a polymer of
an acrylic acid or methacrylic acid which is cross-linked with
polyalkenyl ethers of sugars or polyalcohols.
8. The kit of claim 1, wherein said immunogenic composition further
comprises a carbomer.
9. The kit of claim 8, wherein said carbomer is included in an
amount from about 500 .mu.g to about 5 mg carbomer per dose.
10. The kit of claim 1, wherein said immunogenic composition
further comprises a pharmaceutical acceptable salt, preferably
saline.
11. The kit of claim 1, wherein one dose of said immunogenic
composition comprises at least 4 .mu.g of recombinant PCV2 ORF2
protein.
12. The kit of claim 1, wherein one dose of said immunogenic
composition comprises at least 8 .mu.g of recombinant PCV2 ORF2
protein.
13. The kit of claim 1, wherein one dose of said immunogenic
composition comprises at least 16 .mu.g of recombinant PCV2 ORF2
protein.
14. The kit of claim 1, wherein said immunogenic composition is a
vaccine.
15. The kit of claim 1, wherein said immunogenic composition is
effective for lessening the severity of clinical symptoms
associated with PCV2 infection comprising a single dose
administration of said recombinant PCV2 ORF2 protein.
16. The kit of claim 15, wherein the clinical symptoms are selected
from the group consisting of lung lesions, nasal shedding, cough,
diarrhea, and combinations thereof.
17. The kit of claim 1, wherein one dose of said immunogenic
composition of PCV2 ORF2 has a volume of at least 1 ml.
18. The kit of claim 1, wherein one dose of said immunogenic
composition of PCV2 ORF2 has a volume of at least 2 ml.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
11/319,975, filed Dec. 29, 2005, which claims the benefit of
provisional application Ser. No. 60/640,510, filed on Dec. 30,
2004, and application Ser. No. 11/034,737, filed on Jan. 13, 2005,
the teachings and contents all of which are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] This application contains a sequence listing in paper format
and in computer readable format, the teachings and content of which
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] One aspect of the present invention is concerned with the
recovery of a protein expressed by open reading frame 2 (ORF2) of
porcine circovirus type 2 (PCV2). More particularly, the protein is
a recombinant protein expressed by a transfected virus containing
recombinant coding sequences for porcine circovirus type 2, open
reading frame 2. Still more particularly, the transfected virus is
permitted to infect cells in growth media and the protein expressed
by open reading frame 2 is recovered in the supernate, rather than
from inside the cells. Even more particularly, the method involves
the steps of amplifying the open reading frame 2 gene from porcine
circovirus type 2, cloning this amplified portion into a first
vector, excising the open reading frame 2 portion from this first
vector and cloning it into a transfer vector, cotransfecting the
transfer vector with a viral vector into cells in growth media,
causing the cells to become infected by the viral vector and
thereby express open reading frame 2, and recovering the expressed
recombinant protein coded for by open reading frame 2 in the
supernate.
[0005] In another aspect, the present invention is concerned with
an immunogenic composition effective for inducing an immune
response against PCV2, and methods for producing those immunogenic
compositions. More particularly, the present invention is concerned
with an immunological composition effective for providing an immune
response that protects an animal receiving the composition and
reduces, or lessens the severity, of the clinical symptoms
associated with PCV2 infection. Still more particularly, the
present invention is concerned with a protein-based immunological
composition that confers effective protection against infection by
PCV2. Even more particularly, the present invention is concerned
with an immunological composition comprising ORF2 of PCV2, wherein
administration of PCV2-ORF2 results in protection against infection
by PCV2. Most particularly, the present invention is concerned with
an immunological composition effective for conferring effective
immunity to a swine receiving the immunological composition, and
wherein the composition comprises the protein expressed by ORF2 of
PCV2.
[0006] 2. Description of the Prior Art
[0007] Porcine circovirus type 2 (PCV2) is a small (17-22 nm in
diameter), icosahedral, non-enveloped DNA virus, which contains a
single-stranded circular genome. PCV2 shares approximately 80%
sequence identity with porcine circovirus type 1 (PCV1). However,
in contrast with PCV1, which is generally non-virulent, swine
infected with PCV2 exhibit a syndrome commonly referred to as
Post-weaning Multisystemic Wasting Syndrome (PMWS). PMWS is
clinically characterized by wasting, paleness of the skin,
unthriftiness, respiratory distress, diarrhea, icterus, and
jaundice. In some affected swine, a combination of all symptoms
will be apparent while other swine will only have one or two of
these symptoms. During necropsy, microscopic and macroscopic
lesions also appear on multiple tissues and organs, with lymphoid
organs being the most common site for lesions. A strong correlation
has been observed between the amount of PCV2 nucleic acid or
antigen and the severity of microscopic lymphoid lesions. Mortality
rates for swine infected with PCV2 can approach 80%. In addition to
PMWS, PCV2 has been associated with several other infections
including pseudorabies, porcine reproductive and respiratory
syndrome (PRRS), Glasser's disease, streptococcal meningitis,
salmonellosis, postweaning colibacillosis, dietetic hepatosis, and
suppurative bronchopneumonia.
[0008] Open reading frame 2 (ORF2) protein of PCV2, having an
approximate molecular weight of 30 kDa when run on SDS-PAGE gel,
has been utilized in the past as an antigenic component in vaccines
for PCV2. Typical methods of obtaining ORF2 for use in such
vaccines generally consist of amplifying the PCV2 DNA coding for
ORF2, transfecting a viral vector with the ORF2 DNA, infecting
cells with the viral vector containing the ORF2 DNA, permitting the
virus to express ORF2 protein within the cell, and extracting the
ORF2 protein from the cell via cell lysis. These procedures
generally take up to about four days after infection of the cells
by the viral vector. However, these procedures have a disadvantage
in that the extraction procedures are both costly and
time-consuming. Additionally, the amount of ORF2 recovered from the
cells is not very high; consequently, a large number of cells need
to be infected by a large number of viral vectors in order to
obtain sufficient quantities of the recombinant expressed protein
for use in vaccines and the like.
[0009] Current approaches to PCV2 immunization include DNA-based
vaccines, such as those described in U.S. Pat. No. 6,703,023.
However, such vaccines have been ineffective at conferring
protective immunity against PCV2 infection and the clinical signs
associated therewith.
[0010] Accordingly, what is needed in the art is a method of
obtaining ORF2 protein, which does not require extraction of the
ORF2 protein from within infected cells. What is further needed are
methods of obtaining recombinant ORF2 protein in quantities
sufficient for efficiently preparing vaccine compositions. What is
still further needed are methods for obtaining ORF2 protein which
do not require the complicated and labor-intensive methods required
by the current ORF2 protein extraction protocols. Finally, with
respect to compositions, what is needed in the art is an
immunogenic composition which does confer protective immunity
against PCV2 infection and lessens the severity of or prevents the
clinical signs associated therewith.
SUMMARY OF THE INVENTION
[0011] The present invention overcomes the problems inherent in the
prior art and provides a distinct advance in the state of the art.
Specifically, one aspect of the present invention provides improved
methods of producing and/or recovering recombinant PCV2 ORF2
protein, i) by permitting infection of susceptible cells in culture
with a recombinant viral vector containing PCV2 ORF2 DNA coding
sequences, wherein ORF2 protein is expressed by the recombinant
viral vector, and ii) thereafter recovering the ORF2 in the
supernate. It has been unexpectedly discovered that ORF2 is
released into the supernate in large quantities if the infection
and subsequent incubation of the infected cells is allowed to
progress past the typical prior PCV 2 ORF2 recovery process, which
extracts the PCV2 ORF2 from within cells. It furthermore has been
surprisingly found, that PCV ORF2 protein is robust against
prototypical degradation outside of the production cells. Both
findings together allow a recovery of high amounts of PCV2 ORF2
protein from the supernate of cell cultures infected with
recombinant viral vectors containing a PCV2 ORF2 DNA and expressing
the PCV2 ORF2 protein. High amounts of PCV2 ORF2 protein means more
than about 20 .mu.g/mL supernate, preferably more than about 25
.mu.g/mL, even more preferred more than about 30 .mu.g/mL, even
more preferred more than about 40 .mu.g/mL, even more preferred
more than about 50 .mu.g/mL, even more preferred more than about 60
.mu.g/mL, even more preferred more than about 80 .mu.g/mL, even
more preferred more than about 100 .mu.g/mL, even more preferred
than about 150 .mu.g/mL, most preferred than about 190 .mu.g/mL.
Those expression rates can also be achieved for example by the
methods as described in Examples 1 to 3.
[0012] Preferred cell cultures have a cell count between about
0.3-2.0.times.10.sup.6 cells/mL, more preferably from about
0.35-1.9.times.10.sup.6 cells/mL, still more preferably from about
0.4-1.8.times.10.sup.6 cells/mL, even more preferably from about
0.45-1.7.times.10.sup.6 cells/mL, and most preferably from about
0.5-1.5.times.10.sup.6 cells/mL. Preferred cells are determinable
by those of skill in the art. Preferred cells are those susceptible
for infection with an appropriate recombinant viral vector,
containing a PCV2 ORF2 DNA and expressing the PCV2 ORF2 protein.
Preferably the cells are insect cells, and more preferably, they
include the insect cells sold under the trademark Sf+ insect cells
(Protein Sciences Corporation, Meriden, Conn.).
[0013] Appropriate growth media will also be determinable by those
of skill in the art with a preferred growth media being serum-free
insect cell media such as Excell 420 (JRH Biosciences, Inc.,
Lenexa, Kans.) and the like. Preferred viral vectors include
baculovirus such as BaculoGold (BD Biosciences Pharmingen, San
Diego, Calif.), in particular if the production cells are insect
cells. Although the baculovirus expression system is preferred, it
is understood by those of skill in the art that other expression
systems will work for purposes of the present invention, namely the
expression of PCV2 ORF2 into the supernatant of a cell culture.
Such other expression systems may require the use of a signal
sequence in order to cause ORF2 expression into the media. It has
been surprisingly discovered that when ORF2 is produced by a
baculovirus expression system, it does not require any signal
sequence or further modification to cause expression of ORF2 into
the media. It is believed that this protein can independently form
virus-like particles (Journal of General Virology Vol. 81, pp.
2281-2287 (2000) and be secreted into the culture supernate. The
recombinant viral vector containing the PCV2 ORF2 DNA sequences has
a preferred multiplicity of infection (MOI) of between about
0.03-1.5, more preferably from about 0.05-1.3, still more
preferably from about 0.09-1.1, and most preferably from about
0.1-1.0, when used for the infection of the susceptible cells.
Preferably the MOIs mentioned above relates to one mL of cell
culture fluid. Preferably, the method described herein comprises
the infection of 0.35-1.9.times.10.sup.6 cells/mL, still more
preferably of about 0.4-1.8.times.10.sup.6 cells/mL, even more
preferably of about 0.45-1.7.times.10.sup.6 cells/mL, and most
preferably of about 0.5-1.5.times.10.sup.6 cells/mL with a
recombinant viral vector containing a PCV2 ORF2 DNA and expressing
the PCV2 ORF protein having a MOI (multiplicity of infection) of
between about 0.03-1.5, more preferably from about 0.05-1.3, still
more preferably from about 0.09-1.1, and most preferably from about
0.1-1.0.
[0014] The infected cells are then incubated over a period of up to
ten days, more preferably from about two days to about ten days,
still more preferably from about four days to about nine days, and
most preferably from about five days to about eight days. Preferred
incubation conditions include a temperature between about
22-32.degree. C., more preferably from about 24-30.degree. C.,
still more preferably from about 25-29.degree. C., even more
preferably from about 26-28.degree. C., and most preferably about
27.degree. C. Preferably, the Sf+ cells are observed following
inoculation for characteristic baculovirus-induced changes. Such
observation may include monitoring cell density trends and the
decrease in viability during the post-infection period. It was
found that peak viral titer is observed 3-5 days after infection
and peak ORF2 release from the cells into the supernate is obtained
between days 5 and 8, and/or when cell viability decreases to less
than 10%.
[0015] Thus, one aspect of the present invention provides an
improved method of producing and/or recovering recombinant PCV2
ORF2 protein, preferably in amounts described above, by i)
permitting infection of a number of susceptible cells (see above)
in culture with a recombinant viral vector with a MOI as defined
above, ii) expressing PCV2 ORF2 protein by the recombinant viral
vector, and iii) thereafter recovering the PCV2 ORF2 in the
supernate of cells obtained between days 5 and 8 after infection
and/or cell viability decreases to less then 10%. Preferably, the
recombinant viral vector is a recombinant baculovirus containing
PCV2 ORF2 DNA coding sequences and the cells are Sf+ cells.
Additionally, it is preferred that the culture be periodically
examined for macroscopic and microscopic evidence of contamination
or for atypical changes in cell morphology during the
post-infection period. Any culture exhibiting any contamination
should be discarded. Preferably, the expressed ORF2 recombinant
protein is secreted by the cells into the surrounding growth media
that maintains cell viability. The ORF2 is then recovered in the
supernate surrounding the cells rather than from the cells
themselves.
[0016] The recovery process preferably begins with the separation
of cell debris from the expressed ORF2 in media via a separation
step. Preferred separation steps include filtration, centrifugation
at speeds up to about 20,000.times.g, continuous flow
centrifugation, chromatographic separation using ion exchange or
gel filtration, and conventional immunoaffinity methods. Those
methods are known to persons skilled in the art for example by
(Harris and Angel (eds.), Protein purification methods--a practical
approach, IRL press Oxford 1995). The most preferred separation
methods include centrifugation at speeds up to about 20,000.times.g
and filtration. Preferred filtration methods include dead-end
microfiltration and tangential flow (or cross flow) filtration
including hollow fiber filtration dead-end micro filtration. Of
these, dead-end microfiltration is preferred. Preferred pore sizes
for dead-end microfiltration are between about 0.30-1.35 .mu.m,
more preferably between about 0.35-1.25 .mu.m, still more
preferably between about 0.40-1.10 .mu.m, and most preferably
between about 0.45-1.0 .mu.m. It is believed that any conventional
filtration membrane will work for purposes of the present invention
and polyethersulfone membranes are preferred. Any low weight
nucleic acid species are removed during the filtration step.
[0017] Thus, one further aspect of the present invention provides
an improved method of producing and/or recovering recombinant PCV2
ORF2 protein, preferably in amounts described above, by i)
permitting infection of a number of susceptible cells (see above)
in culture with a recombinant viral vector with a MOI as defined
above, ii) expressing PCV ORF2 protein by the recombinant viral
vector, iii) recovering the PCV2 ORF2 in the supernate of cells
obtained between days 5 and 8 after infection and/or cell viability
decreases to less then 10%, and, iv) separating cell debris from
the expressed PCV2 ORF2 via a separation step. Preferably, the
recombinant viral vector is a baculovirus containing ORF2 DNA
coding sequences and the cells are SF+ cells. Preferred separation
steps are those described above. Most preferred is a dead-end
microfiltration using a membrane having a pore size between about
0.30-1.35 .mu.m, more preferably between about 0.35-1.25 .mu.m,
still more preferably between about 0.40-1.10 .mu.m, and most
preferably between about 0.45-1.0 .mu.m.
[0018] For recovery of PCV2 ORF2 that will be used in an
immunogenic or immunological composition such as a vaccine, the
inclusion of an inactivation step is preferred in order to
inactivate the viral vector. An "immunogenic or immunological
composition" refers to a composition of matter that comprises at
least one antigen which elicits an immunological response in the
host of a cellular and/or antibody-mediated immune response to the
composition or vaccine of interest. Usually, an "immunological
response" includes but is not limited to one or more of the
following effects: the production or activation of antibodies, B
cells, helper T cells, suppressor T cells, and/or cytotoxic T cells
and/or yd T cells, directed specifically to an antigen or antigens
included in the composition or vaccine of interest. Preferably, the
host will display either a therapeutic or protective immunological
response such that resistance to new infection will be enhanced
and/or the clinical severity of the disease reduced. Such
protection will be demonstrated by either a reduction or lack of
symptoms normally displayed by an infected host, a quicker recovery
time and/or a lowered viral titer in the infected host. Thus, the
present invention also relates to method of producing and/or
recovering recombinant PCV2 ORF2 protein, preferably in amounts
described above, by i) permitting infection of a number of
susceptible cells (see above) in culture with a recombinant viral
vector with a MOI as defined above, ii) expressing PCV ORF2 protein
by the recombinant viral vector, iii) recovering the PCV2 ORF2 in
the supernate of cells obtained between days 5 and 8 after
infection and/or cell viability decreases to less then 10%, iv)
separating cell debris from the expressed PCV2 ORF2 via a
separation step, and v) inactivating the recombinant viral
vector.
[0019] Preferably, this inactivation is done either just before or
just after the filtration step, with after the filtration step
being the preferred time for inactivation. Any conventional
inactivation method can be used for purposes of the present
invention. Thus, inactivation can be performed by chemical and/or
physical treatments. In preferred forms, the volume of harvest
fluids is determined and the temperature is brought to between
about 32-42.degree. C., more preferably between about 34-40.degree.
C., and most preferably between about 35-39.degree. C. Preferred
inactivation methods include the addition cyclized binary
ethylenimine (BEI), preferably in a concentration of about 1 to
about 20 mM, preferably of about 2 to about 10 mM, still more
preferably of about 2 to about 8 mM, still more preferably of about
3 to about 7 mM, most preferably of about 5 mM. For example the
inactivation includes the addition of a solution of
2-bromoethyleneamine hydrobromide, preferably of about 0.4M, which
has been cyclized to 0.2M binary ethylenimine (BEI) in 0.3N NaOH,
to the fluids to give a final concentration of about 5 mM BEI.
Preferably, the fluids are then stirred continuously for 72-96
hours and the inactivated harvest fluids can be stored frozen at
-40.degree. C. or below or between about 1-7.degree. C. After
inactivation is completed a sodium thiosulfate solution, preferably
at 1.0M is added to neutralize any residual BEI. Preferably, the
sodium thiosulfate is added in equivalent amount as compared to the
BEI added prior to for inactivation. For example, in the event BEI
is added to a final concentration of 5 mM, a 1.0M sodium
thiosulfate solution is added to give a final minimum concentration
of 5 mM to neutralize any residual BEI.
[0020] Thus, one further aspect of the present invention relates to
a method of producing recombinant PCV2 ORF2 protein, preferably in
amounts described above, by i) permitting infection of a number of
susceptible cells (see above) in culture with a recombinant viral
vector with a MOI as defined above, ii) expressing PCV ORF2 protein
by the recombinant viral vector, iii) recovering the PCV2 ORF2 in
the supernate of cells obtained between days 5 and 8 after
infection and/or cell viability decreases to less then 10%, iv)
separating cell debris from the expressed PCV2 ORF2 via a
separation step, and v) inactivating the recombinant viral vector.
Preferably, the recombinant viral vector is a baculovirus
containing ORF2 DNA coding sequences and the cells are SF+ cells.
Preferred separation steps are those described above, most
preferred is the filtration step. Preferred inactivation steps are
those described above. Preferably, inactivation is performed
between about 35-39.degree. C. and in the presence of 2 to 8 mM
BEI, still more preferred in the presence of about 5 mM BEI. It has
been surprisingly found, that higher concentrations of BEI
negatively affect the PCV2 ORF2 protein.
[0021] According to one further aspect of the present invention,
the method described above also includes an neutralization step
after step v). This step vi) comprises adding of an equivalent
amount of an agent that neutralizes the inactivation agent within
the solution. Preferably, if the inactivation agent is BEI,
addition of sodium thiosulfate to an equivalent amount is
preferred. Thus, according to a further aspect, step vi) comprises
adding of a sodium thiosulfate solution to a final concentration of
about 1 to about 20 mM, preferably of about 2 to about 10 mM, still
more preferably of about 2 to about 8 mM, still more preferably of
about 3 to about 7 mM most preferably of about 5 mM, when the
inactivation agent is BEI.
[0022] In preferred forms and especially in forms that will use the
recombinant PCV2 ORF2 protein in an immunogenic composition such as
a vaccine, each lot of harvested ORF2 will be tested for
inactivation by passage in the anchorage dependent, baculovirus
susceptible Sf+ cells. In a preferred form of this testing, 150
cm.sup.2 of appropriate cell culture monolayer is inoculated with
1.0 mL of inactivated PCV2 fluids and maintained at 25-29.degree.
C. for 14 days with at least two passages. At the end of the
maintenance period, the cell monolayers are examined for
cytopathogenic effect (CPE) typical of PCV2 ORF2 baculovirus.
Preferably, positive virus controls are also used. Such controls
can consist of one culture of Sf+ cells inoculated with a
non-inactivated reference PCV2 ORF2 baculovirus and one flask of
Sf+ cells that remain uninoculated. After incubation and passage,
the absence of virus-infected cells in the BEI treated viral fluids
would constitute a satisfactory inactivation test. The control
cells inoculated with the reference virus should exhibit CPE
typical of PCV2 ORF2 baculovirus and the uninoculated flask should
not exhibit any evidence of PCV2 ORF2 baculovirus CPE.
Alternatively, at the end of the maintenance period, the
supernatant samples could be collected and inoculated onto a Sf+ 96
well plate, which has been loaded with Sf+ cells, and then
maintained at 25-29.degree. C. for 5-6 days. The plate is then
fixed and stained with anti-PCV2 ORF2 antibody conjugated to FITC.
The absence of CPE and ORF2 expression, as detected by IFA
micoscopy, in the BEI treated viral fluids constitutes a
satisfactory inactivation test. The control cells inoculated with
the reference virus should exhibit CPE and IFA activity and the
uninoculated flask should not exhibit any evidence of PCV2 ORF2
baculovirus CPE and contain no IFA activity.
[0023] Thus a further aspect of the present invention relates to an
inactivation test for determining the effectiveness of the
inactivation of the recombination viral vector, comprises the
steps: i) contacting at least a portion of the culture fluid
containing the recombinant viral vector with an inactivating agent,
preferably as described above, ii) adding a neutralization agent to
neutralize the inactivation agent, preferably as described above,
and iii) determining the residual infectivity by the assays as
described above.
[0024] After inactivation, the relative amount of recombinant PCV2
ORF2 protein in a sample can be determined in a number of ways.
Preferred methods of quantitation include SDS-PAGE densitometry,
ELISA, and animal vaccination studies that correlate known
quantities of vaccine with clinical outcomes (serology, etc.). When
SDS-PAGE is utilized for quantitation, the sample material
containing an unknown amount of recombinant PCV2 ORF2 protein is
run on a gel, together with samples that contain different known
amounts of recombinant PCV2 ORF2 protein. A standard curve can then
be produced based on the known samples and the amount of
recombinant PCV2 ORF2 in the unknown sample can be determined by
comparison with this standard curve. Because ELISAs are generally
recognized as the industry standard for antigen quantitation, they
are preferred for quantitation.
[0025] Thus, according to a further aspect, the present invention
also relates to an ELISA for the quantification of recombinant PCV2
ORF2 protein. A preferred ELISA as provided herewith will generally
begin with diluting the capture antibody 1:6000 or an appropriate
working dilution in coating buffer. A preferred capture antibody is
Swine anti-PCV2 PAb Prot. G purified, and a preferred coating
buffer is 0.05M Carbonate buffer, which can be made by combining
2.93 g NaHCO.sub.3 (Sigma Cat. No. S-6014, or equivalent) and 1.59
g NaCO.sub.3 (Sigma Cat. No. S-6139, or equivalent). The mixture is
combined with distilled water, or equivalent, to make one liter at
a pH of 9.6.+-.0.1. Next, the capture antibody is diluted 1:6000,
or any other appropriate working dilution, in coating buffer. For
example, for four plates, one would need 42 mLs of coating buffer
and seven .mu.L of capture antibody. Using a reverse pipetting
method, 100 .mu.L of diluted capture antibody is added to all of
the wells. In order to obtain an even coating, the sides of each
plate should be gently tapped. The plates are then sealed with
plate sealers, prior to stacking the plates and capping the stack
with an empty 96-well plate. The plates are incubated overnight
(14-24 hours) at 35-39.degree. C. Each plate is then washed three
times with wash buffer using the ultra wash plus micro titer plate
washer set at 250 .mu.L/wash with three washes and no soak time.
After the last wash, the plates should be tapped onto a paper
towel. Again, using the reverse pipetting technique, 250 .mu.L of
blocking solution should be added to all of the wells. The test
plates should be sealed and incubated for approximately one hour
(.+-.five minutes) at 35-37.degree. C. Preferably, the plates will
not be stacked after this step. During the blocking step, all test
samples should be pulled out and thawed at room temperature. Next,
four separate dilution plates should be prepared by adding 200
.mu.L of diluent solution to all of the remaining wells except for
row A and row H, columns 1-3. Next, six test tubes should be
labeled as follows, low titer, medium titer, high titer,
inactivated/filtered (1:240), inactivated/filtered (1:480), and
internal control. In the designated tubes, an appropriate dilution
should be prepared for the following test samples. The thawed test
samples should be vortexed prior to use. For four plates, the
following dilutions will be made: A) the low titer will not be
pre-diluted: 3.0 mLs of low titer; B) negative control at a 1:30
dilution (SF+ cells): 3.77 mLs of diluent+130 .mu.L of the negative
control; C) medium titer at a 1:30 dilution (8 .mu.g/mL): 3.77 mLs
of diluent+130 .mu.L of the medium titer; D) high titer at a 1:90
dilution (16 .mu.g/mL): 2.967 mLs of diluent+33 .mu.L of high
titer; E) inactivated/filtered at a 1:240 dilution: 2.39 mLs of
diluent+10 .mu.L of inactivated/filtered sample; F)
inactivated/filtered at a 1:480 dilution: 1.0 mL of diluent+1.0 mL
of inact/filtered (1:240) prepared sample from E above; G) internal
control at 1:30 dilution: 3.77 mLs of diluent+130 .mu.L of the
internal control. Next, add 300 .mu.L of the prepared samples to
corresponding empty wells in the dilution plates for plates 1
through 4. The multichannel pipettor is then set to 100 .mu.L, and
the contents in Row A are mixed by pipetting up and down for at
least 5 times and then 100 .mu.L is transferred to Row B using the
reverse pipetting technique. The tips should be changed and this
same procedure is followed down the plate to Row G. Samples in
these dilution plates are now ready for transfer to the test plates
once the test plates have been washed 3 times with wash buffer
using the ultrawash plus microtiter plate washer (settings at 250
.mu.L/wash, 3 washes, no soak time). After the last wash, the
plates should be tapped onto a paper towel. Next, the contents of
the dilution plate are transferred to the test plate using a simple
transfer procedure. More specifically, starting at row H, 100
.mu.L/well is transferred from the dilution plate(s) to
corresponding wells of the test plate(s) using reverse pipetting
technique. After each transfer, the pipette tips should be changed.
From Row G, 100 .mu.L/well in the dilution plate(s) is transferred
to corresponding wells of the test plate(s) using reverse pipetting
technique. The same set of pipette tips can be used for the
remaining transfer. To ensure a homogenous solution for the
transfer, the solution should be pipetted up and down at least 3
times prior to transfer. Next, the test plate(s) are sealed and
incubated for 1.0 hour .+-.5 minutes at 37.degree.
C..+-.2.0.degree. C. Again, it is preferable not to stack the
plates. The plates are then washed 3 times with wash buffer using
the ultrawash plus microtiter plate washer (settings at 250
.mu.L/wash, 3 washes, and no soak time). After the last wash, the
plates are tapped onto a paper towel. Using reverse pipetting
technique, 100 .mu.L of detection antibody diluted 1:300, or
appropriate working dilution, in diluent solution is added to all
of the wells of the test plate(s). For example, for four plates,
one will need 42 mLs of diluent solution with 140 .mu.L of capture
antibody. The test plate(s) are then sealed and incubated for 1.0
hour .+-.5 minutes at 37.degree. C..+-.2.0.degree. C. Again, the
plates are washed 3 times with wash buffer using the ultrawash plus
microtiter plate washer (settings at 250 .mu.L/wash, 3 washes, and
no soak time). After the last wash, the plates are tapped onto a
paper towel. Next, the conjugate diluent is prepared by adding 1%
normal rabbit serum to the diluent. For example, for four plates,
420 .mu.L of normal rabbit serum is added to 42 mL of diluent. The
conjugate antibody is diluted to 1:10,000, or any other appropriate
working dilution, in a freshly prepared conjugate diluent solution
to all wells of the test plate(s). Using a reverse pipetting
technique, 100 .mu.L of this diluted conjugate antibody is added to
all the wells. The test plate(s) are then sealed and incubated for
45.+-.5 minutes at 37.degree. C..+-.2.0.degree. C. Preferably, the
plates are not stacked. The plates are then washed 3 times with
wash buffer using the ultrawash plus microtiter plate washer
(settings at 250 .mu.L/wash, 3 washes, and no soak time). After the
last wash, the plates are tapped onto a paper towel. Next, equal
volumes of TMB Peroxidase Substrate (Reagent A) with Peroxidase
Solution B (Reagent B) are mixed immediately prior to use. The
amount mixed will vary depending upon the quantity of plates but
each plate will require 10 mL/plate+2 mLs. Therefore, for 4 plates,
it will be 21 mL of Reagent A+21 mL of Reagent B. Using a reverse
pipetting technique, 100 .mu.L of substrate is added to all wells
of the test plate(s). The plates are then incubated at room
temperature for 15 minutes .+-.15 seconds. The reaction is stopped
by the addition of 100 .mu.L of 1N HCl solution to all wells using
a reverse pipetting technique. The ELISA plate reader is then
turned on and allowed to proceed through its diagnostics and
testing phases in a conventional manner.
[0026] A further aspect of the invention relates to a method for
constructing a recombinant viral vector containing PCV2 ORF2 DNA
and expressing PCV2 ORF2 protein in high amounts, when infected
into susceptible cells. It has been surprisingly found that the
recombinant viral vector as provided herewith expresses high
amounts, as defined above, of PCV2 ORF2 after infecting susceptible
cells. Therefore, the present invention also relates to an improved
method for producing and/or recovering of PCV2 ORF2 protein,
preferably comprises the step: constructing a recombinant viral
vector containing PCV2 ORF2 DNA and expressing PCV2 ORF2 protein.
Preferably, the viral vector is a recombinant baculorvirus. Details
of the method for constructing recombinant viral vectors containing
PCV2 ORF2 DNA and expressing PCV2 ORF2 protein, as provided
herewith, are described to the following: In preferred forms the
recombinant viral vector containing PCV2 ORF 2 DNA and expressing
PCV2 ORF2 protein used to infect the cells is generated by
transfecting a transfer vector that has had an ORF2 gene cloned
therein into a viral vector. Preferably, only the portion of the
transfer vector is transfected into the viral vector, that contains
the ORF2 DNA. The term "transfected into a viral vector" means, and
is used as a synonym for "introducing" or "cloning" a heterologous
DNA into a viral vector, such as for example into a baculovirus
vector. The viral vector is preferably but not necessarily a
baculovirus.
[0027] Thus, according to a further aspect of the present
invention, the recombinant viral vector is generated by
recombination between a transfer vector containing the heterologous
PCV2 ORF2 DNA and a viral vector, preferably a baculorvirus, even
more preferably a linearized replication-deficient baculovirus
(such as Baculo Gold DNA). A "transfer vector" means a DNA
molecule, that includes at least one origin of replication, the
heterologous gene, in the present case PCV2 ORF2, and DNA sequences
which allows the cloning of said heterologous gene into the viral
vector. Preferably the sequences which allow cloning of the
heterologous gene into the viral vector are flanking the
heterologous gene. Even more preferably those flanking sequences
are at least homologous in parts with sequences of the viral
vector. The sequence homology then allows recombination of both
molecules, the viral vector and the transfer vector to generate a
recombinant viral vector containing the heterologous gene. One
preferred transfer vector is the pVL1392 vector (BD Biosciences
Pharmingen), which is designed for co-transfection with the
BaculoGold DNA into the preferred Sf+ cell line. Preferably, said
transfer vector comprises a PCV2 ORF2 DNA. The construct
co-transfected is approximately 10,387 base pairs in length.
[0028] In more preferred forms, the methods of the present
invention will begin with the isolation of PCV2 ORF2 DNA.
Generally, this can be from a known or unknown strain as the ORF2
DNA appears to be highly conserved with at least about 95% sequence
identity between different isolates. Any PCV2 ORF2 gene known in
the art can be used for purposes of the present invention as each
would be expressed into the supernate. The PCV ORF2 DNA is
preferably amplified using PCR methods, even more preferred
together with the introduction of a 5' flanking Kozak's consensus
sequence (CCGCCAUG) (SEQ ID NO 1) and/or a 3' flanking EcoR1 site
(GAATTC) (SEQ ID NO 2). Such introduction of a 5' Kozak's consensus
preferably removes the naturally occurring start codon AUG of PCV2
ORF2. The 3' EcoR1 site is preferably introduced downstream of the
stop codon of the PCV2 ORF2. More preferably it is introduced
downstream of a poly A transcription termination sequence, that
itself is located downstream of the PCV2 ORF2 stop codon. It has
been found, that the use of a Kozak consensus sequence, in
particular as described above, increases the expression level of
the subsequent PCV2 ORF2 protein. The amplified PCV2 ORF2 DNA, with
these additional sequences, is cloned into a vector. A preferred
vector for this initial cloning step is the pGEM-T-Easy Vector
(Promega, Madison, Wis.). The PCV2 ORF2 DNA including some pGEM
vector sequences (SEQ ID NO: 7) is preferably excised from the
vector at the Not1 restriction site. The resulting DNA is then
cloned into the transfer vector.
[0029] Thus, in one aspect of the present invention, a method for
constructing a recombinant viral vector containing PCV2 ORF2 DNA is
provided. This method comprises the steps: i) cloning a recombinant
PCV2 ORF2 into a transfer vector; and ii) transfecting the portion
of the transfer vector containing the recombinant PCV2 ORF2 into a
viral vector, to generate the recombinant viral vector. Preferably,
the transfer vector is that described above or is constructed as
described above or as exemplarily shown in FIG. 1. Thus according
to a further aspect, the transfer vector, used for the construction
of the recombinant viral vector as described herein, contains the
sequence of SEQ ID NO: 7.
[0030] According to a further aspect, this method further comprises
prior to step i) the following step: amplifying the PCV2 ORF2 DNA
in vitro, wherein the flanking sequences of the PCV2 ORF2 DNA are
modified as described above. In vitro methods for amplifying the
PCV2 ORF2 DNA and modifying the flanking sequences, cloning in
vitro amplified PCV2 ORF2 DNA into a transfer vector and suitable
transfer vectors are described above, exemplarily shown in FIG. 1,
or known to a person skilled in the art. Thus according to a
further aspect, the present invention relates to a method for
constructing a recombinant viral vector containing PCV2 ORF2 DNA
and expressing PCV2 ORF2 protein comprises the steps of: i)
amplifying PCV2 ORF2 DNA in vitro, wherein the flanking sequences
of said PCV2 ORF2 DNA are modified, ii) cloning the amplified PCV2
ORF2 DNA into a transfer vector; and iii) transfecting the transfer
vector or a portion thereof containing the recombinant PCV2 ORF2
DNA into a viral vector to generate the recombinant viral vector.
Preferably, the modification of the flanking sequences of the PCV2
ORF2 DNA is performed as described above, e.g. by introducing a 5'
Kozak sequence and/or a EcoR 1 site, preferably as described
above.
[0031] According to a further aspect, a method of producing and/or
recovering recombinant protein expressed by open reading frame 2 of
PCV2 is provided. The method generally comprises the steps of: i)
cloning a recombinant PCV2 ORF2 into a transfer vector; ii)
transfecting the portion of the transfer vector containing the
recombinant PCV2 ORF2 into a virus; iii) infecting cells in media
with the transfected virus; iv) causing the transfected virus to
express the recombinant protein from PCV2 ORF2; v) separating cells
from the supernate; and vi) recovering the expressed PCV2 ORF2
protein from the supernate.
[0032] Methods of how to clone a recombinant PCV2 ORF2 DNA into a
transfer vector are described above. Preferably, the transfer
vector contains the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:7. However, the transfer vector can contain any PCV2 ORF2 DNA,
unmodified or modified, as long as the PCV2 ORF2 DNA, when
transfected into a recombinant viral vector, is expressed in cell
culture. Preferably, the recombinant viral vector comprises the
sequence of SEQ ID NO:8. Moreover, methods of how to infect cells,
preferably how to infect insect cells with a defined number of
recombinant baculovirus containing PCV2 ORF2 DNA and expressing
PCV2 ORF2 protein are described above in detail. Moreover, steps of
separating cells from the supernate as well as steps for recovering
the expressed PCV2 ORF2 protein are also described above in detail.
Any of these specific process steps, as described herein, are part
of the method of producing and/or recovering recombinant protein
expressed by open reading frame 2 of PCV2 as described above.
Preferably, the cells are SF+ cells. Still more preferably, cell
cultures have a cell count between about 0.3-2.0.times.10.sup.6
cells/mL, more preferably from about 0.35-1.9.times.10.sup.6
cells/mL, still more preferably from about 0.4-1.8.times.10.sup.6
cells/mL, even more preferably from about 0.45-1.7.times.10.sup.6
cells/mL, and most preferably from about 0.5-1.5.times.10.sup.6
cells/mL. Preferably, the recombinant viral vector containing the
PCV2 ORF2 DNA has a preferred multiplicity of infection (MOI) of
between about 0.03-1.5, more preferably from about 0.05-1.3, still
more preferably from about 0.09-1.1, still more preferably from
about 0.1-1.0, and most preferably to about 0.5, when used for the
infection of the susceptible cells. Preferably, recovering of the
PCV2 ORF2 protein in the supernate of cells obtained between days 5
and 8 after infection and/or cell viability decreases to less then
10%. Preferably, for producing PCV2 ORF2 protein, cells are
cultivated at 25 to 29.degree. C. Preferably, the separation step
is a centrifugation or a filtration step.
[0033] Optionally, this method can include the step of amplifying
the PCV2 ORF2 DNA from a strain of PCV2 prior to cloning the PCV2
ORF2 DNA into the transfer vector. In preferred forms, a 5' Kozak's
sequence, a 3' EcoR1 site, and combinations thereof can also be
added to the amplified sequence, preferably prior to or during
amplification. A preferred 5' Kozak's sequence comprises SEQ ID NO:
1. A preferred 3' EcoR1 site comprises SEQ ID NO: 2. Preferred PCV2
ORF2 DNA comprises the nucleotide sequence Genbank Accession No.
AF086834 (SEQ ID NO: 3) and SEQ ID NO: 4. Preferred recombinant
PCV2 ORF2 protein comprises the amino acid sequence of SEQ ID NO:
5, which is the protein encoded by SEQ ID NO: 3 (Genbank Accession
No. AF086834) and SEQ ID No: 6, which is the protein encoded by SEQ
ID NO: 4. A preferred media comprises serum-free insect cell media,
still more preferably Excell 420 media. When the optional
amplification step is performed, it is preferable to first clone
the amplified open reading frame 2 into a first vector, excise the
open reading frame 2 from the first vector, and use the excised
open reading frame for cloning into the transfer vector. A
preferred cell line for cotransfection is the SF+ cell line. A
preferred virus for cotransfection is baculovirus. In preferred
forms of this method, the transfected portion of the transfer
vector comprises SEQ ID NO: 8. Finally, for this method, it is
preferred to recover the PCV2 open reading frame 2 (ORF2) protein
in the cell culture supernate at least 5 days after infecting the
cells with the virus.
[0034] Thus, a further aspect of the invention relates to a method
for producing and/or recovering the PCV2 open reading frame 2,
comprises the steps: i) amplifying the PCV2 ORF2 DNA in vitro,
preferably by adding a 5' Kozak sequence and/or by adding a 3'
EcoR1 restriction site, ii) cloning the amplified PCV2 ORF2 into a
transfer vector; iii) transfecting the portion of the transfer
vector containing the recombinant PCV2 ORF2 into a virus; iv)
infecting cells in media with the transfected virus; v) causing the
transfected virus to express the recombinant protein from PCV2
ORF2; vi) separating cells from the supernate; and vii) recovering
the expressed PCV2 ORF2 protein from the supernate.
[0035] A further aspect of the present invention relates to a
method for preparing a composition comprising PCV2 ORF2 protein,
and inactivated viral vector. This method comprises the steps: i)
cloning the amplified PCV2 ORF2 into a transfer vector; ii)
transfecting the portion of the transfer vector containing the
recombinant PCV2 ORF2 into a virus; iii) infecting cells in media
with the transfected viral vector; iv) causing the transfected
viral vector to express the recombinant protein from PCV2 ORF2; v)
separating cells from the supernate; iv) recovering the expressed
PCV2 ORF2 protein from the supernate; and vii) inactivating the
recombinant viral vector. Preferably, the recombinant viral vector
is a baculovirus containing ORF2 DNA coding sequences and the cells
are SF+ cells. Preferred separation steps are those described
above, most preferred is the filtration step. Preferred
inactivation steps are those described above. Preferably,
inactivation is performed between about 35-39.degree. C. and in the
presence of 2 to 8 mM BEI, still more preferred in the presence of
about 5 mM BEI. It has been surprisingly found, that higher
concentrations of BEI negatively affect the PCV2 ORF2 protein, and
lower concentrations are not effective to inactivate the viral
vector within 24 to 72 hours of inactivation. Preferably,
inactivation is performed for at least 24 hours, even more
preferred for 24 to 72 hours.
[0036] According to a further aspect, the method for preparing a
composition comprising PCV2 ORF2 protein, and inactivated viral
vector, as described above, also includes an neutralization step
after step vii). This step viii) comprises adding of an equivalent
amount of an agent that neutralizes the inactivation agent within
the solution. Preferably, if the inactivation agent is BEI,
addition of sodium thiosulfate to an equivalent amount is
preferred. Thus, according to a further aspect, step viii)
comprises adding of a sodium thiosulfate solution to a final
concentration of about 1 to about 20 mM, preferably of about 2 to
about 10 mM, still more preferably of about 2 to about 8 mM, still
more preferably of about 3 to about 7 mM, most preferably of about
5 mM, when the inactivation agent is BEI.
[0037] According to a further aspect, the method for preparing a
composition comprising PCV2 ORF2 protein, and inactivated viral
vector, as described above, comprises prior to step i) the
following step: amplifying the PCV2 ORF2 DNA in vitro, wherein the
flanking sequences of the PCV2 ORF2 DNA are modified as described
above. In vitro methods for amplifying the PCV2 ORF2 DNA and
modifying the flanking sequences, cloning in vitro amplified PCV2
ORF2 DNA into a transfer vector and suitable transfer vectors are
described above, exemplarily shown in FIG. 1, or known to a person
skilled in the art. Thus according to a further aspect, this method
comprises the steps: i) amplifying PCV2 ORF2 DNA in vitro, wherein
the flanking sequences of said PCV2 ORF2 DNA are modified, ii)
cloning the amplified PCV2 ORF2 DNA into a transfer vector; and
iii) transfecting the transfer vector or a portion thereof
containing the recombinant PCV2 ORF2 DNA into a viral vector to
generate the recombinant viral vector, iv) infecting cells in media
with the transfected virus; v) causing the transfected virus to
express the recombinant protein from PCV2 ORF2; vi) separating
cells from the supernate; vii) recovering the expressed PCV2 ORF2
protein from the supernate; viii) inactivating the recombinant
viral vector, preferably, in the presence of about 1 to about 20 mM
BEI, most preferred in the presence of about 5 mM BEI; and ix)
adding of an equivalent amount of an agent that neutralizes the
inactivation agent within the solution, preferably, adding of a
sodium thiosulfate solution to a final concentration of about 1 to
about 20 mM, preferably of about 5 mM, when the inactivation agent
is BEI.
[0038] In another aspect of the present invention, a method for
preparing a composition, preferably an antigenic composition, such
as for example a vaccine, for invoking an immune response against
PCV2 is provided. Generally, this method includes the steps of
transfecting a construct into a virus, wherein the construct
comprises i) recombinant DNA from ORF2 of PCV2, ii) infecting cells
in growth media with the transfected virus, iii) causing the virus
to express the recombinant protein from PCV2 ORF2, iv) recovering
the expressed ORF2 protein from the supernate, v) and preparing the
composition by combining the recovered protein with a suitable
adjuvant and/or other pharmaceutically acceptable carrier.
[0039] "Adjuvants" as used herein, can include aluminum hydroxide
and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge
Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals,
Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water
emulsion, water-in-oil-in-water emulsion. The emulsion can be based
in particular on light liquid paraffin oil (European Pharmacopea
type); isoprenoid oil such as squalane or squalene; oil resulting
from theoligomerization of alkenes, in particular of isobutene or
decene; esters of acids or of alcohols containing a linear alkyl
group, more particularly plant oils, ethyl oleate, propylene glycol
di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or
propylene glycol dioleate; esters of branched fatty acids or
alcohols, in particular isostearic acid esters. The oil is used in
combination with emulsifiers to form the emulsion. The emulsifiers
are preferably nonionic surfactants, in particular esters of
sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of
polyglycerol, of propylene glycol and of oleic, isostearic,
ricinoleic or hydroxystearic acid, which are optionally
ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks,
in particular the Pluronic products, especially L121. See Hunter et
al., The Theory and Practical Application of Adjuvants (Ed.
Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995)
and Todd et al., Vaccine 15:564-570 (1997).
[0040] For example, it is possible to use the SPT emulsion
described on page 147 of "Vaccine Design, The Subunit and Adjuvant
Approach" edited by M. Powell and M. Newman, Plenum Press, 1995,
and the emulsion MF59 described on page 183 of this same book.
[0041] A further instance of an adjuvant is a compound chosen from
the polymers of acrylic or methacrylic acid and the copolymers of
maleic anhydride and alkenyl derivative. Advantageous adjuvant
compounds are the polymers of acrylic or methacrylic acid which are
cross-linked, especially with polyalkenyl ethers of sugars or
polyalcohols. These compounds are known by the term carbomer
(Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art
can also refer to U.S. Pat. No. 2,909,462 which describes such
acrylic polymers cross-linked with a polyhydroxylated compound
having at least 3 hydroxyl groups, preferably not more than 8, the
hydrogen atoms of at least three hydroxyls being replaced by
unsaturated aliphatic radicals having at least 2 carbon atoms. The
preferred radicals are those containing from 2 to 4 carbon atoms,
e.g. vinyls, allyls and other ethylenically unsaturated groups. The
unsaturated radicals may themselves contain other substituents,
such as methyl. The products sold under the name Carbopol; (BF
Goodrich, Ohio, USA) are particularly appropriate. They are
cross-linked with an allyl sucrose or with allyl pentaerythritol.
Among then, there may be mentioned Carbopol 974P, 934P and 971P.
Most preferred is the use of Cabopol 971P. Among the copolymers of
maleic anhydride and alkenyl derivative, the copolymers EMA
(Monsanto) which are copolymers of maleic anhydride and ethylene.
The dissolution of these polymers in water leads to an acid
solution that will be neutralized, preferably to physiological pH,
in order to give the adjuvant solution into which the immunogenic,
immunological or vaccine composition itself will be
incorporated.
[0042] Further suitable adjuvants include, but are not limited to,
the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx,
Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl
lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin
from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or
muramyl dipeptide among many others.
[0043] Preferably, the adjuvant is added in an amount of about 100
.mu.g to about 10 mg per dose. Even more preferred the adjuvant is
added in an amount of about 100 .mu.g to about 10 mg per dose. Even
more preferred the adjuvant is added in an amount of about 500
.mu.g to about 5 mg per dose. Even more preferred the adjuvant is
added in an amount of about 750 .mu.g to about 2.5 mg per dose.
Most preferred the adjuvant is added in an amount of about 1 mg per
dose.
[0044] Thus, according to a further aspect, the method for
preparing an antigenic composition, such as for example a vaccine,
for invoking an immune response against PCV2 comprises i) preparing
and recovering PCV2 ORF2 protein, and ii) admixing this with a
suitable adjuvant. Preferably, the adjuvant is Carbopol 971P. Even
more preferred, Carbopol 971P is added in an amount of about 500
.mu.g to about 5 mg per dose, even more preferred in an amount of
about 750 .mu.g to about 2.5 mg per dose and most preferred in an
amount of about 1 mg per dose. Preferably, the process step i)
includes the process steps as described for the preparation and
recovery of PCV2 ORF2. For example, in preferred forms of this
method, the construct comprising PCV2 ORF2 DNA is obtained in a
transfer vector. Suitable transfer vectors and methods of preparing
them are described above. Optionally, the method may include the
step of amplifying the ORF2 from a strain of PCV2 through PCR prior
to cloning the ORF2 into the transfer vector. Preferred open
reading frame sequences, Kozak's sequences, 3' EcoR1 site
sequences, recombinant protein sequences, transfected construct
sequences, media, cells, and viruses are as described in the
previous methods. Another optional step for this method includes
cloning the amplified PCV2 ORF2 DNA into a first vector, excising
the ORF2 DNA from this first vector, and using this excised PCV2
ORF2 DNA for cloning into the transfer vector. As with the other
methods, it is preferred to wait for at least 5 days after
infection of the cells by the transfected baculovirus prior to
recovery of recombinant ORF2 protein from the supernate.
Preferably, the recovery step of this method also includes the step
of separating the media from the cells and cell debris. This can be
done in a variety of ways but for ease and convenience, it is
preferred to filter the cells, cell debris, and growth media
through a filter having pores ranging in size from about 0.45 .mu.M
to about 1.0 .mu.M. Finally, for this method, it is preferred to
include a virus inactivation step prior to combining the recovered
recombinant PCV2 ORF2 protein in a composition. This can be done in
a variety of ways, but it is preferred in the practice of the
present invention to use BEI.
[0045] Thus according to a further aspect, this method comprises
the steps: i) amplifying PCV2 ORF2 DNA in vitro, wherein the
flanking sequences of said PCV2 ORF2 DNA are modified, ii) cloning
the amplified PCV2 ORF2 DNA into a transfer vector; and iii)
transfecting the transfer vector or a portion thereof containing
the recombinant PCV2 ORF2 DNA into a viral vector to generate the
recombinant viral vector, iv) infecting cells in media with the
transfected virus; v) causing the transfected virus to express the
recombinant protein from PCV2 ORF2; vi) separating cells from the
supernate; vii) recovering the expressed PCV2 ORF2 protein from the
supernate; viii) inactivating the recombinant viral vector,
preferably, in the presence of about 1 to about 20 mM BEI, most
preferred in the presence of about 5 mM BEI; ix) adding of an
equivalent amount of an agent that neutralizes the inactivation
agent within the solution, preferably, adding of a sodium
thiosulfate solution to a final concentration of about 1 to about
20 mM, preferably of about 5 mM, when the inactivation agent is
BEI, and x) adding a suitable amount of an adjuvant, preferably
adding Carbopol, more preferably Carbopol 971P, even more preferred
in amounts as described above (e.g. of about 500 .mu.g to about 5
mg per dose, even more preferred in an amount of about 750 .mu.g to
about 2.5 mg per dose and most preferred in an amount of about 1 mg
per dose).
[0046] Additionally, the composition can include one or more
pharmaceutical-acceptable carriers. As used herein, "a
pharmaceutical-acceptable carrier" includes any and all solvents,
dispersion media, coatings, stabilizing agents, diluents,
preservatives, antibacterial and antifungal agents, isotonic
agents, adsorption delaying agents, and the like. Most preferred,
the composition provided herewith, contains PCV2 ORF2 protein
recovered from the supernate of in vitro cultured cells, wherein
said cells were infected with a recombinant viral vector containing
PCV2 ORF2 DNA and expressing PCV2 ORF2 protein, and wherein said
cell culture were treated with about 2 to about 8 mM BEI,
preferably with about 5 mM BEI to inactivate the viral vector, and
an equivalent concentration of a neutralization agent, preferably
sodium thiosulfate solution to a final concentration of about 2 to
about 8 mM, preferably of about 5 mM, Carbopol, more preferably
Carbopol 971P, preferably in amounts of about 500 .mu.g to about 5
mg per dose, even more preferred in an amount of about 750 .mu.g to
about 2.5 mg per dose and most preferred in an amount of about 1 mg
per dose, and physiological saline, preferably in an amount of
about 50 to about 90% (v/v), more preferably to about 60 to 80%
(v/v), still more preferably of about 70% (v/v).
[0047] Thus, a further aspect relates to a method for preparing an
antigenic composition, such as for example a vaccine, for invoking
an immune response against PCV2 comprising the steps: i) amplifying
PCV2 ORF2 DNA in vitro, wherein the flanking sequences of said PCV2
ORF2 DNA are modified, ii) cloning the amplified PCV2 ORF2 DNA into
a transfer vector; and iii) transfecting the transfer vector or a
portion thereof containing the recombinant PCV2 ORF2 DNA into a
viral vector to generate the recombinant viral vector, iv)
infecting cells in media with the transfected virus; v) causing the
transfected virus to express the recombinant protein from PCV2
ORF2; vi) separating cells from the supernate; vii) recovering the
expressed PCV2 ORF2 protein from the supernate; viii) inactivating
the recombinant viral vector, preferably, in the presence of about
2 to about 20 mM BEI, most preferred in the presence of about 5 mM
BEI; ix) adding of an equivalent amount of an agent that neutralize
the inactivation agent within the solution, preferably, adding of a
sodium thiosulfate solution to a final concentration of about 0.5
to about 20 mM, preferably of about 5 mM, when the inactivation
agent is BEI, x) adding a suitable amount of an adjuvants,
preferably adding Carbopol, more preferably Carbopol 971P, still
more preferred in amounts as described above (e.g. of about 500
.mu.g to about 5 mg per dose, even more preferred in an amount of
about 750 .mu.g to about 2.5 mg per dose and most preferred in an
amount of about 1 mg per dose); and xi) adding physiological
saline, preferably in an amount of about 50 to about 90% (v/v),
more preferably to about 60 to 80% (v/v), still more preferably of
about 70% (v/v). Optionally, this method can also include the
addition of a protectant. A protectant as used herein, refers to an
anti-microbiological active agent, such as for example Gentamycin,
Merthiolate, and the like. In particular adding of a protectant is
most preferred for the preparation of a multi-dose composition.
Those anti-microbiological active agents are added in
concentrations effective to prevent the composition of interest for
any microbiological contamination or for inhibition of any
microbiological growth within the composition of interest.
[0048] Moreover, this method can also comprise addition of any
stabilizing agent, such as for example saccharides, trehalose,
mannitol, saccharose and the like, to increase and/or maintain
product shelf-life. However, it has been surprisingly found, that
the resulting formulation is immunologically effective over a
period of at least 24 months, without adding any further
stabilizing agent.
[0049] A further aspect of the present invention relates to the
products result from the methods as described above. In particular,
the present invention relates to a composition of matter comprises
recombinantly expressed PCV2 ORF2 protein. Moreover, the present
invention also relates to a composition of matter that comprises
recombinantly expressed PCV2 ORF2 protein, recovered from the
supernate of an insect cell culture. Moreover, the present
invention also relates to a composition of matter comprises
recombinantly expressed PCV2 ORF2 protein, recovered from the
supernate of an insect cell culture. Preferably, this composition
of matter also comprises an agent suitable for the inactivation of
viral vectors. Preferably, said inactivation agent is BEI.
Moreover, the present invention also relates to a composition of
matter that comprises recombinantly expressed PCV2 ORF2 protein,
recovered from the supernate of an insect cell culture, and
comprises an agent, suitable for the inactivation of viral vectors,
preferably BEI and a neutralization agent for neutralization of the
inactivation agent. Preferably, that neutralization agent is sodium
thiosulfate, when BEI is used as an inactivation agent.
[0050] In yet another aspect of the present invention, an
immunogenic composition that induces an immune response and, more
preferably, confers protective immunity against the clinical signs
of PCV2 infection, is provided. The composition generally comprises
the polypeptide, or a fragment thereof, expressed by Open Reading
Frame 2 (ORF2) of PCV2, as the antigenic component of the
composition.
[0051] PCV2 ORF2 DNA and protein, as used herein for the
preparation of the compositions and also as used within the
processes provided herein is a highly conserved domain within PCV2
isolates and thereby, any PCV2 ORF2 would be effective as the
source of the PCV ORF2 DNA and/or polypeptide as used herein. A
preferred PCV2 ORF2 protein is that of SEQ ID NO. 11. A preferred
PCV ORF2 polypeptide is provided herein as SEQ ID NO. 5, but it is
understood by those of skill in the art that this sequence could
vary by as much as 6-10% in sequence homology and still retain the
antigenic characteristics that render it useful in immunogenic
compositions. The antigenic characteristics of an immunological
composition can be, for example, estimated by the challenge
experiment as provided by Example 4. Moreover, the antigenic
characteristic of an modified antigen is still retained, when the
modified antigen confers at least 70%, preferably 80%, more
preferably 90% of the protective immunity as compared to the PCV2
ORF 2 protein, encoded by the polynucleotide sequence of SEQ ID
NO:3 or SEQ ID NO:4. An "immunogenic composition" as used herein,
means a PCV2 ORF2 protein which elicits an "immunological response"
in the host of a cellular and/or antibody-mediated immune response
to PCV2 ORF2 protein. Preferably, this immunogenic composition is
capable to confer protective immunity against PCV2 infection and
the clinical signs associated therewith. In some forms, immunogenic
portions of PCV2 ORF2 protein are used as the antigenic component
in the composition. The term "immunogenic portion" as used herein
refers to truncated and/or substituted forms, or fragments of PCV2
ORF2 protein and/or polynucleotide, respectively. Preferably, such
truncated and/or substituted forms, or fragments will comprise at
least 6 contiguous amino acids from the full-length ORF2
polypeptide. More preferably, the truncated or substituted forms,
or fragments will have at least 10, more preferably at least 15,
and still more preferably at least 19 contiguous amino acids from
the full-length ORF2 polypeptide. Two preferred sequences in this
respect are provided herein as SEQ ID NOs. 9 and 10. It is further
understood that such sequences may be a part of larger fragments or
truncated forms. A further preferred PCV2 ORF2 polypeptide provided
herein is encoded by the nucleotide sequences of SEQ ID NO: 3 or
SEQ ID NO: 4. But it is understood by those of skill in the art
that this sequence could vary by as much as 6-20% in sequence
homology and still retain the antigenic characteristics that render
it useful in immunogenic compositions. In some forms, a truncated
or substituted form, or fragment of ORF2 is used as the antigenic
component in the composition. Preferably, such truncated or
substituted forms, or fragments will comprise at least 18
contiguous nucleotides from the full-length ORF2 nucleotide
sequence, e.g. of SEQ ID NO: 3 or SEQ ID NO: 4. More preferably,
the truncated or substituted forms, or fragments will have at least
30, more preferably at least 45, and still more preferably at least
57 contiguous nucleotides the full-length ORF2 nucleotide sequence,
e.g. of SEQ ID NO: 3 or SEQ ID NO: 4.
[0052] "Sequence Identity" as it is known in the art refers to a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, namely a reference sequence and a
given sequence to be compared with the reference sequence. Sequence
identity is determined by comparing the given sequence to the
reference sequence after the sequences have been optimally aligned
to produce the highest degree of sequence similarity, as determined
by the match between strings of such sequences. Upon such
alignment, sequence identity is ascertained on a
position-by-position basis, e.g., the sequences are "identical" at
a particular position if at that position, the nucleotides or amino
acid residues are identical. The total number of such position
identities is then divided by the total number of nucleotides or
residues in the reference sequence to give % sequence identity.
Sequence identity can be readily calculated by known methods,
including but not limited to, those described in Computational
Molecular Biology, Lesk, A. N., ed., Oxford University Press, New
York (1988), Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York (1993); Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey (1994); Sequence Analysis in Molecular
Biology, von Heinge, G., Academic Press (1987); Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New
York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied
Math., 48: 1073 (1988), the teachings of which are incorporated
herein by reference. Preferred methods to determine the sequence
identity are designed to give the largest match between the
sequences tested. Methods to determine sequence identity are
codified in publicly available computer programs which determine
sequence identity between given sequences. Examples of such
programs include, but are not limited to, the GCG program package
(Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)),
BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol.,
215:403-410 (1990). The BLASTX program is publicly available from
NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM
NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,
215:403-410 (1990), the teachings of which are incorporated herein
by reference). These programs optimally align sequences using
default gap weights in order to produce the highest level of
sequence identity between the given and reference sequences. As an
illustration, by a polynucleotide having a nucleotide sequence
having at least, for example, 85%, preferably 90%, even more
preferably 95% "sequence identity" to a reference nucleotide
sequence, it is intended that the nucleotide sequence of the given
polynucleotide is identical to the reference sequence except that
the given polynucleotide sequence may include up to 15, preferably
up to 10, even more preferably up to 5 point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
in a polynucleotide having a nucleotide sequence having at least
85%, preferably 90%, even more preferably 95% identity relative to
the reference nucleotide sequence, up to 15%, preferably 10%, even
more preferably 5% of the nucleotides in the reference sequence may
be deleted or substituted with another nucleotide, or a number of
nucleotides up to 15%, preferably 10%, even more preferably 5% of
the total nucleotides in the reference sequence may be inserted
into the reference sequence. These mutations of the reference
sequence may occur at the 5' or 3' terminal positions of the
reference nucleotide sequence or anywhere between those terminal
positions, interspersed either individually among nucleotides in
the reference sequence or in one or more contiguous groups within
the reference sequence. Analogously, by a polypeptide having a
given amino acid sequence having at least, for example, 85%,
preferably 90%, even more preferably 95% sequence identity to a
reference amino acid sequence, it is intended that the given amino
acid sequence of the polypeptide is identical to the reference
sequence except that the given polypeptide sequence may include up
to 15, preferably up to 10, even more preferably up to 5 amino acid
alterations per each 100 amino acids of the reference amino acid
sequence. In other words, to obtain a given polypeptide sequence
having at least 85%, preferably 90%, even more preferably 95%
sequence identity with a reference amino acid sequence, up to 15%,
preferably up to 10%, even more preferably up to 5% of the amino
acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up
to 15%, preferably up to 10%, even more preferably up to 5% of the
total number of amino acid residues in the reference sequence may
be inserted into the reference sequence. These alterations of the
reference sequence may occur at the amino or the carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in the one or more contiguous
groups within the reference sequence. Preferably, residue positions
which are not identical differ by conservative amino acid
substitutions. However, conservative substitutions are not included
as a match when determining sequence identity.
[0053] "Sequence homology", as used herein, refers to a method of
determining the relatedness of two sequences. To determine sequence
homology, two or more sequences are optimally aligned, and gaps are
introduced if necessary. However, in contrast to "sequence
identity", conservative amino acid substitutions are counted as a
match when determining sequence homology. In other words, to obtain
a polypeptide or polynucleotide having 95% sequence homology with a
reference sequence, 85%, preferably 90%, even more preferably 95%
of the amino acid residues or nucleotides in the reference sequence
must match or comprise a conservative substitution with another
amino acid or nucleotide, or a number of amino acids or nucleotides
up to 15%, preferably up to 10%, even more preferably up to 5% of
the total amino acid residues or nucleotides, not including
conservative substitutions, in the reference sequence may be
inserted into the reference sequence. Preferably the homolog
sequence comprises at least a stretch of 50, even more preferred of
100, even more preferred of 250, even more preferred of 500
nucleotides.
[0054] A "conservative substitution" refers to the substitution of
an amino acid residue or nucleotide with another amino acid residue
or nucleotide having similar characteristics or properties
including size, hydrophobicity, etc., such that the overall
functionality does not change significantly.
[0055] Isolated" means altered "by the hand of man" from its
natural state, i.e., if it occurs in nature, it has been changed or
removed from its original environment, or both. For example, a
polynucleotide or polypeptide naturally present in a living
organism is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural
state is "isolated", as the term is employed herein.
[0056] Thus, a further aspect of the present invention relates to
an immunogenic composition effective for lessening the severity of
clinical symptoms associated with PCV2 infection comprising PCV2
ORF2 protein. Preferably, the PCV2 ORF2 protein is anyone of those,
described above. Preferably, said PCV2 ORF2 protein is [0057] i) a
polypeptide comprising the sequence of SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; [0058] ii) any
polypeptide that is at least 80% homologous to the polypeptide of
i), [0059] iii) any immunogenic portion of the polypeptides of i)
and/or ii) [0060] iv) the immunogenic portion of iii), comprising
at least 10 contiguous amino acids included in the sequences of SEQ
ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:
11, [0061] v) a polypeptide that is encoded by a DNA comprising the
sequence of SEQ ID NO: 3 or SEQ ID NO: 4. [0062] iv) any
polypeptide that is encoded by a polynucleotide that is at least
80% homolog to the polynucleotide of v), [0063] vii) any
immunogenic portion of the polypeptides encoded by the
polynucleotide of v) and/or vi) [0064] viii) the immunogenic
portion of vii), wherein polynucleotide coding for said immunogenic
portion comprises at least 30 contiguous nucleotides included in
the sequences of SEQ ID NO: 3, or SEQ ID NO: 4.
[0065] Preferably any of those immunogenic portions having the
immunogenic characteristics of PCV2 ORF2 protein that is encoded by
the sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
[0066] According to a further aspect, PCV2 ORF2 protein is provided
in the immunological composition at an antigen inclusion level
effective for inducing the desired immune response, namely reducing
the incidence of or lessening the severity of clinical signs
resulting from PCV2 infection. Preferably, the PCV2 ORF2 protein
inclusion level is at least 0.2 .mu.g antigen/ml of the final
immunogenic composition (.mu.g/ml), more preferably from about 0.2
to about 400 .mu.g/ml, still more preferably from about 0.3 to
about 200 .mu.g/ml, even more preferably from about 0.35 to about
100 .mu.g/ml, still more preferably from about 0.4 to about 50
.mu.g/ml, still more preferably from about 0.45 to about 30
.mu.g/ml, still more preferably from about 0.6 to about 15
.mu.g/ml, even more preferably from about 0.75 to about 8 .mu.g/ml,
even more preferably from about 1.0 to about 6 .mu.g/ml, still more
preferably from about 1.3 to about 3.0 .mu.g/ml, even more
preferably from about 1.4 to about 2.5 .mu.g/ml, even more
preferably from about 1.5 to about 2.0 .mu.g/ml, and most
preferably about 1.6 .mu.g/ml.
[0067] According to a further aspect, the ORF2 antigen inclusion
level is at least 0.2 .mu.g PCV2 ORF2 protein as described above
per dose of the final antigenic composition (.mu.g/dose), more
preferably from about 0.2 to about 400 .mu.g/dose, still more
preferably from about 0.3 to about 200 .mu.g/dose, even more
preferably from about 0.35 to about 100 .mu.g/dose, still more
preferably from about 0.4 to about 50 .mu.g/dose, still more
preferably from about 0.45 to about 30 .mu.g/dose, still more
preferably from about 0.6 to about 15 .mu.g/dose, even more
preferably from about 0.75 to about 8 .mu.g/dose, even more
preferably from about 1.0 to about 6 .mu.g/dose, still more
preferably from about 1.3 to about 3.0 .mu.g/dose, even more
preferably from about 1.4 to about 2.5 .mu.g/dose, even more
preferably from about 1.5 to about 2.0 .mu.g/dose, and most
preferably about 1.6 .mu.g/dose.
[0068] The PCV2 ORF2 polypeptide used in an immunogenic composition
in accordance with the present invention can be derived in any
fashion including isolation and purification of PCV2 ORF2, standard
protein synthesis, and recombinant methodology. Preferred methods
for obtaining PCV2 ORF2 polypeptide are described herein above and
are also provided in U.S. patent application Ser. No. 11/034,797,
the teachings and content of which are hereby incorporated by
reference. Briefly, susceptible cells are infected with a
recombinant viral vector containing PCV2 ORF2 DNA coding sequences,
PCV2 ORF2 polypeptide is expressed by the recombinant virus, and
the expressed PCV2 ORF2 polypeptide is recovered from the supernate
by filtration and inactivated by any conventional method,
preferably using binary ethylenimine, which is then neutralized to
stop the inactivation process.
[0069] Thus, according to a further aspect the immunogenic
composition comprises i) any of the PCV2 ORF2 protein described
above, preferably in concentrations described above, and ii) at
least a portion of the viral vector expressing said PCV2 ORF2
protein, preferably of a recombinant baculovirus. Moreover,
according to a further aspect, the immunogenic composition
comprises i) any of the PCV2 ORF2 protein described above,
preferably in concentrations described above, ii) at least a
portion of the viral vector expressing said PCV2 ORF2 protein,
preferably of a recombinant baculovirus, and iii) a portion of the
cell culture supernate.
[0070] According to one specific embodiment of the production and
recovery process for PCV2 ORF2 protein, the cell culture supernate
is filtered through a membrane having a pore size, preferably
between about 0.45 to 1 .mu.m. Thus, a further aspect relates to an
immunogenic composition that comprises i) any of the PCV2 ORF2
protein described above, preferably in concentrations described
above, ii) at least a portion of the viral vector expressing said
PCV2 ORF2 protein, preferably of a recombinant baculovirus, and
iii) a portion of the cell culture; wherein about 90% of the
components have a size smaller than 1 .mu.m.
[0071] According to a further aspect, the present invention relates
to an immunogenic composition that comprises i) any of the PCV2
ORF2 protein described above, preferably in concentrations
described above, ii) at least a portion of the viral vector
expressing said PCV2 ORF2 protein, iii) a portion of the cell
culture, iv) and inactivating agent to inactivate the recombinant
viral vector preferably BEI, wherein about 90% of the components i)
to iii) have a size smaller than 1 .mu.m. Preferably, BEI is
present in concentrations effective to inactivate the baculovirus.
Effective concentrations are described above.
[0072] According to a further aspect, the present invention relates
to an immunogenic composition that comprises i) any of the PCV2
ORF2 protein described above, preferably in concentrations
described above, ii) at least a portion of the viral vector
expressing said PCV2 ORF2 protein, iii) a portion of the cell
culture, iv) an inactivating agent to inactivate the recombinant
viral vector preferably BEI, and v) an neutralization agent to stop
the inactivation mediated by the inactivating agent, wherein about
90% of the components i) to iii) have a size smaller than 1 .mu.m.
Preferably, if the inactivating agent is BEI, said composition
comprises sodium thiosulfate in equivalent amounts to BEI.
[0073] The polypeptide is incorporated into a composition that can
be administered to an animal susceptible to PCV2 infection. In
preferred forms, the composition may also include additional
components known to those of skill in the art (see also Remington's
Pharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton).
Additionally, the composition may include one or more
veterinary-acceptable carriers. As used herein, "a
veterinary-acceptable carrier" includes any and all solvents,
dispersion media, coatings, adjuvants, stabilizing agents,
diluents, preservatives, antibacterial and antifungal agents,
isotonic agents, adsorption delaying agents, and the like.
[0074] In a preferred embodiment, the immunogenic composition
comprises PCV2 ORF2 protein as provided herewith, preferably in
concentrations described above as an antigenic component, which is
mixed with an adjuvant, preferably Carbopol, and physiological
saline.
[0075] Those of skill in the art will understand that the
composition herein may incorporate known injectable,
physiologically acceptable sterile solutions. For preparing a
ready-to-use solution for parenteral injection or infusion, aqueous
isotonic solutions, such as e.g. saline or corresponding plasma
protein solutions are readily available. In addition, the
immunogenic and vaccine compositions of the present invention can
include diluents, isotonic agents, stabilizers, or adjuvants.
Diluents can include water, saline, dextrose, ethanol, glycerol,
and the like. Isotonic agents can include sodium chloride,
dextrose, mannitol, sorbitol, and lactose, among others.
Stabilizers include albumin and alkali salts of
ethylendiamintetracetic acid, among others. Suitable adjuvants, are
those described above. Most preferred is the use of Carbopol, in
particular the use of Carbopol 971P, preferably in amounts as
described above (e.g. of about 500 .mu.g to about 5 mg per dose,
even more preferred in an amount of about 750 .mu.g to about 2.5 mg
per dose and most preferred in an amount of about 1 mg per
dose).
[0076] Thus, the present invention also relates to an immunogenic
composition that comprises i) any of the PCV2 ORF2 proteins
described above, preferably in concentrations described above, ii)
at least a portion of the viral vector expressing said PCV2 ORF2
protein, iii) a portion of the cell culture, iv) an inactivating
agent to inactivate the recombinant viral vector preferably BEI,
and v) an neutralization agent to stop the inactivation mediated by
the inactivating agent, preferably sodium thiosulfate in equivalent
amounts to BEI; and vi) a suitable adjuvant, preferably Carbopol
971 in amounts described above; wherein about 90% of the components
i) to iii) have a size smaller than 1 .mu.m. According to a further
aspect, this immunogenic composition further comprises a
pharmaceutical acceptable salt, preferably a phosphate salt in
physiologically acceptable concentrations. Preferably, the pH of
said immunogenic composition is adjusted to a physiological pH,
meaning between about 6.5 and 7.5.
[0077] Thus, the present invention also relates to an immunogenic
composition comprises per one ml i) at least 1.6 .mu.g of PCV2 ORF2
protein described above, ii) at least a portion of baculovirus
expressing said PCV2 ORF2 protein iii) a portion of the cell
culture, iv) about 2 to 8 mM BEI, v) sodium thiosulfate in
equivalent amounts to BEI; and vi) about 1 mg Carbopol 971, and
vii) phosphate salt in a physiologically acceptable concentration;
wherein about 90% of the components i) to iii) have a size smaller
than 1 .mu.m and the pH of said immunogenic composition is adjusted
to about 6.5 to 7.5.
[0078] The immunogenic compositions can further include one or more
other immunomodulatory agents such as, e.g., interleukins,
interferons, or other cytokines. The immunogenic compositions can
also include Gentamicin and Merthiolate. While the amounts and
concentrations of adjuvants and additives useful in the context of
the present invention can readily be determined by the skilled
artisan, the present invention contemplates compositions comprising
from about 50 .mu.g to about 2000 .mu.g of adjuvant and preferably
about 250 .mu.g/ml dose of the vaccine composition. In another
preferred embodiment, the present invention contemplates vaccine
compositions comprising from about 1 ug/ml to about 60 .mu.g/ml of
antibiotics, and more preferably less than about 30 .mu.g/ml of
antibiotics.
[0079] Thus, the present invention also relates to an immunogenic
composition that comprises i) any of the PCV2 ORF2 proteins
described above, preferably in concentrations described above, ii)
at least a portion of the viral vector expressing said PCV2 ORF2
protein, iii) a portion of the cell culture, iv) an inactivating
agent to inactivate the recombinant viral vector preferably BEI,
and v) an neutralization agent to stop the inactivation mediated by
the inactivating agent, preferably sodium thiosulfate in equivalent
amounts to BEI; vi) a suitable adjuvant, preferably Carbopol 971 in
amounts described above; vii) a pharmaceutical acceptable
concentration of a saline buffer, preferably of a phosphate salt,
and viii) an anti-microbiological active agent; wherein about 90%
of the components i) to iii) have a size smaller than 1 .mu.m.
[0080] It has been surprisingly found, that the immunogenic
composition provided herewith comprises was highly stable over a
period of 24 months. It has also been found the immunogenic
compositions provided herewith, comprising recombinant, baculovirus
expressed PCV2 ORF2 protein as provided herewith are very effective
in reducing the clinical symptoms associated with PCV2 infections.
It has been surprisingly found, that the immunogenic compositions
comprising the recombinant baculovirus expressed PCV2 ORF2 protein
as provided herewith, are more effective than an immunogenic
composition comprising the whole PCV2 virus in an inactivated form,
or isolated viral PCV2 ORF2 antigen. In particular, it has been
surprisingly found, that the recombinant baculovirus expressed PCV2
ORF2 protein is effective is in very low concentrations, which
means in concentrations up to 0.25 .mu.g/dose. This unexpected high
immunogenic potential of the PCV2 ORF2 protein could be further
increased by the addition of Carbopol.
[0081] A further aspect relates to a container comprises at least
one dose of the immunogenic composition of PCV2 ORF2 protein as
provided herewith, wherein one dose comprises at least 2 .mu.g PCV2
ORF2 protein, preferably 2 to 16 .mu.g PCV2 ORF2 protein. Said
container can comprises 1 to 250 doses of the immunogenic
composition, preferably it contains 1, 10, 25, 50, 100, 150, 200,
or 250 doses of the immunogenic composition of PCV2 ORF2 protein.
Preferably, each of the containers comprising more than one dose of
the immunogenic composition of PCV2 ORF2 protein further comprises
an anti-microbiological active agent. Those agents are for example
antibiotics including Gentamicin and Merthiolate and the like.
Thus, one aspect of the present invention relates to a container
that comprises 1 to 250 doses of the immunogenic composition of
PCV2 ORF2 protein, wherein one dose comprises at least 2 .mu.g PCV2
ORF2 protein, and Gentamicin and/or Merthiolate, preferably from
about 1 .mu.g/ml to about 60 .mu.g/ml of antibiotics, and more
preferably less than about 30 .mu.g/ml.
[0082] A further aspect relates to a kit, comprising any of the
containers, described above, and an instruction manual, including
the information for the intramuscular application of at least one
dose of the immunogenic composition of PCV2 ORF2 protein into
piglets to lessening the severity of clinical symptoms associated
with PCV2 infection. Moreover, according to a further aspect, said
instruction manual comprises the information of a second or further
administration(s) of at least one dose of the immunogenic
composition of PCV2 ORF2, wherein the second administration or any
further administration is at least 14 days beyond the initial or
any former administration. Preferably, said instruction manual also
includes the information, to administer an immune stimulant.
Preferably, said immune stimulant shall be given at least twice.
Preferably, at least 3, more preferably at least 5, even more
preferably at least 7 days are between the first and the second or
any further administration of the immune stimulant. Preferably, the
immune stimulant is given at least 10 days, preferably 15, even
more preferably 20, even more preferably at least 22 days beyond
the initial administration of the immunogenic composition of PCV2
ORF2 protein. A preferred immune stimulant is for example is
keyhole limpet hemocyanin (KLH), still preferably emulsified with
incomplete Freund's adjuvant (KLH/ICFA). However, it is herewith
understood, that any other immune stimulant known to a person
skilled in the art can also be used. "Immune stimulant" as used
herein, means any agent or composition that can trigger the immune
response, preferably without initiating or increasing a specific
immune response, for example the immune response against a specific
pathogen. It is further instructed to administer the immune
stimulant in a suitable dose. Moreover, the kit may also comprises
a container, including at least one dose of the immune stimulant,
preferably one dose of KLH, or KLH/ICFA.
[0083] Moreover, it has also been surprisingly found that the
immunogenic potential of the immunogenic compositions comprising
recombinant baculovirus expressed PCV2 ORF2 protein, preferably in
combination with Carbopol, can be further enhanced by the
administration of the IngelVac PRRS MLV vaccine (see Example 5).
PCV2 clinical signs and disease manifestations are greatly
magnified when PRRS infection is present. However, the immunogenic
compositions and vaccination strategies as provided herewith
lessened this effect greatly, and more than expected. In other
words, an unexpected synergistic effect was observed when animals,
preferably pigs are treated with any of the PCV2 ORF2 immunogenic
composition, as provided herewith, and the Ingelvac PRRS MLV
vaccine (Boehringer Ingelheim).
[0084] Thus, a further aspect of the present invention relates to
the kit as described above, comprising the immunogenic composition
of PCV2 ORF2 as provided herewith and the instruction manual,
wherein the instruction manual further include the information to
administer the PCV2 ORF2 immunogenic composition together with
immunogenic composition that comprises PRRS antigen, preferably
adjuvanted PRRS antigen. Preferably, the PRRS antigen is adjuvanted
with Carbopol. Preferably, the PRRS antigen is IngelVac.RTM. PRRS
MLV (Boehringer Ingelheim).
[0085] A further aspect of the present invention also relates to a
kit comprising i) a container containing at least one dose of an
immunogenic composition of PCV2 ORF2 as provided herewith, and ii)
a container containing an immunogenic composition comprising PRRS
antigen, preferably adjuvanted PRRS antigen. Preferably, the PRRS
antigen is adjuvanted with Carbopol. Preferably the PRRS antigen is
IngelVac.RTM. PRRS MLV (Boehringer Ingelheim). More preferably, the
kit further comprises an instruction manual, including the
information to administer both pharmaceutical compositions.
Preferably, it contains the information that the PCV2 ORF2
containing composition is administered temporally prior to the PRRS
containing composition.
[0086] A further aspect, relates to the use of any of the
compositions provided herewith as a medicament, preferably as a
veterinary medicament, even more preferred as a vaccine. Moreover,
the present invention also relates to the use of any of the
compositions described herein, for the preparation of a medicament
for lessening the severity of clinical symptoms associated with
PCV2 infection. Preferably, the medicament is for the prevention of
a PCV2 infection, even more preferably in piglets.
[0087] A further aspect relates to a method for (i) the prevention
of an infection, or re-infection with PCV2 or (ii) the reduction or
elimination of clinical symptoms caused by PCV2 in a subject,
comprising administering any of the immunogenic compositions
provided herewith to a subject in need thereof. Preferably, the
subject is a pig. Preferably, the immunogenic composition is
administered intramuscular. Preferably, one dose or two doses of
the immunogenic composition is/are administered, wherein one dose
preferably comprises at least about 2 .mu.g PCV2 ORF2 protein, even
more preferably about 2 to about 16 .mu.g, and at least about 0.1
to about 5 mg Carbopol, preferably about 1 mg Carbopol. A further
aspect relates to the method of treatment as described above,
wherein a second application of the immunogenic composition is
administered. Preferably, the second administration is done with
the same immunogenic composition, preferably having the same amount
of PCV2 ORF2 protein. Preferably the second administration is also
given intramuscular. Preferably, the second administration is done
at least 14 days beyond the initial administration, even more
preferably at least 4 weeks beyond the initial administration.
[0088] According to a further aspect, the method of treatment also
comprises the administration of an immune stimulant. Preferably,
said immune stimulant is administered at least twice. Preferably,
at least 3, more preferably at least 5 days, even more preferably
at least 7 days are between the first and the second administration
of the immune stimulant. Preferably, the immune stimulant is
administered at least 10 days, preferably 15, even more preferably
20, even more preferably at least 22 days beyond the initial
administration of the PCV2 ORF2 immunogenic composition. A
preferred immune stimulant is for example is keyhole limpet
hemocyanin (KLH), still preferably emulsified with incomplete
Freund's adjuvant (KLH/ICFA). However, it is herewith understood,
that any other immune stimulant known to a person skilled in the
art can also be used. It is within the general knowledge of a
person skilled in the art to administer the immune stimulant in a
suitable dose.
[0089] According to a further aspect, the method of treatments
described above also comprises the administration of PRRS antigen.
Preferably, the PRRS antigen is adjuvanted with Carbopol.
Preferably the PRRS antigen is IngelVac.RTM. PRRS MLV (Boehringer
Ingelheim). Preferably, said PRRS antigen is administered
temporally beyond the administration of the immunogenic composition
of PCV2 ORF2 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0090] FIG. 1 is a schematic flow diagram of a preferred
construction of PCV2 ORF2 recombinant baculovirus; and
[0091] FIGS. 2a and 2b are a schematic flow diagram of how to
produce a composition in accordance with the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0092] The following examples set forth preferred materials and
procedures in accordance with the present invention. It is to be
understood, however, that these examples are provided by way of
illustration only, and nothing therein should be deemed a
limitation upon the overall scope of the invention.
Example 1
[0093] This example compares the relative yields of ORF2 using
methods of the present invention with methods that are known in the
prior art. Four 1000 mL spinner flasks were each seeded with
approximately 1.0.times.10.sup.6 Sf+ cells/ml in 300 mL of insect
serum free media, Excell 420 (JRH Biosciences, Inc., Lenexa,
Kans.). The master cell culture is identified as SF+(Spodoptera
frugiperda) Master Cell Stock, passage 19, Lot#N112-095W. The cells
used to generate the SF+ Master Cell Stock were obtained from
Protein Sciences Corporation, Inc., Meriden, Conn. The SF+ cell
line for this example was confined between passages 19 and 59.
Other passages will work for purposes of the present invention, but
in order to scale the process up for large scale production, at
least 19 passages will probably be necessary and passages beyond 59
may have an effect on expression, although this was not
investigated. In more detail, the initial SF+ cell cultures from
liquid nitrogen storage were grown in Excell 420 media in
suspension in sterile spinner flasks with constant agitation. The
cultures were grown in 100 mL to 250 mL spinner flasks with 25 to
150 mL of Excell 420 serum-free media. When the cells had
multiplied to a cell density of 1.0-8.0.times.10.sup.6 cells/mL,
they were split to new vessels with a planting density of
0.5-1.5.times.10.sup.6 cells/mL. Subsequent expansion cultures were
grown in spinner flasks up to 36 liters in size or in stainless
steel bioreactors of up to 300 liters for a period of 2-7 days at
25-29.degree. C.
[0094] After seeding, the flasks were incubated at 27.degree. C.
for four hours. Subsequently, each flask was seeded with a
recombinant baculovirus containing the PCV2 ORF2 gene (SEQ ID NO:
4). The recombinant baculovirus containing the PCV2 ORF2 gene was
generated as follows: the PCV2 ORF2 gene from a North American
strain of PCV2 was PCR amplified to contain a 5' Kozak's sequence
(SEQ ID NO: 1) and a 3' EcoR1 site (SEQ ID NO: 2), cloned into the
pGEM-T-Easy vector (Promega, Madison, Wis.). Then, it was
subsequently excised and subcloned into the transfer vector pVL1392
(BD Biosciences Pharmingen, San Diego, Calif.). The subcloned
portion is represented herein as SEQ ID NO: 7. The pVL1392 plasmid
containing the PCV2 ORF2 gene was designated N47-064Y and then
co-transfected with BaculoGold.RTM. (BD Biosciences Pharmingen)
baculovirus DNA into Sf+ insect cells (Protein Sciences, Meriden,
Conn.) to generate the recombinant baculovirus containing the PCV2
ORF2 gene. The new construct is provided herein as SEQ ID NO: 8.
The recombinant baculovirus containing the PCV2 ORF2 gene was
plaque-purified and Master Seed Virus (MSV) was propagated on the
SF+ cell line, aliquotted, and stored at -70.degree. C. The MSV was
positively identified as PCV2 ORF2 baculovirus by PCR-RFLP using
baculovirus specific primers. Insect cells infected with PCV2 ORF2
baculovirus to generate MSV or Working Seed Virus express PCV2 ORF2
antigen as detected by polyclonal serum or monoclonal antibodies in
an indirect fluorescent antibody assay. Additionally, the identity
of the PCV2 ORF2 baculovirus was confirmed by N-terminal amino acid
sequencing. The PCV2 ORF2 baculovirus MSV was also tested for
purity in accordance with 9 C.F.R. 113.27 (c), 113.28, and 113.55.
Each recombinant baculovirus seeded into the spinner flasks had
varying multiplicities of infection (MOIs). Flask 1 was seeded with
7.52 mL of 0.088 MOI seed; flask 2 was seeded with 3.01 mL of 0.36
MOI seed; flask 3 was seeded with 1.5 mL of 0.18 MOI seed; and
flask 4 was seeded with 0.75 mL of 0.09 MOI seed. A schematic flow
diagram illustrating the basic steps used to construct a PCV2 ORF2
recombinant baculovirus is provided herein as FIG. 1.
[0095] After being seeded with the baculovirus, the flasks were
then incubated at 27.+-.2.degree. C. for 7 days and were also
agitated at 100 rpm during that time. The flasks used ventilated
caps to allow for air flow. Samples from each flask were taken
every 24 hours for the next 7 days. After extraction, each sample
was centrifuged, and both the pellet and the supernatant were
separated and then microfiltered through a 0.45-1.0 .mu.m pore size
membrane.
[0096] The resulting samples then had the amount of ORF2 present
within them quantified via an ELISA assay. The ELISA assay was
conducted with capture antibody Swine anti-PCV2 Pab IgG Prot. G
purified (diluted 1:250 in PBS) diluted to 1:6000 in 0.05M
Carbonate buffer (pH 9.6). 100 .mu.L of the antibody was then
placed in the wells of the mictrotiter plate, sealed, and incubated
overnight at 37.degree. C. The plate was then washed three times
with a wash solution which comprised 0.5 mL of Tween 20 (Sigma, St.
Louis, Mo.), 100 mL of 10.times. D-PBS (Gibco Invitrogen, Carlsbad,
Calif.) and 899.5 mL of distilled water. Subsequently, 250 .mu.L of
a blocking solution (5 g Carnation Non-fat dry milk (Nestle,
Glendale, Calif.) in 10 mL of D-PBS QS to 100 mL with distilled
water) was added to each of the wells. The next step was to wash
the test plate and then add pre-diluted antigen. The pre-diluted
antigen was produced by adding 200 .mu.L of diluent solution (0.5
mL Tween 20 in 999.5 mL D-PBS) to each of the wells on a dilution
plate. The sample was then diluted at a 1:240 ratio and a 1:480
ratio, and 100 .mu.L of each of these diluted samples was then
added to one of the top wells on the dilution plate (i.e. one top
well received 100 .mu.L of the 1:240 dilution and the other
received 100 .mu.L of the 1:480 dilution). Serial dilutions were
then done for the remainder of the plate by removing 100 .mu.L form
each successive well and transferring it to the next well on the
plate. Each well was mixed prior to doing the next transfer. The
test plate washing included washing the plate three times with the
wash buffer. The plate was then sealed and incubated for an hour at
37.degree. C. before being washed three more times with the wash
buffer. The detection antibody used was monoclonal antibody to PCV
ORF2. It was diluted to 1:300 in diluent solution, and 100 .mu.L of
the diluted detection antibody was then added to the wells. The
plate was then sealed and incubated for an hour at 37.degree. C.
before being washed three times with the wash buffer. Conjugate
diluent was then prepared by adding normal rabbit serum (Jackson
Immunoresearch, West Grove, Pa.) to the diluent solution to 1%
concentration. Conjugate antibody Goat anti-mouse (H+1)-HRP
(Jackson Immunoresearch) was diluted in the conjugate diluent to
1:10,000. 100 .mu.L of the diluted conjugate antibody was then
added to each of the wells. The plate was then sealed and incubated
for 45 minutes at 37.degree. C. before being washed three times
with the wash buffer. 100 .mu.L of substrate (TMB Peroxidase
Substrate, Kirkgaard and Perry Laboratories (KPL), Gaithersburg,
Md.), mixed with an equal volume of Peroxidase Substrate B (KPL)
was added to each of the wells. The plate was incubated at room
temperature for 15 minutes. 100 .mu.L of 1N HCL solution was then
added to all of the wells to stop the reaction. The plate was then
run through an ELISA reader. The results of this assay are provided
in Table 1 below:
TABLE-US-00001 TABLE 1 ORF2 in pellet ORF2 in supernatant Day Flask
(.mu.g) (.mu.g) 3 1 47.53 12 3 2 57.46 22 3 3 53.44 14 3 4 58.64 12
4 1 43.01 44 4 2 65.61 62 4 3 70.56 32 4 4 64.97 24 5 1 31.74 100 5
2 34.93 142 5 3 47.84 90 5 4 55.14 86 6 1 14.7 158 6 2 18.13 182 6
3 34.78 140 6 4 36.88 146 7 1 6.54 176 7 2 12.09 190 7 3 15.84 158
7 4 15.19 152
[0097] These results indicate that when the incubation time is
extended, expression of ORF2 into the supernatant of the
centrifuged cells and media is greater than expression in the
pellet of the centrifuged cells and media. Accordingly, allowing
the ORF2 expression to proceed for at least 5 days and recovering
it in the supernate rather than allowing expression to proceed for
less than 5 days and recovering ORF2 from the cells, provides a
great increase in ORF2 yields, and a significant improvement over
prior methods.
Example 2
[0098] This example provides data as to the efficacy of the
invention claimed herein. A 1000 mL spinner flask was seeded with
approximately 1.0.times.10.sup.6 Sf+ cells/ml in 300 mL of Excell
420 media. The flask was then incubated at 27.degree. C. and
agitated at 100 rpm. Subsequently, the flask was seeded with 10 mL
of PCV2 ORF2/Bac p+6 (the recombinant baculovirus containing the
PCV2 ORF2 gene passaged 6 additional times in the Sf9 insect cells)
virus seed with a 0.1 MOI after 24 hours of incubation.
[0099] The flask was then incubated at 27.degree. C. for a total of
6 days. After incubation, the flask was then centrifuged and three
samples of the resulting supernatant were harvested and
inactivated. The supernatant was inactivated by bringing its
temperature to 37.+-.2.degree. C. To the first sample, a 0.4M
solution of 2-bromoethyleneamine hydrobromide which had been
cyclized to 0.2M binary ethlylenimine (BEI) in 0.3N NaOH is added
to the supernatant to give a final concentration of BEI of 5 mM. To
the second sample, 10 mM BEI was added to the supernatant. To the
third sample, no BEI was added to the supernatant. The samples were
then stirred continuously for 48 hrs. A 1.0 M sodium thiosulfate
solution to give a final minimum concentration of 5 mM was added to
neutralize any residual BEI. The quantity of ORF2 in each sample
was then quantified using the same ELISA assay procedure as
described in Example 1. The results of this may be seen in Table 2
below:
TABLE-US-00002 TABLE 2 Sample ORF2 in supernatant (.mu.g) 1 78.71 2
68.75 3 83.33
[0100] This example demonstrates that neutralization with BEI does
not remove or degrade significant amounts of the recombinant PCV2
ORF2 protein product. This is evidenced by the fact that there is
no large loss of ORF2 in the supernatant from the BEI or elevated
temperatures. Those of skill in the art will recognize that the
recovered ORF2 is a stable protein product.
Example 3
[0101] This example demonstrates that the present invention is
scalable from small scale production of recombinant PCV2 ORF2 to
large scale production of recombinant PCV2 ORF2. 5.0.times.10.sup.5
cells/ml of SF+ cells/ml in 7000 mL of ExCell 420 media was planted
in a 20000 mL Applikon Bioreactor. The media and cells were then
incubated at 27.degree. C. and agitated at 100 RPM for the next 68
hours. At the 68.sup.th hour, 41.3 mL of PCV2 ORF2 Baculovirus
MSV+3 was added to 700 mL of ExCell 420 medium. The resultant
mixture was then added to the bioreactor. For the next seven days,
the mixture was incubated at 27.degree. C. and agitated at 100 RPM.
Samples from the bioreactor were extracted every 24 hours beginning
at day 4, post-infection, and each sample was centrifuged. The
supernatant of the samples were preserved and the amount of ORF2
was then quantified using SDS-PAGE densitometry. The results of
this can be seen in Table 3 below:
TABLE-US-00003 TABLE 3 Day after infection: ORF2 in supernatant
(.mu.g/mL) 4 29.33 5 41.33 6 31.33 7 60.67
Example 4
[0102] This example tests the efficacy of seven PCV2 candidate
vaccines and further defines efficacy parameters following exposure
to a virulent strain of PCV2. One hundred and eight (108) cesarean
derived colostrum deprived (CDCD) piglets, 9-14 days of age, were
randomly divided into 9 groups of equal size. Table 4 sets forth
the General Study Design for this Example.
TABLE-US-00004 TABLE 4 General Study Design Challenged KLH/ICFA
with on Day Virulent No. Of Day of 21 and PCV2 on Necropsy Group
Pigs Treatment Treatment Day 27 Day 24 on Day 49 1 12 PCV2 Vaccine
No. 1 - 0 + + + (vORF2 16 .mu.g) 2 12 PCV2 Vaccine No. 2 - 0 + + +
(vORF2 8 .mu.g) 3 12 PCV2 Vaccine No. 3 - 0 + + + (vORF2 4 .mu.g) 4
12 PCV2 Vaccine No. 4 - 0 + + + (rORF2 16 .mu.g) 5 12 PCV2 Vaccine
No. 5 - 0 + + + (rORF2 8 .mu.g) 6 12 PCV2 Vaccine No. 6 - 0 + + +
(rORF2 4 .mu.g) 7 12 PCV2 Vaccine No. 7 - 0 + + + (Killed whole
cell virus) 8 12 None - Challenge N/A + + + Controls 9 12 None -
Strict N/A + - + Negative Control Group vORF2 = isolated viral
ORF2; rORF2 = recombinant baculovirus expressed ORF2; killed whole
cell virus = PCV2 virus grown in suitable cell culture
[0103] Seven of the groups (Groups 1-7) received doses of PCV2 ORF2
polypeptide, one of the groups acted as a challenge control and
received no PCV2 ORF2, and another group acted as the strict
negative control group and also received no PCV2 ORF2. On Day 0,
Groups 1 through 7 were treated with assigned vaccines. Piglets in
Group 7 were given a booster treatment on Day 14. Piglets were
observed for adverse events and injection site reactions following
vaccination and on Day 19, piglets were moved to the second study
site. At the second study site, Groups 1-8 were group housed in one
building while Group 9 was housed in a separate building. All pigs
received keyhole limpet hemocyanin (KLH)/incomplete Freund's
adjuvant (ICFA) on Days 21 and 27 and on Day 24, Groups 1-8 were
challenged with a virulent PCV2.
[0104] Pre- and post-challenge, blood samples were collected for
PCV2 serology. Post-challenge, body weight data for determination
of average daily weight gain (ADWG), and clinical symptoms, as well
as nasal swab samples to determine nasal shedding of PCV2, were
collected. On Day 49, all surviving pigs were necropsied, lungs
were scored for lesions, and selected tissues were preserved in
formalin for Immunohistochemistry (IHC) testing at a later
date.
Materials and Methods
[0105] This was a partially blinded vaccination-challenge
feasibility study conducted in CDCD pigs, 9 to 14 days of age on
Day 0. To be included in the study, PCV2 IFA titers of sows were
.ltoreq.1:1000. Additionally, the serologic status of sows were
from a known PRRS-negative herd. Twenty-eight (28) sows were tested
for PCV2 serological status. Fourteen (14) sows had a PCV2 titer of
.ltoreq.1000 and were transferred to the first study site. One
hundred ten (110) piglets were delivered by cesarean section
surgeries and were available for this study on Day -4. On Day -3,
108 CDCD pigs at the first study site were weighed, identified with
ear tags, blocked by weight and randomly assigned to 1 of 9 groups,
as set forth above in table 4. If any test animal meeting the
inclusion criteria was enrolled in the study and was later excluded
for any reason, the Investigator and Monitor consulted in order to
determine the use of data collected from the animal in the final
analysis. The date of which enrolled piglets were excluded and the
reason for exclusion was documented. Initially, no sows were
excluded. A total of 108 of an available 110 pigs were randomly
assigned to one of 9 groups on Day -3. The two smallest pigs (No.
17 and 19) were not assigned to a group and were available as
extras, if needed. During the course of the study, several animals
were removed. Pig 82 (Group 9) on Day -1, Pig No. 56 (Group 6) on
Day 3, Pig No. 53 (Group 9) on Day 4, Pig No. 28 (Group 8) on Day
8, Pig No. 69 (Group 8) on Day 7, and Pig No. 93 (Group 4) on Day
9, were each found dead prior to challenge. These six pigs were not
included in the final study results. Pig no 17 (one of the extra
pigs) was assigned to Group 9. The remaining extra pig, No. 19, was
excluded from the study.
[0106] The formulations given to each of the groups were as
follows: Group 1 was designed to administer 1 ml of viral ORF2
(vORF2) containing 16 .mu.g ORF2/ml. This was done by mixing 10.24
ml of viral ORF2 (256 .mu.g/25 .mu.g/ml=10.24 ml vORF2) with 3.2 ml
of 0.5% Carbopol and 2.56 ml of phosphate buffered saline at a pH
of 7.4. This produced 16 ml of formulation for group 1. Group 2 was
designed to administer 1 ml of vORF2 containing 8 .mu.g vORF2/ml.
This was done by mixing 5.12 ml of vORF2 (128 .mu.g/25
.mu.g/ml=5.12 ml vORF2) with 3.2 ml of 0.5% Carbopol and 7.68 ml of
phosphate buffered saline at a pH of 7.4. This produced 16 ml of
formulation for group 2. Group 3 was designed to administer 1 ml of
vORF2 containing 4 .mu.g vORF2/ml. This was done by mixing 2.56 ml
of vORF2 (64 .mu.g/25 .mu.g/ml=2.56 ml vORF2) with 3.2 ml of 0.5%
Carbopol and 10.24 ml of phosphate buffered saline at a pH of 7.4.
This produced 16 ml of formulation for group 3. Group 4 was
designed to administer 1 ml of recombinant ORF2 (rORF2) containing
16 .mu.g rORF2/ml. This was done by mixing 2.23 ml of rORF2 (512
.mu.g/230 .mu.g/ml=2.23 ml rORF2) with 6.4 ml of 0.5% Carbopol and
23.37 ml of phosphate buffered saline at a pH of 7.4. This produced
32 ml of formulation for group 4. Group 5 was designed to
administer 1 ml of rORF2 containing 8 .mu.g rORF2/ml. This was done
by mixing 1.11 ml of rORF2 (256 .mu.g/230 .mu.g/ml=1.11 ml rORF2)
with 6.4 ml of 0.5% Carbopol and 24.49 ml of phosphate buffered
saline at a pH of 7.4. This produced 32 ml of formulation for group
5. Group 6 was designed to administer 1 ml of rORF2 containing 8
.mu.g rORF2/ml. This was done by mixing 0.56 ml of rORF2 (128
.mu.g/230 .mu.g/ml=0.56 ml rORF2) with 6.4 ml of 0.5% Carbopol and
25.04 ml of phosphate buffered saline at a pH of 7.4. This produced
32 ml of formulation for group 6. Group 7 was designed to
administer 2 ml of PCV2 whole killed cell vaccine (PCV2 KV)
containing the MAX PCV2 KV. This was done by mixing 56 ml of PCV2
KV with 14 ml of 0.5% Carbopol. This produced 70 ml of formulation
for group 7. Finally group 8 was designed to administer KLH at 0.5
.mu.g/ml or 1.0 .mu.g/ml per 2 ml dose. This was done by mixing
40.71 ml KLH (7.0 .mu.g protein/ml at 0.5 .mu.g/ml=570 ml (7.0
.mu.g/ml)(x)=(0.5)(570 ml)), 244.29 ml phosphate buffered saline at
a pH of 7.4, and 285 ml Freunds adjuvant. Table 5 describes the
time frames for the key activities of this Example.
TABLE-US-00005 TABLE 5 Study Activities Study Day Study Activity
-4, 0 to General observations for overall health and clinical
symptoms 49 -3 Weighed; Randomized to groups; Collected blood
samples from all pigs 0 Health examination; Administered IVP Nos.
1-7 to Groups 1-7, respectively 0-7 Observed pigs for injection
site reactions 14 Boostered Group 7 with PCV2 Vaccine No. 7; Blood
samples from all pigs 14-21 Observed Group 7 for injection site
reactions 16-19 Treated all pigs with antibiotics (data missing) 19
Pigs transported from the first test site to a second test site 21
Treated Groups 1-9 with KLH/ICFA 24 Collected blood and nasal swab
samples from all pigs; Weighed all pigs; Challenged Groups 1-8 with
PCV2 challenge material 25, 27, Collected nasal swab samples from
all pigs 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 27 Treated Groups
1-9 with KLH/ICFA 31 Collected blood samples from all pigs 49
Collected blood and nasal swab samples from all pigs; Weighed all
pigs; Necropsy all pigs; Gross lesions noted with emphasis placed
on icterus and gastric ulcers; Lungs evaluated for lesions; Fresh
and formalin fixed tissue samples saved; In- life phase of the
study completed
[0107] Following completion of the in-life phase of the study,
formalin fixed tissues were examined by Immunohistochemistry (IHC)
for detection of PCV2 antigen by a pathologist, blood samples were
evaluated for PCV2 serology, nasal swab samples were evaluated for
PCV2 shedding, and average daily weight gain (ADWG) was determined
from Day 24 to Day 49.
[0108] Animals were housed at the first study site in individual
cages in five rooms from birth to approximately 11 days of age
(approximately Day 0 of the study). Each room was identical in
layout and consisted of stacked individual stainless steel cages
with heated and filtered air supplied separately to each isolation
unit. Each room had separate heat and ventilation, thereby
preventing cross-contamination of air between rooms. Animals were
housed in two different buildings at the second study site. Group 9
(The Strict negative control group) was housed separately in a
converted finisher building and Groups 1-8 were housed in converted
nursery building. Each group was housed in a separate pen (11-12
pigs per pen) and each pen provided approximately 3.0 square feet
per pig. Each pen was on an elevated deck with plastic slatted
floors. A pit below the pens served as a holding tank for excrement
and waste. Each building had its own separate heating and
ventilation systems, with little likelihood of cross-contamination
of air between buildings.
[0109] At the first study site, piglets were fed a specially
formulated milk ration from birth to approximately 3 weeks of age.
All piglets were consuming solid, special mixed ration by Day 19
(approximately 41/2 weeks of age). At the second study site, all
piglets were fed a custom non-medicated commercial mix ration
appropriate for their age and weight, ad libitum. Water at both
study sites was also available ad libitum.
[0110] All test pigs were treated with Vitamin E on Day -2, with
iron injections on Day -1 and with NAXCEL.RTM. (1.0 mL, 1M, in
alternating hams) on Days 16, 17, 18 and 19. In addition, Pig No.
52 (Group 9) was treated with an iron injection on Day 3, Pig 45
(Group 6) was treated with an iron injection on Day 11, Pig No. 69
(Group 8) was treated with NAXCEL.RTM. on Day 6, Pig No. 74 (Group
3) was treated with dexamethazone and penicillin on Day 14, and Pig
No. 51 (Group 1) was treated with dexamethazone and penicillin on
Day 13 and with NAXCEL.RTM. on Day 14 for various health
reasons.
[0111] While at both study sites, pigs were under veterinary care.
Animal health examinations were conducted on Day 0 and were
recorded on the Health Examination Record Form. All animals were in
good health and nutritional status before vaccination as determined
by observation on Day 0. All test animals were observed to be in
good health and nutritional status prior to challenge. Carcasses
and tissues were disposed of by rendering. Final disposition of
study animals was records on the Animal Disposition Record.
[0112] On Day 0, pigs assigned to Groups 1-6 received 1.0 mL of
PCV2 Vaccines 1-6, respectively, IM in the left neck region using a
sterile 3.0 mL Luer-lock syringe and a sterile 20 g.times.1/2''
needle. Pigs assigned to Group 7 received 2.0 mL of PCV2 Vaccine
No. 7 IM in the left neck region using a sterile 3.0 mL Luer-lock
syringe and a sterile 20 g.times.1/2'' needle. On Day 14, pigs
assigned to Group 7 received 2.0 mL of PCV2 Vaccine No. 7 IM in the
right neck region using a sterile 3.0 mL Luer-lock syringe and a
sterile 20 g.times.1/2'' needle.
[0113] On Day 21 all test pigs received 2.0 mL of KLH/ICFA IM in
the right ham region using a sterile 3.0 mL Luer-lock syringe and a
sterile 20 g.times.1'' needle. On Day 27 all test pigs received 2.0
mL of KLH/ICFA in the left ham region using a sterile 3.0 mL
Luer-lock syringe and a sterile 20 g.times.1'' needle.
[0114] On Day 24, pigs assigned to Groups 1-8 received 1.0 mL of
PCV2 ISUVDL challenge material (5.11 log.sub.10 TCID.sub.50/mL) IM
in the left neck region using a sterile 3.0 mL Luer-lock syringe
and a sterile 20 g.times.1'' needle. An additional 1.0 mL of the
same material was administered IN to each pig (0.5 mL per nostril)
using a sterile 3.0 mL Luer-lock syringe and nasal canula.
[0115] Test pigs were observed daily for overall health and adverse
events on Day -4 and from Day 0 to Day 19. Observations were
recorded on the Clinical Observation Record. All test pigs were
observed from Day 0 to Day 7, and Group 7 was further observed from
Day 14 to 21, for injection site reactions. Average daily weight
gain was determined by weighing each pig on a calibrated scale on
Days -3, 24 and 49, or on the day that a pig was found dead after
challenge. Body weights were recorded on the Body Weight Form. Day
-3 body weights were utilized to block pigs prior to randomization.
Day 24 and Day 49 weight data was utilized to determine the average
daily weight gain (ADWG) for each pig during these time points. For
pigs that died after challenge and before Day 49, the ADWG was
adjusted to represent the ADWG from Day 24 to the day of death.
[0116] In order to determine PCV2 serology, venous whole blood was
collected from each piglet from the orbital venous sinus on Days -3
and 14. For each piglet, blood was collected from the orbital
venous sinus by inserting a sterile capillary tube into the medial
canthus of one of the eyes and draining approximately 3.0 mL of
whole blood into a 4.0 mL Serum Separator Tube (SST). On Days 24,
31, and 49, venous whole blood from each pig was collected from the
anterior vena cava using a sterile 18 g.times.11/2'' Vacutainer
needle (Becton Dickinson and Company, Franklin Lakes, N.J.), a
Vacutainer needle holder and a 13 mL SST. Blood collections at each
time point were recorded on the Sample Collection Record. Blood in
each SST was allowed to clot, each SST was then spun down and the
serum harvested. Harvested serum was transferred to a sterile snap
tube and stored at -70.+-.10.degree. C. until tested at a later
date. Serum samples were tested for the presence of PCV2 antibodies
by BIVI-R&D personnel.
[0117] Pigs were observed once daily from Day 20 to Day 49 for
clinical symptoms and clinical observations were recorded on the
Clinical Observation Record.
[0118] To test for PCV2 nasal shedding, on Days 24, 25, and then
every other odd numbered study day up to and including Day 49, a
sterile dacron swab was inserted intra nasally into either the left
or right nostril of each pig (one swab per pig) as aseptically as
possible, swished around for a few seconds and then removed. Each
swab was then placed into a single sterile snap-cap tube containing
1.0 mL of EMEM media with 2% IFBS, 500 units/mL of Penicillin, 500
.mu.g/mL of Streptomycin and 2.5 .mu.g/mL of Fungizone. The swab
was broken off in the tube, and the snap tube was sealed and
appropriately labeled with animal number, study number, date of
collection, study day and "nasal swab." Sealed snap tubes were
stored at -40.+-.10.degree. C. until transported overnight on ice
to BIVI-St. Joseph. Nasal swab collections were recorded on the
Nasal Swab Sample Collection Form. BIVI-R&D conducted
quantitative virus isolation (VI) testing for PCV2 on nasal swab
samples. The results were expressed in log.sub.10 values. A value
of 1.3 logs or less was considered negative and any value greater
than 1.3 logs was considered positive.
[0119] Pigs that died (Nos. 28, 52, 56, 69, 82, and 93) at the
first study site were necropsied to the level necessary to
determine a diagnosis. Gross lesions were recorded and no tissues
were retained from these pigs. At the second study site, pigs that
died prior to Day 49 (Nos. 45, 23, 58, 35), pigs found dead on Day
49 prior to euthanasia (Nos. 2, 43) and pigs euthanized on Day 49
were necropsied. Any gross lesions were noted and the percentages
of lung lobes with lesions were recorded on the Necropsy Report
Form.
[0120] From each of the 103 pigs necropsied at the second study
site, a tissue sample of tonsil, lung, heart, liver, mesenteric
lymph node, kidney and inguinal lymph node was placed into a single
container with buffered 10% formalin; while another tissue sample
from the same aforementioned organs was placed into a Whirl-pak
(M-Tech Diagnostics Ltd., Thelwall, UK) and each Whirl-pak was
placed on ice. Each container was properly labeled. Sample
collections were recorded on the Necropsy Report Form. Afterwards,
formalin-fixed tissue samples and a Diagnostic Request Form were
submitted for IHC testing. IHC testing was conducted in accordance
with standard ISU laboratory procedures for receiving samples,
sample and slide preparation, and staining techniques. Fresh
tissues in Whirl-paks were shipped with ice packs to the Study
Monitor for storage (-70.degree..+-.10.degree. C.) and possible
future use. Formalin-fixed tissues were examined by a pathologist
for detection of PCV2 by IHC and scored using the following scoring
system: 0=None; 1=Scant positive staining, few sites; 2=Moderate
positive staining, multiple sites; and 3=Abundant positive
staining, diffuse throughout the tissue. Due to the fact that the
pathologist could not positively differentiate inguinal LN from
mesenteric LN, results for these tissues were simply labeled as
Lymph Node and the score given the highest score for each of the
two tissues per animal.
Results
[0121] Results for this example are given below. It is noted that
one pig from Group 9 died before Day 0, and 5 more pigs died
post-vaccination (1 pig from Group 4; 1 pig from Group 6; 2 pigs
from Group 8; and 1 pig from Group 9). Post-mortem examination
indicated all six died due to underlying infections that were not
associated with vaccination or PMWS. Additionally, no adverse
events or injection site reactions were noted with any groups.
[0122] Average daily weight gain (ADWG) results are presented below
in Table 6. Group 9, the strict negative control group, had the
highest ADWG (1.06.+-.0.17 lbs/day), followed by Group 5
(0.94.+-.0.22 lbs/day), which received one dose of 8 .mu.g of
rORF2. Group 3, which received one dose of 4 .mu.g of vORF2, had
the lowest ADWG (0.49.+-.0.21 lbs/day), followed by Group 7
(0.50.+-.0.15 lbs/day), which received 2 doses of killed
vaccine.
TABLE-US-00006 TABLE 6 Summary of Group Average Daily Weight Gain
(ADWG) ADWG - lbs/day (Day 24 to Day 49) or adjusted Group
Treatment N for pigs dead before Day 29 1 vORF2 - 16 .mu.g (1 dose)
12 0.87 .+-. 0.29 lbs/day 2 vORF2 - 8 .mu.g (1 dose) 12 0.70 .+-.
0.32 lbs/day 3 vORF2 - 4 .mu.g (1 dose) 12 0.49 .+-. 0.21 lbs/day 4
rORF2 - 16 .mu.g (1 dose) 11 0.84 .+-. 0.30 lbs/day 5 rORF2 - 8
.mu.g (1 dose) 12 0.94 .+-. 0.22 lbs/day 6 rORF2 - 4 .mu.g (1 dose)
11 0.72 .+-. 0.25 lbs/day 7 KV (2 doses) 12 0.50 .+-. 0.15 lbs/day
8 Challenge Controls 10 0.76 .+-. 0.19 lbs/day 9 Strict Negative
Controls 11 1.06 .+-. 0.17 lbs/day vORF2 = isolated viral ORF2;
rORF2 = recombinant baculovirus expressed ORF2; killed whole cell
virus = PCV2 virus grown in suitable cell culture
[0123] PCV2 serology results are presented below in Table 7. All
nine groups were seronegative for PCV2 on Day -3. On Day 14, Groups
receiving vORF2 vaccines had the highest titers, which ranged from
187.5 to 529.2. Pigs receiving killed viral vaccine had the next
highest titers, followed by the groups receiving rORF2 vaccines.
Groups 8 and 9 remained seronegative at this time. On Day 24 and
Day 31, pigs receiving vORF2 vaccines continued to demonstrate a
strong serological response, followed closely by the group that
received two doses of a killed viral vaccine. Pigs receiving rORF2
vaccines were slower to respond serologically and Groups 8 and 9
continued to remain seronegative. On Day 49, pigs receiving vORF2
vaccine, 2 doses of the killed viral vaccine and the lowest dose of
rORF2 demonstrated the strongest serological responses. Pigs
receiving 16 .mu.g and 8 .mu.g of rORF2 vaccines had slightly
higher IFA titers than challenge controls. Group 9 on Day 49
demonstrated a strong serological response.
TABLE-US-00007 TABLE 7 Summary of Group PCV2 IFA Titers AVERAGE IFA
TITER Group Treatment Day -3 Day 14 Day 24 Day 31** Day 49*** 1
vORF2 - 16 .mu.g (1 dose) 50.0 529.2 4400.0 7866.7 11054.5 2 vORF2
- 8 .mu.g (1 dose) 50.0 500.0 3466.7 6800.0 10181.8 3 vORF2 - 4
.mu.g (1 dose) 50.0 187.5 1133.3 5733.3 9333.3 4 rORF2 - 16 .mu.g
(1 dose) 50.0 95.5 1550.0 3090.9 8000.0 5 rORF2 - 8 .mu.g (1 dose)
50.0 75.0 887.5 2266.7 7416.7 6 rORF2 - 4 .mu.g (1 dose) 50.0 50.0
550.0 3118.2 10570.0 7 KV (2 doses) 50.0 204.2 3087.5 4620.8 8680.0
8 Challenge Controls 50.0 55.0 50.0 50.0 5433.3 9 Strict Negative
Controls 50.0 59.1 59.1 54.5 6136.4 vORF2 = isolated viral ORF2;
rORF2 = recombinant baculovirus expressed ORF2; killed whole cell
virus = PCV2 virus grown in suitable cell culture *For calculation
purposes, a .ltoreq. 100 IFA titer was designated as a titer of
"50"; a .gtoreq. 6400 IFA titer was designated as a titer of
"12,800". **Day of Challenge ***Day of Necropsy
[0124] The results from the post-challenge clinical observations
are presented below in Table 8. This summary of results includes
observations for Abnormal Behavior, Abnormal Respiration, Cough and
Diarrhea. Table 9 includes the results from the Summary of Group
Overall Incidence of Clinical Symptoms and Table 10 includes
results from the Summary of Group Mortality Rates Post-challenge.
The most common clinical symptom noted in this study was abnormal
behavior, which was scored as mild to severe lethargy. Pigs
receiving the 2 lower doses of vORF2, pigs receiving 16 .mu.g of
rORF2 and pigs receiving 2 doses of KV vaccine had incidence rates
of .gtoreq.27.3%. Pigs receiving 8 .mu.g of rORF2 and the strict
negative control group had no abnormal behavior. None of the pigs
in this study demonstrated any abnormal respiration. Coughing was
noted frequently in all groups (0 to 25%), as was diarrhea (0-20%).
None of the clinical symptoms noted were pathognomic for PMWS.
[0125] The overall incidence of clinical symptoms varied between
groups. Groups receiving any of the vORF2 vaccines, the group
receiving 16 .mu.g of rORF2, the group receiving 2 doses of KV
vaccine and the challenge control group had the highest incidence
of overall clinical symptoms (.gtoreq.36.4%). The strict negative
control group, the group receiving 8 .mu.g of rORF2 and the group
receiving 4 .mu.g of rORF2 had overall incidence rates of clinical
symptoms of 0%, 8.3% and 9.1%, respectively.
[0126] Overall mortality rates between groups varied as well. The
group receiving 2 doses of KV vaccine had the highest mortality
rate (16.7%); while groups that received 4 .mu.g of vORF2, 16 .mu.g
of rORF2, or 8 .mu.g of rORF2 and the strict negative control group
all had 0% mortality rates.
TABLE-US-00008 TABLE 8 Summary of Group Observations for Abnormal
Behavior, Abnormal Respiration, Cough, and Diarrhea Abnormal
Abnormal Group Treatment N Behavior.sup.1 Behavior.sup.2
Cough.sup.3 Diarrhea.sup.4 1 vORF2 - 16 .mu.g (1 dose) 12 2/12 0/12
3/12 2/12 (16.7% (0%) (25%) (16/7%) 2 vORF2 - 8 .mu.g (1 dose) 12
4/12 0/12 1/12 1/12 (33.3%) (0%) (8.3% (8.3%) 3 vORF2 - 4 .mu.g (1
dose) 12 8/12 0/12 2/12 1/12 (66.7%) (0%) (16.7%) (8.3%) 4 rORF2 -
16 .mu.g (1 dose) 11 3/11 0/11 0/11 2/11 (27.3%) (0%) (0%) (18.2%)
5 rORF2 - 8 .mu.g (1 dose) 12 0/12 0/12 1/12 0/12 (0%) (0%) (8.3%)
(0%) 6 rORF2 - 4 .mu.g (1 dose) 11 1/11 0/11 0/11 0/12 (9.1%) (0%)
(0%) (0%) 7 KV (2 doses) 12 7/12 0/12 0/12 1/12 (58.3) (0%) (0%)
(8.3%) 8 Challenge Controls 10 1/10 0/10 2/10 2/10 (10%) (0%) (20%)
(20%) 9 Strict Negative Controls 11 0/11 0/11 0/11 0/11 (0%) (0%)
(0%) (0%) vORF2 = isolated viral ORF2; rORF2 = recombinant
baculovirus expressed ORF2; killed whole cell virus = PCV2 virus
grown in suitable cell culture .sup.1Total number of pigs in each
group that demonstrated any abnormal behavior for at least one day
.sup.2Total number of pigs in each group that demonstrated any
abnormal respiration for at least one day .sup.3Total number of
pigs in each group that demonstrated a cough for at least one day
.sup.4Total number of pigs in each group that demonstrated diarrhea
for at least one day
TABLE-US-00009 TABLE 9 Summary of Group Overall Incidence of
Clinical Symptoms Incidence of pigs with Incidence Group Treatment
N Clinical Symptoms.sup.1 Rate 1 vORF2 - 16 .mu.g (1 dose) 12 5
41.7% 2 vORF2 - 8 .mu.g (1 dose) 12 5 41.7% 3 vORF2 - 4 .mu.g (1
dose) 12 8 66.7% 4 rORF2 - 16 .mu.g (1 dose) 11 4 36.4% 5 rORF2 - 8
.mu.g (1 dose) 12 1 8.3% 6 rORF2 - 4 .mu.g (1 dose) 11 1 9.1% 7 KV
(2 doses) 12 7 58.3% 8 Challenge Controls 10 4 40% 9 Strict
Negative Controls 11 0 0% vORF2 = isolated viral ORF2; rORF2 =
recombinant baculovirus expressed ORF2; killed whole cell virus =
PCV2 virus grown in suitable cell culture .sup.1Total number of
pigs in each group that demonstrated any clinical symptom for at
least one day
TABLE-US-00010 TABLE 10 Summary of Group Mortality Rates
Post-challenge Dead Post- Group Treatment N challenge Mortality
Rate 1 vORF2 - 16 .mu.g (1 dose) 12 1 8.3% 2 vORF2 - 8 .mu.g (1
dose) 12 1 8.3% 3 vORF2 - 4 .mu.g (1 dose) 12 0 0% 4 rORF2 - 16
.mu.g (1 dose) 11 0 0% 5 rORF2 - 8 .mu.g (1 dose) 12 0 0% 6 rORF2 -
4 .mu.g (1 dose) 11 1 9.1% 7 KV (2 doses) 12 2 16.7% 8 Challenge
Controls 10 1 10% 9 Strict Negative Controls 11 0 0% vORF2 =
isolated viral ORF2; rORF2 = recombinant baculovirus expressed
ORF2; killed whole cell virus = PCV2 virus grown in suitable cell
culture
[0127] PCV2 nasal shedding results are presented below in Table 11.
Following challenge on Day 24, 1 pig in Group 7 began shedding PCV2
on Day 27. None of the other groups experienced shedding until Day
33. The bulk of nasal shedding was noted from Day 35 to Day 45.
Groups receiving any of the three vORF2 vaccines and groups
receiving either 4 or 8 .mu.g of rORF2 had the lowest incidence of
nasal shedding of PCV2 (.ltoreq.9.1%). The challenge control group
(Group 8) had the highest shedding rate (80%), followed by the
strict negative control group (Group 9), which had an incidence
rate of 63.6%.
TABLE-US-00011 TABLE 11 Summary of Group Incidence of Nasal
Shedding of PCV2 No. Of pigs that shed for Group Treatment N at
least one day Incidence Rate 1 vORF2 - 16 .mu.g (1 dose) 12 1 8.3%
2 vORF2 - 8 .mu.g (1 dose) 12 1 8.3% 3 vORF2 - 4 .mu.g (1 dose) 12
1 8.3% 4 rORF2 - 16 .mu.g (1 dose) 11 2 18.2% 5 rORF2 - 8 .mu.g (1
dose) 12 1 8.3% 6 rORF2 - 4 .mu.g (1 dose) 11 1 9.1% 7 KV (2 doses)
12 5 41.7% 8 Challenge Controls 10 8 80% 9 Strict Negative Controls
11 7 63.6% vORF2 = isolated viral ORF2; rORF2 = recombinant
baculovirus expressed ORF2; killed whole cell virus = PCV2 virus
grown in suitable cell culture
[0128] The Summary of Group Incidence of Icterus, Group Incidence
of Gastric Ulcers, Group Mean Lung Lesion Scores, and Group
Incidence of Lung Lesions are shown below in Table 12. Six pigs
died at the first test site during the post-vaccination phase of
the study (Group 4, N=1; Group 6, N=1; Group 8, N=2; Group 9, N=2).
Four out of six pigs had fibrinous lesions in one or more body
cavities, one pig (Group 6) had lesions consistent with clostridial
disease, and one pig (Group 9) had no gross lesions. None of the
pigs that died during the post-vaccination phased of the study had
lesions consistent with PMWS.
[0129] Pigs that died post-challenge and pigs euthanized on Day 49
were necropsied. At necropsy, icterus and gastric ulcers were not
present in any group. With regard to mean % lung lesions, Group 9
had lowest mean % lung lesions (0%), followed by Group 1 with
0.40.+-.0.50% and Group 5 with 0.68.+-.1.15%. Groups 2, 3, 7 and 8
had the highest mean % lung lesions (.gtoreq.7.27%). Each of these
four groups contained one pig with % lung lesions .gtoreq.71.5%,
which skewed the results higher for these four groups. With the
exception of Group 9 with 0% lung lesions noted, the remaining 8
groups had .ltoreq.36% lung lesions. Almost all lung lesions noted
were described as red/purple and consolidated.
TABLE-US-00012 TABLE 12 Summary of Group Incidence of Icterus,
Group Incidence of Gastric Ulcers, Group Mean % Lung Lesion Scores,
and Group Incidence of Lung Lesions Noted Incidence of Gastric Mean
% Lung Lung Lesions Group Treatment Icterus Ulcers Lesions Noted 1
vORF2 - 16 .mu.g (1 0/12 (0%) 0/12 0.40 .+-. 0.50% 10/12 dose) (0%)
(83%) 2 vORF2 - 8 .mu.g (1 dose) 0/12 (0%) 0/12 7.41 .+-. 20.2%
10/12 (0%) (83%) 3 vORF2 - 4 .mu.g (1 dose) 0/12 (0%) 0/12 9.20
.+-. 20.9% 10/12 (0%) (83%) 4 rORF2 - 16 .mu.g (1 0/11 (0%) 0/11
1.5 .+-. 4.74% 4/11 dose) (0%) (36%) 5 rORF2 - 8 .mu.g (1 dose)
0/12 (0%) 0/12 0.68 .+-. 1.15% 9/12 (0%) (75%) 6 rORF2 - 4 .mu.g (1
dose) 0/11 (0%) 0/11 2.95 .+-. 5.12% 7/11 (0%) (64%) 7 KV (2 doses)
0/12 (0%) 0/12 7.27 .+-. 22.9% 9/12 (0%) (75%) 8 Challenge Controls
0/10 (0%) 0/10 9.88 .+-. 29.2% 8/10 (0%) (80%) 9 Strict Negative
0/11 (0%) 0/11 0/11 0/11 Controls (0%) (0%) (0%) vORF2 = isolated
viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or
killed whole cell virus = PCV2 virus grown in suitable cell
culture
[0130] The Summary of Group IHC Positive Incidence Results are
shown in Table 13. Group 1 (vORF2-16 .mu.g) and Group 5 (rORF2-8
.mu.g) had the lowest rate of IHC positive results (16.7%). Group 8
(Challenge Controls) and Group 9 (Strict Negative Controls) had the
highest rate of IHC positive results, 90% and 90.9%,
respectively.
TABLE-US-00013 TABLE 13 Summary of Group IHC Positive Incidence
Rate No. Of pigs that had at least one tissue Incidence Group
Treatment N positive for PCV2 Rate 1 vORF2 - 16 .mu.g (1 dose) 12 2
16.7% 2 vORF2 - 8 .mu.g (1 dose) 12 3 25.0% 3 vORF2 - 4 .mu.g (1
dose) 12 8 66.7% 4 rORF2 - 16 .mu.g (1 dose) 11 4 36.3% 5 rORF2 - 8
.mu.g (1 dose) 12 2 16.7% 6 rORF2 - 4 .mu.g (1 dose) 11 4 36.4% 7
KV (2 doses) 12 5 41.7% 8 Challenge Controls 10 9 90.0% 9 Strict
Negative Controls 11 10 90.9% vORF2 = isolated viral ORF2; rORF2 =
recombinant baculovirus expressed ORF2; KV or killed whole cell
virus = PCV2 virus grown in suitable cell culture
[0131] Post-challenge, Group 5, which received one dose of 8 .mu.g
of rORF2 antigen, outperformed the other 6 vaccine groups. Group 5
had the highest ADWG (0.94.+-.0.22 lbs/day), the lowest incidence
of abnormal behavior (0%), the second lowest incidence of cough
(8.3%), the lowest incidence of overall clinical symptoms (8.3%),
the lowest mortality rate (0%), the lowest rate of nasal shedding
of PCV2 (8.3%), the second lowest rate for mean % lung lesions
(0.68.+-.1.15%) and the lowest incidence rate for positive tissues
(16.7%). Groups receiving various levels of rORF2 antigen overall
outperformed groups receiving various levels of vORF2 and the group
receiving 2 doses of killed whole cell PCV2 vaccine performed the
worst. Tables 14 and 15 contain summaries of group post-challenge
data.
TABLE-US-00014 TABLE 14 Summary of Group Post-Challenge Data - Part
1 Overall Incidence of ADWG Abnormal Clinical Group N Treatment
(lbs/day) Behavior Cough Symptoms 1 12 vORF2 - 16 .mu.g 0.87 .+-.
0.29 2/12 (16.7%) 3/12 (25%) 41.7% (1 dose) 2 12 vORF2 - 8 .mu.g
0.70 .+-. 0.32 4/12 (33.3% 1/12 (8.3% 41.7% (1 dose) 3 12 vORF2 - 4
.mu.g 0.49 .+-. 0.21 8/12 (66.7%) 2/12 (16.7% 66.7% (1 dose) 4 11
rORF2 - 16 .mu.g 0.84 .+-. 0.30 3/11 (27.3%) 0/11 (0%) 36.4% (1
dose) 5 12 rORF2 - 8 .mu.g 0.94 .+-. 0.22 0/12 (0%) 1/12 (8.3% 8.3%
(1 dose) 6 11 rORF2 - 4 .mu.g 0.72 .+-. 0.25 1/11 (9.1% 0/11 (0%)
9.1% (1 dose) 7 12 KV 0.50 .+-. 0.15 7/12 (58.3) 0/12 (0%) 58.3% (2
doses) 8 10 Challenge 0.76 .+-. 0.19 1/10 (10%) 2/10 (20% 40%
Controls 9 11 Strict Negative 1.06 .+-. 0.17 0/11 (0%) 0/11 (0%) 0%
Controls vORF2 = isolated viral ORF2; rORF2 = recombinant
baculovirus expressed ORF2; KV or killed whole cell virus = PCV2
virus grown in suitable cell culture
TABLE-US-00015 TABLE 15 Summary of Group Post-Challenge Data - Part
2 Mean % Incidence Rate of at Mortality Nasal Lung least one tissue
IHC Group N Treatment Rate Shedding Lesions positive for PCV2 1 12
vORF2 - 16 .mu.g 8.3% 8.3% 0.40 .+-. 0.50% 16.7% (1 dose) 2 12
vORF2 - 8 .mu.g 8.3% 8.3% 7.41 .+-. 20.2% 25.0% (1 dose) 3 12 vORF2
- 4 .mu.g 0% 8.3% 9.20 .+-. 20.9% 66.7% (1 dose) 4 11 rORF2 - 16
.mu.g 0% 18.2% 1.50 .+-. 4.74% 36.3% (1 dose) 5 12 rORF2 - 8 .mu.g
0% 8.3% 0.68 .+-. 1.15% 16.7% (1 dose) 6 11 rORF2 - 4 .mu.g 9.1%
9.1% 2.95 .+-. 5.12% 36.4% (1 dose) 7 12 KV 16.7% 41.7% 7.27 .+-.
22.9% 41.7% (2 doses) 8 10 Challenge 10% 80% 9.88 .+-. 29.2% 90.0%
Controls 9 11 Strict Negative 0% 63.6% 0/11 90.9% Controls (0%)
vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus
expressed ORF2; KV or killed whole cell virus = PCV2 virus grown in
suitable cell culture
[0132] Results of this study indicate that all further vaccine
efforts should focus on a rORF2 vaccine. Overall, nasal shedding of
PCV2 was detected post-challenge and vaccination with a PCV2
vaccine resulted in a reduction of shedding. Immunohistochemistry
of selected lymphoid tissues also served as a good parameter for
vaccine efficacy, whereas large differences in ADWG, clinical
symptoms, and gross lesions were not detected between groups. This
study was complicated by the fact that extraneous PCV2 was
introduced at some point during the study, as evidenced by nasal
shedding of PCV2, PCV2 seroconversion and positive IHC tissues in
Group 9, the strict negative control group.
Discussion
[0133] Seven PCV2 vaccines were evaluated in this study, which
included three different dose levels of vORF2 antigen administered
once on Day 0, three different dose levels of rORF2 antigen
administered once on Day 0 and one dose level of killed whole cell
PCV2 vaccine administered on Day 0 and Day 14. Overall, Group 5,
which received 1 dose of vaccine containing 8 .mu.g of rORF2
antigen, had the best results. Group 5 had the highest ADWG, the
lowest incidence of abnormal behavior, the lowest incidence of
abnormal respiration, the second lowest incidence of cough, the
lowest incidence of overall clinical symptoms, the lowest mortality
rate, the lowest rate of nasal shedding of PCV2, the second lowest
rate for mean % lung lesions and the lowest incidence rate for
positive IHC tissues.
[0134] Interestingly, Group 4, which received a higher dose of
rORF2 antigen than Group 5, did not perform as well or better than
Group 5. Group 4 had a slightly lower ADWG, a higher incidence of
abnormal behavior, a higher incidence of overall clinical symptoms,
a higher rate of nasal shedding of PCV2, a higher mean % lung
lesions, and a higher rate for positive IHC tissues than Group 5.
Statistical analysis, which may have indicated that the differences
between these two groups were not statistically significant, was
not conducted on these data, but there was an observed trend that
Group 4 did not perform as well as Group 5.
[0135] Post-vaccination, 6 pigs died at the first study site. Four
of the six pigs were from Group 8 or Group 9, which received no
vaccine. None of the six pigs demonstrated lesions consistent with
PMWS, no adverse events were reported and overall, all seven
vaccines appeared to be safe when administered to pigs
approximately 11 days of age. During the post-vaccination phase of
the study, pigs receiving either of three dose levels of vORF2
vaccine or killed whole cell vaccine had the highest IFAT levels,
while Group 5 had the lowest IFAT levels just prior to challenge,
of the vaccine groups.
[0136] Although not formally proven, the predominant route of
transmission of PCV2 to young swine shortly after weaning is
believed to be by oronasal direct contact and an efficacious
vaccine that reduces nasal shedding of PCV2 in a production setting
would help control the spread of infection. Groups receiving one of
three vORF2 antigen levels and the group receiving 8 .mu.g of rORF2
had the lowest incidence rate of nasal shedding of PCV2 (8.3%).
Expectedly, the challenge control group had the highest incidence
rate of nasal shedding (80%).
[0137] Gross lesions in pigs with PMWS secondary to PCV2 infection
typically consist of generalized lymphadenopathy in combination
with one or a multiple of the following: (1) interstitial pneumonia
with interlobular edema, (2) cutaneous pallor or icterus, (3)
mottled atrophic livers, (4) gastric ulcers and (5) nephritis. At
necropsy, icterus, hepatitis, nephritis, and gastric ulcers were
not noted in any groups and lymphadenopathy was not specifically
examined for. The mean % lung lesion scores varied between groups.
The group receiving 16 .mu.g of vORF2 antigen had the lowest mean %
lung lesion score (0.40.+-.0.50%), followed by the group that
received 8 .mu.g of rORF2 (0.68.+-.1.15%). As expected, the
challenge control group had the highest mean % lung lesion score
(9.88.+-.29.2%). In all four groups, the mean % lung lesion scores
were elevated due to one pig in each of these groups that had very
high lung lesion scores. Most of the lung lesions were described as
red/purple and consolidated. Typically, lung lesions associated
with PMWS are described as tan and non-collapsible with
interlobular edema. The lung lesions noted in this study were
either not associated with PCV2 infection or a second pulmonary
infectious agent may have been present. Within the context of this
study, the % lung lesion scores probably do not reflect a true
measure of the amount of lung infection due to PCV2.
[0138] Other researchers have demonstrated a direct correlation
between the presence of PCV2 antigen by IHC and histopathology.
Histopathology on select tissues was not conducted with this study.
Group 1 (16 .mu.g of vORF2) and Group 5 (8 .mu.g of rORF2) had the
lowest incidence rate of pigs positive for PCV2 antigen (8.3%),
while Group 9 (the strict negative control group -90.9%) and Group
8 (the challenge control group -90.0%) had the highest incidence
rates for pigs positive for PCV2 antigen. Due to the non-subjective
nature of this test, IHC results are probably one of the best
parameters to judge vaccine efficacy on.
[0139] Thus, in one aspect of the present invention, the Minimum
Portective Dosage (MPD) of a 1 ml/1 dose recombinant product with
extracted PCV2 ORF2 (rORF2) antigen in the CDCD pig model in the
face of a PCV2 challenge was determined. Of the three groups that
received varying levels of rORF2 antigen, Group 5 (8 .mu.g of rORF2
antigen) clearly had the highest level of protection. Group 5
either had the best results or was tied for the most favorable
results with regard to all of the parameters examined. When Group 5
was compared with the other six vaccine groups post-challenge,
Group 5 had the highest ADWG (0.94.+-.0.22 lbs/day), the lowest
incidence of abnormal behavior (0%), the second lowest incidence of
cough (8.3%), the lowest incidence of overall clinical symptoms
(8.3%), the lowest mortality rate (0%), the lowest rate of nasal
shedding of PCV2 (8.3%), the second lowest rate for mean % lung
lesions (0.68.+-.1.15%) and the lowest incidence rate for positive
IHC tissues (16.7%).
[0140] In another aspect of the present invention, the MPD of a 1
ml/1 dose conventional product that is partially purified PCV2 ORF2
(vORF2) antigen in the CDCD pig model in the face of a PCV2
challenge was determined. Of the three groups that received varying
levels of vORF2 antigen, Group 1 (16 .mu.g of vORF2) had the
highest level of protection. Group 1 outperformed Groups 2 and 3
with respect to ADWG, mean % lung lesions, and IHC. Groups 1 and 2
(8 .mu.g of vORF2 antigen) performed equally with respect to
overall incidence of clinical symptoms, Group 3 (4 .mu.g of vORF2
antigen) had the lowest mortality rate, and all three groups
performed equally with respect to nasal shedding. Overall, vORF
vaccines did not perform as well as rORF vaccines.
[0141] In yet another aspect of the present invention, the efficacy
of a maximum dose of a 2 ml/2 dose Conventional Killed PCV2 vaccine
in the CDCD pig model in the face of a PCV2 challenge was
determined. Of the seven vaccines evaluated in this study, the
killed whole cell PCV2 vaccine performed the worst. Piglets
receiving two doses of killed whole cell PCV2 vaccine had the
lowest ADWG, the second highest rate of abnormal behavior (58.3%),
the second highest overall incidence of clinical symptoms (58.3%),
the highest mortality rate (16.7%), the second highest incidence of
nasal shedding (41.7%), highest mean % lung lesions
(9.88.+-.29.2%), a high incidence of lung lesions noted (75%) and a
moderate IHC incidence rate in tissues (41.7%). However, it was
still effective at invoking an immune response.
[0142] In still another aspect of the present invention, nasal
shedding of PCV2 was assessed as an efficacy parameter and the
previous PCV2 efficacy parameters from previous studies were
reconfirmed. Results from this study indicate that nasal shedding
of PCV2 occurs following intra nasal challenge and that PCV2
vaccines reduce nasal shedding of PCV2 post-challenge. Furthermore,
results from this study and reports in the literature indicate that
IHC should continue to be evaluated in future PCV2 vaccine trials
as well.
[0143] Some additional conclusions arising from this study are that
lymphadenopathy is one of the hallmarks of PMWS. Another one of the
hallmarks of PMWS is lymphoid depletion and multinucleated/giant
histiocytes. Additionally, no adverse events or injection site
reactions were noted for any of the 7 PCV2 vaccines and all 7 PCV2
vaccines appeared to be safe when administered to young pigs.
Example 5
[0144] This example tests the efficacy of eight PCV2 candidate
vaccines and reconfirms PCV2 challenge parameters from earlier
challenge studies following exposure to a virulent strain of PCV2.
One hundred and fifty (150) cesarean derived colostrum deprived
(CDCD) piglets, 6-16 days of age, were blocked by weight and
randomly divided into 10 groups of equal size. Table 16 sets forth
the General Study Design for this Example.
TABLE-US-00016 TABLE 16 General Study Design Challenge with
KLH/ICFA Virulent PRRSV Necropsy No. Of Day of on Day 22 PCV2 on
MLV on on Day Group Pigs Treatment Treatment and Day 28 Day 25 Day
46 50 1 15 PVC2 Vaccine 1 0 & 14 + + + + 16 .mu.g rORF2 - IMS
1314 2 15 PVC2 Vaccine 2 0 & 14 + + + + 16 .mu.g vORF2 -
Carbopol 3 15 PCV2 Vaccine 3 0 & 14 + + + + 16 .mu.g rORF2 -
Carbopol 4 15 PCV2 Vaccine 2 0 + + + + 16 .mu.g vORF2 - Carbopol 5
15 PVC2 Vaccine 3 0 & 14 + + + + 4 .mu.g rORF2 - Carbopol 6 15
PVC2 Vaccine 3 0 & 14 + + + + 1 .mu.g rORF2 - Carbopol 7 15
PVC2 Vaccine 3 0 & 14 + + + + 0.25 .mu.g rORF2 - Carbopol 8 15
PVC2 Vaccine 4 0 & 14 + + + + >8.0 log KV - Carbopol 9 15
Challenge N/A + + + + Controls 10 15 None - Strict N/A + - + +
Negative Control Group vORF2 = isolated viral ORF2; rORF2 =
recombinant baculovirus expressed ORF2; KV or killed whole cell
virus = PCV2 virus grown in suitable cell culture
[0145] The vaccine formulation given to each group were as follows.
PCV2 Vaccine No. 1, administered at 1.times.2 ml dose to Group 1,
was a high dose (16 ug/2 ml dose) of inactivated recombinant ORF2
antigen adjuvanted with IMS 1314 (16 ug rORF2-IMS 1314). PCV2
Vaccine No. 2, administered at 1.times.2 ml dose to Group 2, was a
high dose (16 ug/2 ml dose) of a partially purified VIDO R-1
generated PCV2 ORF2 antigen adjuvanted with Carbopol (16 ug
vORF2-Carbopol). PCV2 Vaccine No. 3, administered at 1.times.2 ml
dose to Group 3, was a high dose (16 ug/2 ml dose) of inactivated
recombinant ORF2 antigen adjuvanted with Carbopol (16 ug
rORF2-Carbopol). PCV2 Vaccine No. 4, administered at 1.times.1 ml
dose to Group 4, was a high dose (16 ug/1 ml dose) of a partially
purified VIDO R-1 generated PCV2 ORF2 antigen adjuvanted with
Carbopol (16 ug vORF2-Carbopol). Vaccine No. 5, administered at
1.times.2 ml dose to Group 5, was a 4 ug/2 ml dose of an
inactivated recombinant ORF2 antigen adjuvanted with Carbopol (4 ug
rORF2-Carbopol). PCV2 Vaccine No. 6, administered at 1.times.2 ml
dose to Group 6, was a 1 ug/2 ml dose of an inactivated recombinant
ORF2 antigen adjuvanted with Carbopol (1 ug rORF2-Carbopol). PCV2
Vaccine No. 7, administered at 1.times.2 ml dose to Group 7, was a
low dose (0.25 ug/2 ml dose) of inactivated recombinant ORF2
antigen adjuvanted with Carbopol (0.25 ug rORF2-Carbopol). PCV2
Vaccine No. 8, administered at 1.times.2 ml dose to Group 8, was a
high dose (pre-inactivation titer>8.0 log/2 ml dose) Inactivated
Conventional Killed VIDO R-1 generated PCV2 Struve antigen
adjuvanted with Carbopol (>8.0 log KV-Carbopol). On Day 0,
Groups 1-8 were treated with their assigned vaccines. Groups 1-3
and 5-8 received boosters of their respective vaccines again on Day
14. The effectiveness of a single dose of 16 .mu.g of
vORF2-Carbopol was tested on Group 4 which did not receive a
booster on Day 14. Piglets were observed for adverse events and
injection site reactions following both vaccinations. On Day 21 the
piglets were moved to a second study site where Groups 1-9 were
group housed in one building and Group 10 was housed in a separate
building. All pigs received keyhole limpet hemocyanin emulsified
with incomplete Freund's adjuvant (KLH/ICFA) on Days 22 and 28. On
Day 25, Groups 1-9 were challenged with approximately 4 logs of
virulent PCV2 virus. By Day 46, very few deaths had occurred in the
challenge control group. In an attempt to immunostimulate the pigs
and increase the virulence of the PCV2 challenge material, all
Groups were treated with INGELVAC.RTM. PRRSV MLV (Porcine
Reproductive and Respiratory Vaccine, Modified Live Virus) on Day
46.
[0146] Pre- and post-challenge blood samples were collected for
PCV2 serology. Post-challenge, body weight data for determination
of average daily weight gain (ADWG) and observations of clinical
signs were collected. On Day 50, all surviving pigs were
necropsied, gross lesions were recorded, lungs were scored for
pathology, and selected tissues were preserved in formalin for
examination by Immunohistochemistry (IHC) for detection of PCV2
antigen at a later date.
Materials and Methods
[0147] This was a partially-blind vaccination-challenge feasibility
study conducted in CDCD pigs, 6 to 16 days of age on Day 0. To be
included in the study, PCV2 IFA titers of sows were .ltoreq.1:1000.
Additionally, the serologic status of sows were from a known
PRRS-negative herd. Sixteen (16) sows were tested for PCV2
serological status and all sixteen (16) had a PCV2 titer of
.ltoreq.1000 and were transferred to the first study site. One
hundred fifty (150) piglets were delivered by cesarean section
surgeries and were available for this study on Day -3. On Day -3,
150 CDCD pigs at the first study site were weighed, identified with
ear tags, blocked by weight and randomly assigned to 1 of 10
groups, as set forth above in table 16. Blood samples were
collected from all pigs. If any test animal meeting the inclusion
criteria was enrolled in the study and was later excluded for any
reason, the Investigator and Monitor consulted in order to
determine the use of data collected from the animal in the final
analysis. The date of which enrolled piglets were excluded and the
reason for exclusion was documented. No sows meeting the inclusion
criteria, selected for the study and transported to the first study
site were excluded. No piglets were excluded from the study, and no
test animals were removed from the study prior to termination.
Table 17 describes the time frames for the key activities of this
Example.
TABLE-US-00017 TABLE 17 Study Activities Study Day Actual Dates
Study Activity -3 Apr. 04, 2003 Weighed pigs; health exam;
randomized to groups; collected blood samples -3, Apr. 04, 2003
Observed for overall health and for adverse events post- 0-21 Apr.
07, 2003 to vaccination May 27, 2003 0 Apr. 07, 2003 Administered
respective IVPs to Groups 1-8 0-7 Apr. 07, 2003 to Observed pigs
for injection site reactions Apr. 14, 2003 14 Apr. 21, 2003
Boostered Groups 1-3, 5-8 with respective IVPs; blood sampled all
pigs 14-21 Apr. 21, 2003 to Observed pigs for injection reactions
Apr. 28, 2003 19-21 Apr. 26, 2003 to Treated all pigs with
antibiotics Apr. 28, 2003 21 Apr. 28, 2003 Pigs transported from
Struve Labs, Inc. to Veterinary Resources, Inc. (VRI) 22-50 Apr.
28, 2003 to Observed pigs for clinical signs post-challenge May 27,
2003 22 Apr. 29, 2003 Treated Groups 1-10 with KLH/ICFA 25 May 02,
2003 Collected blood samples from all pigs; weighed all pigs;
challenged Groups 1-9 with PCV2 challenge material 28 May 05, 2003
Treated Groups 1-10 with KLH/ICFA 32 May 09, 2003 Collected blood
samples from all pigs 46 May 23, 2003 Administered INGELVAC .RTM.
PRRS MLV to all groups 50 May 27, 2003 Collected blood samples,
weighed and necropsied all pigs; gross lesions were recorded; lungs
were evaluated for lesions; fresh and formalin fixed tissue samples
were saved; In-life phase of the study was completed
[0148] Following completion of the in-life phase of the study,
formalin fixed tissues were examined by Immunohistochemistry (IHC)
for detection of PCV2 antigen by a pathologist, blood samples were
evaluated for PCV2 serology, and average daily weight gain (ADWG)
was determined from Day 25 to Day 50.
[0149] Animals were housed at the first study site in individual
cages in seven rooms from birth to approximately 11 days of age
(approximately Day 0 of the study). Each room was identical in
layout and consisted of stacked individual stainless steel cages
with heated and filtered air supplied separately to each isolation
unit. Each room had separate heat and ventilation, thereby
preventing cross-contamination of air between rooms. Animals were
housed in two different buildings at the second study site. Group
10 (The Strict negative control group) was housed separately in a
converted nursery building and Groups 1-9 were housed in a
converted farrowing building. Each group was housed in a separate
pen (14-15 pigs per pen) and each pen provided approximately 2.3
square feet per pig. Groups 2, 4 and 8 were penned in three
adjacent pens on one side of the alleyway and Groups 1, 3, 5, 6, 7,
and 9 were penned in six adjacent pens on the other side of the
alleyway. The Group separation was due to concern by the Study
Monitor that vaccines administered to Groups 2, 4, and 8 had not
been fully inactivated. Each pen was on an elevated deck with
plastic slatted floors. A pit below the pens served as a holding
tank for excrement and waste. Each building had its own separate
heating and ventilation systems, with little likelihood of
cross-contamination of air between buildings.
[0150] At the first study site, piglets were fed a specially
formulated milk ration from birth to approximately 3 weeks of age.
All piglets were consuming solid, special mixed ration by Day 21
(approximately 41/2 weeks of age). At the second study site, all
piglets were fed a custom non-medicated commercial mix ration
appropriate for their age and weight, ad libitum. Water at both
study sites was also available ad libitum.
[0151] All test pigs were treated with 1.0 mL of NAXCEL.RTM., IM,
in alternating hams on Days 19, 20, and 21. In addition, Pig No. 11
(Group 1) was treated with 0.5 mL of NAXCEL.RTM. IM on Day 10, Pig
No. 13 (Group 10) was treated with 1 mL of Penicillin and 1 mL of
PREDEF.RTM. 2.times. on Day 10, Pig No. 4 (Group 9) was treated
with 1.0 mL of NAXCEL.RTM. IM on Day 11, and Pigs 1 (Group 1), 4
and 11 were each treated with 1.0 mL of NAXCEL.RTM. on Day 14 for
various health reasons.
[0152] While at both study sites, pigs were under veterinary care.
Animal health examinations were conducted on Day -3 and were
recorded on the Health Examination Record Form. All animals were in
good health and nutritional status before vaccination as determined
by observation on Day 0. All test animals were observed to be in
good health and nutritional status prior to challenge. Carcasses
and tissues were disposed of by rendering. Final disposition of
study animals was recorded on the Animal Disposition Record.
[0153] On Days 0 and 14, pigs assigned to Groups 1-3 and 5-8
received 2.0 mL of assigned PCV2 Vaccines 1-4, respectively, IM in
the right and left neck region, respectively, using a sterile 3.0
mL Luer-lock syringe and a sterile 20 g.times.1/2'' needle. Pigs
assigned to Group 4 received 1.0 mL of PCV2 Vaccine No. 2, IM in
the right neck region using a sterile 3.0 mL Luer-lock syringe and
a sterile 20 g.times.1/2'' needle on Day 0 only.
[0154] On Day 22 all test pigs received 2.0 mL of KLH/ICFA IM in
the left neck region using a sterile 3.0 mL Luer-lock syringe and a
sterile 20 g.times.1'' needle. On Day 28 all test pigs received 2.0
mL of KLH/ICFA in the right ham region using a sterile 3.0 mL
Luer-lock syringe and a sterile 20 g.times.1'' needle.
[0155] On Day 25, pigs assigned to Groups 1-9 received 1.0 mL of
PCV2 ISUVDL challenge material (3.98 log.sub.10 TCID.sub.50/mL) IM
in the right neck region using a sterile 3.0 mL Luer-lock syringe
and a sterile 20 g.times.1'' needle. An additional 1.0 mL of the
same material was administered IN to each pig (0.5 mL per nostril)
using a sterile 3.0 mL Luer-lock syringe and nasal canula.
[0156] On Day 46, all test pigs received 2.0 mL INGELVAC.RTM. PRRS
MLV, IM, in the right neck region using a sterile 3.0 mL Luer0lock
syringe and a sterile 20 g.times.1'' needle. The PRRSV MLV was
administered in an attempt to increase virulence of the PCV2
challenge material.
[0157] Test pigs were observed daily for overall health and adverse
events on Day -3 and from Day 0 to Day 21. Each of the pigs were
scored for normal or abnormal behavior, respiration or cough.
Observations were recorded on the Clinical Observation Record. All
test pigs were observed from Day 0 to Day 7, and Group 7 was
further observed from Day 14 to 21, for injection site reactions.
Average daily weight gain was determined by weighing each pig on a
calibrated scale on Days -3, 25 and 50, or on the day that a pig
was found dead after challenge. Body weights were recorded on the
Body Weight Form. Day -3 body weights were utilized to block pigs
prior to randomization. Day 25 and Day 50 weight data was utilized
to determine the average daily weight gain (ADWG) for each pig
during these time points. For pigs that died after challenge and
before Day 50, the ADWG was adjusted to represent the ADWG from Day
25 to the day of death.
[0158] In order to determine PCV2 serology, venous whole blood was
collected from each piglet from the orbital venous sinus on Days -3
and 14. For each piglet, blood was collected from the orbital
venous sinus by inserting a sterile capillary tube into the medial
canthus of one of the eyes and draining approximately 3.0 mL of
whole blood into a 4.0 mL Serum Separator Tube (SST). On Days 25,
32, and 50, venous whole blood from each pig was collected from the
anterior vena cava using a sterile 20 g.times.11/2''
Vacutainer.RTM. needle (Becton Dickinson and Company, Franklin
Lakes, N.J.), a Vaccutainer.RTM. needle holder and a 13 mL SST.
Blood collections at each time point were recorded on the Sample
Collection Record. Blood in each SST was allowed to clot, each SST
was then spun down and the serum harvested. Harvested serum was
transferred to a sterile snap tube and stored at -70.+-.10.degree.
C. until tested at a later date. Serum samples were tested for the
presence of PCV2 antibodies by BIVI-R&D personnel.
[0159] Pigs were observed once daily from Day 22 to Day 50 for
clinical symptoms and scored for normal or abnormal behavior,
respiration or cough. Clinical observations were recorded on the
Clinical Observation Record.
[0160] Pigs Nos. 46 (Group 1) and 98 (Groups 9) died at the first
study site. Both of these deaths were categorized as bleeding
deaths and necropsies were not conducted on these two pigs. At the
second study site, pigs that died after challenge and prior to Day
50, and pigs euthanized on Day 50, were necropsied. Any gross
lesions were noted and the percentages of lung lobes with lesions
were recorded on the Necropsy Report Form.
[0161] From each of the pigs necropsied at the second study site, a
tissue sample of tonsil, lung, heart, and mesenteric lymph node was
placed into a single container with buffered 10% formalin; while
another tissue sample from the same aforementioned organs was
placed into a Whirl-pak.RTM. (M-Tech Diagnostics Ltd., Thelwall,
UK) and each Whirl-pak.RTM. was placed on ice. Each container was
properly labeled. Sample collections were recorded on the Necropsy
Report Form. Afterwards, formalin-fixed tissue samples and a
Diagnostic Request Form were submitted for IHC testing. IHC testing
was conducted in accordance with standard laboratory procedures for
receiving samples, sample and slide preparation, and staining
techniques. Fresh tissues in Whirl-paks.RTM. were shipped with ice
packs to the Study Monitor for storage (-70.degree..+-.10.degree.
C.) and possible future use.
[0162] Formalin-fixed tissues were examined by a pathologist for
detection of PCV2 by IHC and scored using the following scoring
system: 0=None; 1=Scant positive staining, few sites; 2=Moderate
positive staining, multiple sites; and 3=Abundant positive
staining, diffuse throughout the tissue. For analytical purposes, a
score of 0 was considered "negative," and a score of greater than 0
was considered "positive."
Results
[0163] Results for this example are given below. It is noted that
Pigs No. 46 and 98 died on days 14 and 25 respectively. These
deaths were categorized as bleeding deaths. Pig No. 11 (Group 1)
was panting with rapid respiration on Day 15. Otherwise, all pigs
were normal for behavior, respiration and cough during this
observation period and no systemic adverse events were noted with
any groups. No injection site reactions were noted following
vaccination on Day 0. Following vaccination on Day 14, seven (7)
out of fourteen (14) Group 1 pigs (50.0%) had swelling with a score
of "2" on Day 15. Four (4) out of fourteen (14) Group 1 (28.6%)
still had a swelling of "2" on Day 16. None of the other groups
experienced injection site reactions following either
vaccination.
[0164] Average daily weight gain (ADWG) results are presented below
in Table 18. Pigs No. 46 and 98 that died from bleeding were
excluded from group results. Group 4, which received one dose of 16
ug vORF2-Carbopol, had the highest ADWG (1.16.+-.0.26 lbs/day),
followed by Groups 1, 2, 3, 5, 6, and 10 which had ADWGs that
ranged from 1.07.+-.0.23 lbs/day to 1.11.+-.0.26 lbs/day. Group 9
had the lowest ADWG (0.88.+-.0.29 lbs/day), followed by Groups 8
and 7, which had ADWGs of 0.93.+-.0.33 lbs/day and 0.99.+-.0.44
lbs/day, respectively.
TABLE-US-00018 TABLE 18 Summary of Group Average Daily Weight Gains
(ADWG) ADWG - lbs/day (Day 25 to Day 50) or adjusted for pigs Group
Treatment N dead before Day 50 1 rORF2 - 16 .mu.g - IMS 1314 2
doses 14 1.08 .+-. 0.30 lbs/day 2 vORF2 - 16 .mu.g - Carbopol 2
doses 15 1.11 .+-. 0.16 lbs/day 3 rORF2 - 16 .mu.g - Carbopol 2
doses 15 1.07 .+-. 0.21 lbs/day 4 vORF2 - 16 .mu.g - Carbopol 1
dose 15 1.16 .+-. 0.26 lbs/day 5 rORF2 - 4 .mu.g - Carbopol 1 dose
15 1.07 .+-. 0.26 lbs/day 6 rORF2 - 1 .mu.g - Carbopol 2 doses 15
1.11 .+-. 0.26 lbs/day 7 rORF2 - 0.25 .mu.g - Carbopol 2 doses 15
0.99 .+-. 0.44 lbs/day 8 KV > 8.0 log - Carbopol 2 doses 15 0.93
.+-. 0.33 lbs/day 9 Challenge Controls 14 0.88 .+-. 0.29 lbs/day 10
Strict Negative Controls 15 1.07 .+-. 0.23 lbs/day vORF2 = isolated
viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or
killed whole cell virus = PCV2 virus grown in suitable cell
culture
[0165] PVC2 serology results are presented below in Table 19. All
ten (10) groups were seronegative for PCV2 on Day -3. On Day 14,
PCV2 titers remained low for all ten (10) groups (range of 50-113).
On Day 25, Group 8, which received the whole cell killed virus
vaccine, had the highest PCV2 titer (4617), followed by Group 2,
which received 16 ug vORF2-Carbopol, Group 4, which received as
single dose of 16 ug vORF2-Carbopol, and Group 3, which received 16
ug rORF2-Carbopol, which had titers of 2507, 1920 and 1503
respectively. On Day 32 (one week post challenge), titers for
Groups 1-6 and Group 8 ranged from 2360 to 7619; while Groups 7
(0.25 ug rORF2-Carbopol), 9 (Challenge Control), and 10 (Strict
negative control) had titers of 382, 129 and 78 respectively. On
Day 50 (day of necropsy), all ten (10) groups demonstrated high
PCV2 titers (.gtoreq.1257).
[0166] On Days 25, 32, and 50, Group 3, which received two doses of
16 ug rORF2-Carbopol had higher antibody titers than Group 1, which
received two doses of 16 ug rORF2-IMS 1314. On Days 25, 32 and 50,
Group 2, which received two doses of 16 ug vORF2 had higher titers
than Group 4, which received only one does of the same vaccine.
Groups 3, 5, 6, 7, which received decreasing levels of
rORF2-Carbopol, of 16, 4, 1, and 0.25 ug respectively, demonstrated
correspondingly decreasing antibody titers on Days 25 and 32.
TABLE-US-00019 TABLE 19 Summary of Group PCV2 IFA Titers Day Group
Treatment Day -3 Day 14** Day 25*** Day 32 50**** 1 rORF2 - 16
.mu.g - 50 64 646 3326 4314 IMS 1314 2 doses 2 vORF2 - 16 .mu.g -
50 110 2507 5627 4005 Carbopol 2 doses 3 rORF2 - 16 .mu.g - 50 80
1503 5120 6720 Carbopol 2 doses 4 vORF2 - 16 .mu.g - 50 113 1920
3720 1257 Carbopol 1 dose 5 rORF2 - 4 .mu.g - 50 61 1867 3933 4533
Carbopol 1 dose 6 rORF2 - 1 .mu.g - 50 70 490 2360 5740 Carbopol 2
doses 7 rORF2 - 0.25 .mu.g - 50 73 63 382 5819 Carbopol 2 doses 8
KV > 8.0 log - Carbopol 50 97 4617 7619 10817 2 doses 9
Challenge Controls 50 53 50 129 4288 10 Strict Negative Controls 50
50 50 78 11205 vORF2 = isolated viral ORF2; rORF2 = recombinant
baculovirus expressed ORF2; KV or killed whole cell virus = PCV2
virus grown in suitable cell culture *For calculation purposes, a
.ltoreq. 100 IFA titer was designated as a titer of "50"; a
.gtoreq. 6400 IFA titer was designated as a titer of "12,800".
**Day of Challenge ***Day of Necropsy
[0167] The results from the post-challenge clinical observations
are presented below. Table 20 includes observations for Abnormal
Behavior, Abnormal Respiration, Cough and Diarrhea. Table 21
includes the results from the Summary of Group Overall Incidence of
Clinical Symptoms and Table 22 includes results from the Summary of
Group Mortality Rates Post-challenge. The incidence of abnormal
behavior, respiration and cough post-challenge were low in pigs
receiving 16 ug rORF2-IMS 1314 (Group 1), 16 ug rORF2-Carbopol
(Group 3), 1 ug rORF2-Carbopol (Group 6), 0.25 ug rORF2-Carbopol
(Group 7), and in pigs in the Challenge Control Group (Group 9).
The incidence of abnormal behavior respiration and cough
post-challenge was zero in pigs receiving 16 ug vORF2-Carbopol
(Group 2), a single dose of 16 ug vORF2-Carbopol (Group 4), 4 ug
rORF2-Carbopol (Group 5), >8 log KV-Carbopol (Group 8), and in
pigs in the strict negative control group (Group 10).
[0168] The overall incidence of clinical symptoms varied between
groups. Pigs receiving 16 ug vORF2-Carbopol (Group 2), a single
dose of 16 ug vORF2-Carbopol (Group 4), and pigs in the Strict
negative control group (Group 10) had incidence rates of 0%; pigs
receiving 16 ug rORF2-Carbopol (Group 3), and 1 ug rORF2-Carbopol
(Group 6) had incidence rates of 6.7%; pigs receiving 16 ug
rORF2-IMS 1314 (Group 1) had an overall incidence rate of 7.1%;
pigs receiving 4 ug rORF2-Carbopol (Group 5), 0.25 ug
rORF2-Carbopol (Group 7), and >8 log KV vaccine had incidence
rates of 13.3%; and pigs in the Challenge Control Group (Group 9)
had an incidence rate of 14.3%.
[0169] Overall mortality rates between groups varied as well. Group
8, which received 2 doses of KV vaccine had the highest mortality
rate of 20.0%; followed by Group 9, the challenge control group,
and Group 7, which received 0.25 ug rORF2-Carbopol and had
mortality rates of 14.3% and 13.3% respectively. Group 4, which
received one dose of 16 ug vORF2-Carbopol had a 6.7% mortality
rate. All of the other Groups, 1, 2, 3, 5, 6, and 10 had a 0%
mortality rate.
TABLE-US-00020 TABLE 20 Summary of Group Observations for Abnormal
Behavior, Abnormal Respiration, and Cough Post-Challenge Abnormal
Abnormal Group Treatment N Behavior.sup.1 Behavior.sup.2
Cough.sup.3 1 rORF2 - 16 .mu.g - 14 0/14 0/14 1/14 IMS 1314 2 doses
(0%) (0%) (7.1%) 2 vORF2 - 16 .mu.g - 15 0/15 0/15 0/15 Carbopol 2
doses (0%) (0%) (0%) 3 rORF2 - 16 .mu.g - 15 0/15 0/15 1/15
Carbopol 2 doses (0%) (0%) (6.7%) 4 vORF2 - 16 .mu.g - 15 0/15 0/15
0/15 Carbopol 1 dose (0%) (0%) (0%) 5 rORF2 - 4 .mu.g - 15 1/15
1/15 0/15 Carbopol 1 dose (6.7%) (6.7%) (0%) 6 rORF2 - 1 .mu.g - 15
0/15 0/15 1/15 Carbopol 2 doses (0%) (0%) (6.7%) 7 rORF2 - 0.25
.mu.g - 15 0/15 1/15 1/15 Carbopol 2 doses (0%) (6.7%) (06.7%) 8 KV
> 8.0 log - 15 1/15 1/15 0/15 Carbopol 2 doses (6.7%) (6.7%)
(0%) 9 Challenge Controls 14 1/14 1/14 2/14 (7.1%) (7.1%) (14/3%)
10 Strict Negative 15 0/15 0/15 0/15 Controls (0%) (0%) (0%)
.sup.1Total number of pigs in each group that demonstrated any
abnormal behavior for at least one day .sup.2Total number of pigs
in each group that demonstrated any abnormal respiration for at
least one day .sup.3Total number of pigs in each group that
demonstrated a cough for at least one day
TABLE-US-00021 TABLE 21 Summary of Group Overall Incidence of
Clinical Symptoms Post- Challenge Incidence of pigs with Clinical
Incidence Group Treatment N Symptoms.sup.1 Rate 1 rORF2 - 16 .mu.g
- 14 1 7.1% IMS 1314 2 doses 2 vORF2 - 16 .mu.g - Carbopol 2 15 0
0.0% doses 3 rORF2 - 16 .mu.g - Carbopol 2 15 1 6.7% doses 4 vORF2
- 16 .mu.g - Carbopol 1 15 0 0.0% dose 5 rORF2 - 4 .mu.g - 15 2
13.3% Carbopol 1 dose 6 rORF2 - 1 .mu.g - 15 1 6.7% Carbopol 2
doses 7 rORF2 - 0.25 .mu.g - Carbopol 15 2 13.3% 2 doses 8 KV >
8.0 log - Carbopol 2 15 2 13.3% doses 9 Challenge Controls 14 2
14.3% 10 Strict Negative Controls 15 0 0.0% vORF2 = isolated viral
ORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or killed
whole cell virus = PCV2 virus grown in suitable cell culture
.sup.1Total number of pigs in each group that demonstrated any
clinical symptom for at least one day
TABLE-US-00022 TABLE 22 Summary of Group Mortality Rates
Post-Challenge Dead Post- Mortality Group Treatment N challenge
Rate 1 rORF2 - 16 .mu.g - 14 0 0.0% IMS 1314 2 doses 2 vORF2 - 16
.mu.g - Carbopol 2 15 0 0.0% doses 3 rORF2 - 16 .mu.g - Carbopol 2
15 0 0.0% doses 4 vORF2 - 16 .mu.g - Carbopol 1 15 1 6.7% dose 5
rORF2 - 4 .mu.g - 15 0 0.0% Carbopol 1 dose 6 rORF2 - 1 .mu.g - 15
0 0.0% Carbopol 2 doses 7 rORF2 - 0.25 .mu.g - Carbopol 2 15 2
13.3% doses 8 KV > 8.0 log - Carbopol 2 15 3 20.0% doses 9
Challenge Controls 14 2 14.3% 10 Strict Negative Controls 15 0 0.0%
vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus
expressed ORF2; KV or killed whole cell virus = PCV2 virus grown in
suitable cell culture
[0170] The Summary of Group Mean Percentage Lung Lesions and
Tentative Diagnosis is given below in Table 23. Group 9, the
challenge control group, had the highest percentage lung lesions
with a mean of 10.81.+-.23.27%, followed by Group 7, which received
0.25 ug rORF2-Carbopol and had a mean of 6.57.+-.24.74%, Group 5,
which received 4 ug rORF2-Carbopol and had a mean of 2.88.+-.8.88%,
and Group 8, which received the KV vaccine and had a mean of
2.01.+-.4.98%. The remaining six (6) groups had lower mean
percentage lung lesions that ranged from 0.11.+-.0.38% to
0.90.+-.0.15%.
[0171] Tentative diagnosis of pneumonia varied among the groups.
Group 3, which received two doses of 16 ug rORF2-Carbopol, had the
lowest tentative diagnosis of pneumonia, with 13.3%. Group 9, the
challenge control group, had 50% of the group tentatively diagnosed
with pneumonia, followed by Group 10, the strict negative control
group and Group 2, which received two doses of 16 ug
vORF2-Carbopol, with 46.7% of 40% respectively, tentatively
diagnosed with pneumonia.
[0172] Groups 1, 2, 3, 5, 9, and 10 had 0% of the group tentatively
diagnosed as PCV2 infected; while Group 8, which received two doses
if KV vaccine, had the highest group rate of tentative diagnosis of
PCV2 infection, which 20%. Group 7, which received two doses of
0.25 ug rORF2-Carbopol, and Group 4, which received one dose of 16
ug vORF2-Carbopol had tentative group diagnoses of PCV2 infection
in 13.3% and 6.7% of each group, respectively.
[0173] Gastric ulcers were only diagnosed in one pig in Group 7
(6.7%); while the other 9 groups remained free of gastric
ulcers.
TABLE-US-00023 TABLE 23 Summary of Group Mean % Lung Lesion and
Tentative Diagnosis No. Of pigs that shed for Group Treatment N at
least one day Incidence Rate 1 rORF2 - 16 .mu.g - 15 0 0% IMS 1314
2 doses 2 vORF2 - 16 .mu.g - 15 1 6.7% Carbopol 2 doses 3 rORF2 -
16 .mu.g - 15 3 20.0% Carbopol 2 doses 4 vORF2 - 16 .mu.g - 15 2
13.3% Carbopol 1 dose 5 rORF2 - 4 .mu.g - 15 3 20.0% Carbopol 1
dose 6 rORF2 - 1 .mu.g - 15 6 40.0% Carbopol 2 doses 7 rORF2 - 0.25
.mu.g - 15 7 46.7% Carbopol 2 doses 8 KV > 8.0 log - Carbopol 15
12 80% 2 doses 9 Challenge Controls 14 14 100.0% 10 Strict Negative
Controls 15 14 93.3% vORF2 = isolated viral ORF2; rORF2 =
recombinant baculovirus expressed ORF2; KV or killed whole cell
virus = PCV2 virus grown in suitable cell culture
[0174] The Summary of Group IHC Positive Incidence Results are
shown below in Table 24. Group 1 (16 ug rORF2-IMS 1314) had the
lowest group rate of IHC positive results with 0% of the pigs
positive for PCV2, followed by Group 2 (16 ug vORF2-Carbopol) and
Group 4 (single dose 16 ug vORF2-Carbopol), which had group IHC
rates of 6.7% and 13.3% respectively. Group 9, the challenge
control group, had the highest IHC positive incidence rate with
100% of the pigs positive for PCV2, followed by Group 10, the
strict negative control group, and Group 8 (KV vaccine), with 93.3%
and 80% of the pigs positive for PCV2, respectively.
TABLE-US-00024 TABLE 24 Summary of Group IHC Positive Incidence
Rate No. Of pigs that shed for Group Treatment N at least one day
Incidence Rate 1 rORF2 - 16 .mu.g - 15 0 0% IMS 1314 2 doses 2
vORF2 - 16 .mu.g - 15 1 6.7% Carbopol 2 doses 3 rORF2 - 16 .mu.g -
15 3 20.0% Carbopol 2 doses 4 vORF2 - 16 .mu.g - 15 2 13.3%
Carbopol 1 dose 5 rORF2 - 4 .mu.g - 15 3 20.0% Carbopol 1 dose 6
rORF2 - 1 .mu.g - 15 6 40.0% Carbopol 2 doses 7 rORF2 - 0.25 .mu.g
- 15 7 46.7% Carbopol 2 doses 8 KV > 8.0 log - Carbopol 15 12
80% 2 doses 9 Challenge Controls 14 14 100.0% 10 Strict Negative
Controls 15 14 93.3% vORF2 = isolated viral ORF2; rORF2 =
recombinant baculovirus expressed ORF2; KV or killed whole cell
virus = PCV2 virus grown in suitable cell culture
Discussion
[0175] Seven PCV2 vaccines were evaluated in this example, which
included a high dose (16 .mu.g) of rORF2 antigen adjuvanted with
IMS 1314 administered twice, a high dose (16 .mu.g) of vORF2
antigen adjuvanted with Carbopol administered once to one group of
pigs and twice to a second group of pigs, a high dose (16 .mu.g) of
rORF2 antigen adjuvanted with Carbopol administered twice, a 4
.mu.g dose of rORF2 antigen adjuvanted with Carbopol administered
twice, a 1 .mu.g dose of rORF2 antigen adjuvanted with Carbopol
administered twice, a low dose (0.25 .mu.g) of rORF2 antigen
adjuvanted with Carbopol administered twice, and a high dose (>8
log) of killed whole cell PCV2 vaccine adjuvanted with Carbopol.
Overall, Group 1, which received two doses of 16 .mu.g rORF2-IMS
1314, performed slightly better than Groups 2 through 7, which
received vaccines containing various levels of either vORF2 or
rORF2 antigen adjuvanted with Carbopol and much better than Group
8, which received two doses of killed whole cell PCV2 vaccine.
Group 1 had the third highest ADWG (1.80.+-.0.30 lbs/day), the
lowest incidence of abnormal behavior (0%), the lowest incidence of
abnormal respiration (0%), a low incidence of cough (7.1%), a low
incidence of overall clinical symptoms (7.1%), was tied with three
other groups for the lowest mortality rate (0%), the second lowest
rate for mean % lung lesions (0.15.+-.0.34%), the second lowest
rate for pneumonia (21.4%) and the lowest incidence rate for
positive IHC tissues (0%). Group 1 was, however, the only group in
which injection site reactions were noted, which included 50% of
the vaccinates 1 day after the second vaccination. The other
vaccines administered to Groups 2 through 7 performed better than
the killed vaccine and nearly as well as the vaccine administered
to Group 1.
[0176] Group 8, which received two doses of killed PCV2 vaccine
adjuvanted with Carbopol, had the worst set of results for any
vaccine group. Group 8 had the lowest ADWG (0.93.+-.0.33 lbs/day),
the second highest rate of abnormal behavior (6.7%), the highest
rate of abnormal respiration (6.7%), was tied with three other
groups for the highest overall incidence rate of clinical symptoms
(13.3%), had the highest mortality rate of all groups (20%), and
had the highest positive IHC rate (80%) of any vaccine group. There
was concern that the killed whole cell PCV2 vaccine may not have
been fully inactivated prior to administration to Group 8, which
may explain this group's poor results. Unfortunately, definitive
data was not available to confirm this concern. Overall, in the
context of this example, a Conventional Killed PCV2 vaccine did not
aid in the reduction of PCV2 associated disease.
[0177] As previously mentioned, no adverse events were associated
with the test vaccines with exception of the vaccine adjuvanted
with IMS 1314. Injection site reactions were noted in 50.0% of the
pigs 1 day after the second vaccination with the vaccine formulated
with IMS 1314 and in 28.6% of the pigs 2 days after the second
vaccination. No reactions were noted in any pigs receiving Carbopol
adjuvanted vaccines. Any further studies that include pigs
vaccinated with IMS 1314 adjuvanted vaccines should continue to
closely monitor pigs for injection site reactions.
[0178] All pigs were sero-negative for PCV2 on Day -3 and only
Group 2 had a titer above 100 on Day 14. On Day 25 (day of
challenge), Group 8 had the highest PCV2 antibody titer (4619),
followed by Group 2 (2507). With the exception of Groups 7, 9 and
10, all groups demonstrated a strong antibody response by Day 32.
By Day 50, all groups including Groups 7, 9 and 10 demonstrated a
strong antibody response.
[0179] One of the hallmarks of late stage PCV2 infection and
subsequent PMWS development is growth retardation in weaned pigs,
and in severe cases, weight loss is noted. Average daily weight
gain of groups is a quantitative method of demonstrating growth
retardation or weight loss. In this example, there was not a large
difference in ADWG between groups. Group 8 had the lowest ADWG of
0.88.+-.0.29 lbs/day, while Group 4 had the highest ADWG of
1.16.+-.0.26 lb/day. Within the context of this study there was not
a sufficient difference between groups to base future vaccine
efficacy on ADWG.
[0180] In addition to weight loss--dyspnea, lethargy, pallor of the
skin and sometimes icterus are clinical symptoms associated with
PMWS. In this example, abnormal behavior and abnormal respiration
and cough were noted infrequently for each group. As evidenced in
this study, this challenge model and challenge strain do not result
in overwhelming clinical symptoms and this is not a strong
parameter on which to base vaccine efficacy.
[0181] Overall, mortality rates were not high in this example and
the lack of a high mortality rate in the challenge control group
limits this parameter on which to base vaccine efficacy. Prior to
Day 46, Groups 4 and 7 each had one out of fifteen pigs die, Group
9 had two out of fourteen pigs die and Group 8 had three out of
fifteen pigs die. Due to the fact that Group 9, the challenge
control group was not demonstrating PCV2 clinical symptoms and only
two deaths had occurred in this group by Day 46, Porcine
Respiratory and Reproductive Syndrome Virus (PRRSV) MLV vaccine was
administered to all pigs on Day 46. Earlier studies had utilized
INGELVAC.RTM. PRRS MLV as an immunostimulant to exasperate
PCV2-associated PMWS disease and mortality rates were higher in
these earlier studies. Two deaths occurred shortly after
administering the PRRS vaccine on Day 46--Group 4 had one death on
Day 46 and Group 7 had one death on Day 47--which were probably not
associated with the administration of the PRRS vaccine. By Day 50,
Group 8, which received two doses of killed vaccine, had the
highest mortality rate (20%), followed by Group 9 (challenge
control) and Group 7 (0.25 ug rORF2-Carbopol), with mortality rates
of 14.3% and 13.3% respectively. Overall, administration of the
PRRS vaccine to the challenge model late in the post-challenge
observation phase of this example did not significantly increase
mortality rates.
[0182] Gross lesions in pigs with PMWS secondary to PCV2 infection
typically consist of generalized lymphadenopathy in combination
with one or more of the following: (1) interstitial pneumonia with
interlobular edema, (2) cutaneous pallor or icterus, (3) mottled
atrophic livers, (4) gastric ulcers and (5) nephritis. At necropsy
(Day 50), icterus, hepatitis, and nephritis were not noted in any
groups. A gastric ulcer was noted in one Group 7 pig, but
lymphadenopathy was not specifically examined for. Based on the
presence of lesions that were consistent with PCV2 infection, three
groups had at least one pig tentatively diagnosed with PCV2 (PMWS).
Group 8, which received two doses of killed vaccine, had 20%
tentatively diagnosed with PCV2, while Group 7 and Group 4 had
13.3% and 6.7%, respectively, tentatively diagnosed with PCV2. The
mean % lung lesion scores varied between groups at necropsy. Groups
1, 2, 3, 4, 6 and 10 had low % lung lesion scores that ranged from
0.11.+-.0.38% to 0.90.+-.0.15%. As expected, Group 9, the challenge
control group, had the highest mean % lung lesion score
(10.81.+-.23.27%). In four groups, the mean % lung lesion scores
were elevated due to one to three pigs in each of these groups
having very high lung lesion scores. The lung lesions were
red/purple and consolidated. Typically, lung lesions associated
with PMWS are described as tan, non-collapsible with interlobular
edema. The lung lesions noted in this study were either not
associated with PCV2 infection or a second pulmonary infectious
agent may have been present. Within the context of this study, the
% lung lesion scores probably do no reflect a true measure of the
amount of lung infection due to PCV2. Likewise, tentative diagnosis
of pneumonia may have been over-utilized as well. Any pigs with
lung lesions, some as small as 0.10% were listed with a tentative
diagnosis of pneumonia. In this example, there was no sufficient
difference between groups with respect to gross lesions and % lung
lesions on which to base vaccine efficacy.
[0183] IHC results showed the largest differences between groups.
Group 1 (16 .mu.g rORF2-IMS 1314) had the lowest positive IHC
results for PCV2 antigen (0%); while Groups 9 and 10 had the
highest positive IHC results with incidence rates of 100% and 93.3%
respectively. Groups 3, 5, 6 and 7, which received 16, 4, 1 or 0.25
.mu.g of rORF2 antigen, respectively, adjuvanted with Carbopol, had
IHC positive rates of 20%, 20%, 40% and 46.7%, respectively. Group
2, which received two doses of 16 .mu.g vORF2 adjuvanted with
Carbopol had an IHC positive rate of 6.7%, while Group 4 which
received only one dose of the same vaccine, had an IHC positive
rate of 13.3%. Due to the objective nature of this test and the
fact that IHC results correlated with expected results, IHC testing
is probably one of the best parameters on which to base vaccine
efficacy.
[0184] Thus in one aspect of the present invention, the Minimum
Protective Dosage (MPD) of PCV2 rORF2 antigen adjuvanted with
Carbopol in the CDCD pig model in the face of a PCV2 challenge is
determined. Groups 3, 5, 6 and 7 each received two doses of rORF2
antigen adjuvanted with Carbopol, but the level of rORF2 antigen
varied for each group. Groups 3, 5, 6 and 7 each received 16, 4, 1
or 0.25 .mu.g of rORF2 antigen respectively. In general, decreasing
the level of rORF2 antigen decreased PCV2 antibody titers, and
increased the mortality rate, mean % lung lesions and the incidence
of IHC positive tissues. Of the four groups receiving varying
levels of rORF2-Carbopol, Groups 3 and 5, which received two doses
of 16 or 4 .mu.g of rORF2 antigen, respectively, each had an IHC
positive rate of only 20%, and each had similar antibody titers.
Overall, based on IHC positive results, the minimum protective
dosage of rORF2 antigen administered twice is approximately 4
.mu.g.
[0185] In another aspect of the present invention, the antigenicity
of recombinant (rORF2) and VIDO R-1 (vORF2) PCV2 antigens were
assessed. Group 2 received two doses of 16 .mu.g vORF2 and Group 3
received two doses of 16 .mu.g rORF2. Both vaccines were adjuvanted
with Carbopol. Both vaccines were found to be safe and both had 0%
mortality rate. Group 2 had a PCV2 antibody titer of 2507 on Day
25, while Group 3 had a PCV2 antibody titer of 1503. Group 3 had a
lower mean % lung lesion score than Group 2 (0.11.+-.0.38% vs.
0.90.+-.0.15%), but Group 2 had a lower IHC positive incidence rate
that Group 3 (6.7% vs. 20%). Overall, both vaccines had similar
antigenicity, but vORF2 was associated with slightly better IHC
results.
[0186] In yet another aspect of the present invention, the
suitability of two different adjuvants (Carbopol and IMS 1314) was
determined. Groups 1 and 3 both received two doses of vaccine
containing 16 ug of rORF2 antigen, but Group 1 received the antigen
adjuvanted with IMS 1314 while Group 3 received the antigen
adjuvanted with Carbopol. Both groups had essentially the same
ADWG, essentially the same incidence of clinical signs
post-challenge, the same mortality rate, and essentially the same
mean % lung lesions; but Group 1 had an IHC positive rate of 0%
while Group 3 had an IHC positive rate of 20%. However, Group 3,
which received the vaccine adjuvanted with Carbopol had higher IFAT
PCV2 titers on Days 25, 32 and 50 than Group 1, which received the
vaccine adjuvanted with IMS 1314. Overall, although the PCV2
vaccine adjuvanted with IMS 1314 did provide better IHC results, it
did not provide overwhelmingly better protection from PCV2
infection and did induce injection site reaction. Whereas the PCV2
vaccine adjuvanted with Carbopol performed nearly as well as the
IMS 1314 adjuvanted vaccine, but was not associated with any
adverse events.
[0187] In still another aspect of the present invention, the
feasibility of PCV2 ORF2 as a 1 ml, 1 dose product was determined.
Groups 2 and 4 both received 16 .mu.g of vORF2 vaccine adjuvanted
with Carbopol on Day 0, but Group 2 received a second dose on Day
14. Group 4 had a slightly higher ADWG and a lower mean % lung
lesions than Group 2, but Group 2 had higher IFAT PCV2 titers on
Day 25, 32 and 50, and a slightly lower incidence rate of IHC
positive tissues. All other results for these two groups were
similar. Overall, one dose of vORF2 adjuvanted with Carbopol
performed similar to two doses of the same vaccine.
Sequence CWU 1
1
1118DNAArtificial SequenceThis is a modified Kozak's sequence
1ccgccatg 826DNAArtificial SequenceThis is a recombinant Eco R1
sequence 2gaattc 63713DNAPorcine circovirus 3cagctatgac gtatccaagg
aggcgttacc gcagaagaag acaccgcccc cgcagccatc 60ttggccagat cctccgccgc
cgcccctggc tcgtccaccc ccgccaccgc taccgttgga 120gaaggaaaaa
tggcatcttc aacacccgcc tctcccgcac cttcggatat actgtggaga
180aggaaaaatg gcatcttcaa cacccgcctc tcccgcacct tcggatatac
tgtgacgact 240ttgttccccc gggagggggg accaacaaaa tctctatacc
ctttgaatac tacagaataa 300gaaaggttaa ggttgaattc tggccctgct
cccccatcac ccagggtgat aggggagtgg 360gctccactgc tgttattcta
gatgataact ttgtaacaaa ggccacagcc ctaacctatg 420acccatatgt
aaactactcc tcccgccata caatccccca acccttctcc taccactccc
480gttacttcac acccaaacct gttcttgact ccactattga ttacttccaa
ccaaataaca 540aaaggaatca gctttggctg aggctacaaa cctctagaaa
tgtggaccac gtaggcctcg 600gcactgcgtt cgaaaacagt aaatacgacc
aggactacaa tatccgtgta accatgtatg 660tacaattcag agaatttaat
cttaaagacc ccccacttaa accctaaatg aat 7134713DNAPorcine circovirus
4ccgccatgac gtatccaagg aggcgttacc gcagaagaag acaccgcccc cgcagccatc
60ttggccagat cctccgccgc cgcccctggc tcgtccaccc ccgccaccgc taccgttgga
120gaaggaaaaa tggcatcttc aacacccgcc tctcccgcac cttcggatat
actgtcaagg 180ctaccacagt cacaacgccc tcctgggcgg tggacatgat
gagatttaat attgacgact 240ttgttccccc gggagggggg accaacaaaa
tctctatacc ctttgaatac tacagaataa 300gaaaggttaa ggttgaattc
tggccctgct cccccatcac ccagggtgat aggggagtgg 360gctccactgc
tgttattcta gatgataact ttgtaacaaa ggccacagcc ctaacctatg
420acccatatgt aaactactcc tcccgccata caatccccca acccttctcc
taccactccc 480gttacttcac acccaaacct gttcttgact ccactattga
ttacttccaa ccaaataaca 540aaaggaatca gctttggctg aggctacaaa
cctctagaaa tgtggaccac gtaggcctcg 600gcactgcgtt cgaaaacagt
aaatacgacc aggactacaa tatccgtgta accatgtatg 660tacaattcag
agaatttaat cttaaagacc ccccacttga accctaagaa ttc 7135233PRTPorcine
circovirus 5Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg
Pro Arg1 5 10 15Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu
Val His Pro20 25 30Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile
Phe Asn Thr Arg35 40 45Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala
Thr Thr Val Thr Thr50 55 60Pro Ser Trp Ala Val Asp Met Met Arg Phe
Asn Ile Asp Asp Phe Val65 70 75 80Pro Pro Gly Gly Gly Thr Asn Lys
Ile Ser Ile Pro Phe Glu Tyr Tyr85 90 95Arg Ile Arg Lys Val Lys Val
Glu Phe Trp Pro Cys Ser Pro Ile Thr100 105 110Gln Gly Asp Arg Gly
Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn115 120 125Phe Val Thr
Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr130 135 140Ser
Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr145 150
155 160Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln
Pro165 170 175Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr
Ser Arg Asn180 185 190Val Asp His Val Gly Leu Gly Thr Ala Phe Glu
Asn Ser Lys Tyr Asp195 200 205Gln Asp Tyr Asn Ile Arg Val Thr Met
Tyr Val Gln Phe Arg Glu Phe210 215 220Asn Leu Lys Asp Pro Pro Leu
Lys Pro225 2306233PRTPorcine circovirus 6Met Thr Tyr Pro Arg Arg
Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 5 10 15Ser His Leu Gly Gln
Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro20 25 30Arg His Arg Tyr
Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg35 40 45Leu Ser Arg
Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Thr Thr50 55 60Pro Ser
Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val65 70 75
80Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr85
90 95Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile
Thr100 105 110Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu
Asp Asp Asn115 120 125Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp
Pro Tyr Val Asn Tyr130 135 140Ser Ser Arg His Thr Ile Pro Gln Pro
Phe Ser Tyr His Ser Arg Tyr145 150 155 160Phe Thr Pro Lys Pro Val
Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro165 170 175Asn Asn Lys Arg
Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn180 185 190Val Asp
His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp195 200
205Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu
Phe210 215 220Asn Leu Lys Asp Pro Pro Leu Glu Pro225
2307756DNAArtificial SequenceThis sequence is from porcine
circovirus type 2, open reading frame 2, together with a portion
from the pGEM T-easy vector. 7gcggccgcgg gaattcgatc cgccatgacg
tatccaagga ggcgttaccg cagaagaaga 60caccgccccc gcagccatct tggccagatc
ctccgccgcc gcccctggct cgtccacccc 120cgccaccgct accgttggag
aaggaaaaat ggcatcttca acacccgcct ctcccgcacc 180ttcggatata
ctgtcaaggc taccacagtc acaacgccct cctgggcggt ggacatgatg
240agatttaata ttgacgactt tgttcccccg ggagggggga ccaacaaaat
ctctataccc 300tttgaatact acagaataag aaaggttaag gttgaattct
ggccctgctc ccccatcacc 360cagggtgata ggggagtggg ctccactgct
gttattctag atgataactt tgtaacaaag 420gccacagccc taacctatga
cccatatgta aactactcct cccgccatac aatcccccaa 480cccttctcct
accactcccg ttacttcaca cccaaacctg ttcttgactc cactattgat
540tacttccaac caaataacaa aaggaatcag ctttggctga ggctacaaac
ctctagaaat 600gtggaccacg taggcctcgg cactgcgttc gaaaacagta
aatacgacca ggactacaat 660atccgtgtaa ccatgtatgt acaattcaga
gaatttaatc ttaaagaccc cccacttgaa 720ccctaagaat tctatcacta
gtgaattcgc ggccgc 756810387DNAArtificial SequenceThis is the
porcine circovirus type 2, ORF2 construct, which includes
baculovirus and pGEM T-easy coding sequences. 8aagctttact
cgtaaagcga gttgaaggat catatttagt tgcgtttatg agataagatt 60gaaagcacgt
gtaaaatgtt tcccgcgcgt tggcacaact atttacaatg cggccaagtt
120ataaaagatt ctaatctgat atgttttaaa acacctttgc ggcccgagtt
gtttgcgtac 180gtgactagcg aagaagatgt gtggaccgca gaacagatag
taaaacaaaa ccctagtatt 240ggagcaataa tcgatttaac caacacgtct
aaatattatg atggtgtgca ttttttgcgg 300gcgggcctgt tatacaaaaa
aattcaagta cctggccaga ctttgccgcc tgaaagcata 360gttcaagaat
ttattgacac ggtaaaagaa tttacagaaa agtgtcccgg catgttggtg
420ggcgtgcact gcacacacgg tattaatcgc accggttaca tggtgtgcag
atatttaatg 480cacaccctgg gtattgcgcc gcaggaagcc atagatagat
tcgaaaaagc cagaggtcac 540aaaattgaaa gacaaaatta cgttcaagat
ttattaattt aattaatatt atttgcattc 600tttaacaaat actttatcct
attttcaaat tgttgcgctt cttccagcga accaaaacta 660tgcttcgctt
gctccgttta gcttgtagcc gatcagtggc gttgttccaa tcgacggtag
720gattaggccg gatattctcc accacaatgt tggcaacgtt gatgttacgt
ttatgctttt 780ggttttccac gtacgtcttt tggccggtaa tagccgtaaa
cgtagtgccg tcgcgcgtca 840cgcacaacac cggatgtttg cgcttgtccg
cggggtattg aaccgcgcga tccgacaaat 900ccaccacttt ggcaactaaa
tcggtgacct gcgcgtcttt tttctgcatt atttcgtctt 960tcttttgcat
ggtttcctgg aagccggtgt acatgcggtt tagatcagtc atgacgcgcg
1020tgacctgcaa atctttggcc tcgatctgct tgtccttgat ggcaacgatg
cgttcaataa 1080actcttgttt tttaacaagt tcctcggttt tttgcgccac
caccgcttgc agcgcgtttg 1140tgtgctcggt gaatgtcgca atcagcttag
tcaccaactg tttgctctcc tcctcccgtt 1200gtttgatcgc gggatcgtac
ttgccggtgc agagcacttg aggaattact tcttctaaaa 1260gccattcttg
taattctatg gcgtaaggca atttggactt cataatcagc tgaatcacgc
1320cggatttagt aatgagcact gtatgcggct gcaaatacag cgggtcgccc
cttttcacga 1380cgctgttaga ggtagggccc ccattttgga tggtctgctc
aaataacgat ttgtatttat 1440tgtctacatg aacacgtata gctttatcac
aaactgtata ttttaaactg ttagcgacgt 1500ccttggccac gaaccggacc
tgttggtcgc gctctagcac gtaccgcagg ttgaacgtat 1560cttctccaaa
tttaaattct ccaattttaa cgcgagccat tttgatacac gtgtgtcgat
1620tttgcaacaa ctattgtttt ttaacgcaaa ctaaacttat tgtggtaagc
aataattaaa 1680tatgggggaa catgcgccgc tacaacactc gtcgttatga
acgcagacgg cgccggtctc 1740ggcgcaagcg gctaaaacgt gttgcgcgtt
caacgcggca aacatcgcaa aagccaatag 1800tacagttttg atttgcatat
taacggcgat tttttaaatt atcttattta ataaatagtt 1860atgacgccta
caactccccg cccgcgttga ctcgctgcac ctcgagcagt tcgttgacgc
1920cttcctccgt gtggccgaac acgtcgagcg ggtggtcgat gaccagcggc
gtgccgcacg 1980cgacgcacaa gtatctgtac accgaatgat cgtcgggcga
aggcacgtcg gcctccaagt 2040ggcaatattg gcaaattcga aaatatatac
agttgggttg tttgcgcata tctatcgtgg 2100cgttgggcat gtacgtccga
acgttgattt gcatgcaagc cgaaattaaa tcattgcgat 2160tagtgcgatt
aaaacgttgt acatcctcgc ttttaatcat gccgtcgatt aaatcgcgca
2220atcgagtcaa gtgatcaaag tgtggaataa tgttttcttt gtattcccga
gtcaagcgca 2280gcgcgtattt taacaaacta gccatcttgt aagttagttt
catttaatgc aactttatcc 2340aataatatat tatgtatcgc acgtcaagaa
ttaacaatgc gcccgttgtc gcatctcaac 2400acgactatga tagagatcaa
ataaagcgcg aattaaatag cttgcgacgc aacgtgcacg 2460atctgtgcac
gcgttccggc acgagctttg attgtaataa gtttttacga agcgatgaca
2520tgacccccgt agtgacaacg atcacgccca aaagaactgc cgactacaaa
attaccgagt 2580atgtcggtga cgttaaaact attaagccat ccaatcgacc
gttagtcgaa tcaggaccgc 2640tggtgcgaga agccgcgaag tatggcgaat
gcatcgtata acgtgtggag tccgctcatt 2700agagcgtcat gtttagacaa
gaaagctaca tatttaattg atcccgatga ttttattgat 2760aaattgaccc
taactccata cacggtattc tacaatggcg gggttttggt caaaatttcc
2820ggactgcgat tgtacatgct gttaacggct ccgcccacta ttaatgaaat
taaaaattcc 2880aattttaaaa aacgcagcaa gagaaacatt tgtatgaaag
aatgcgtaga aggaaagaaa 2940aatgtcgtcg acatgctgaa caacaagatt
aatatgcctc cgtgtataaa aaaaatattg 3000aacgatttga aagaaaacaa
tgtaccgcgc ggcggtatgt acaggaagag gtttatacta 3060aactgttaca
ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag
3120gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat
gcatctttta 3180atcaaatccc aagatgtgta taaaccacca aactgccaaa
aaatgaaaac tgtcgacaag 3240ctctgtccgt ttgctggcaa ctgcaagggt
ctcaatccta tttgtaatta ttgaataata 3300aaacaattat aaatgctaaa
tttgtttttt attaacgata caaaccaaac gcaacaagaa 3360catttgtagt
attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa
3420aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt
tcacataaac 3480tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt
ttcatttttc tcctcataaa 3540aattaacata gttattatcg tatccatata
tgtatctatc gtatagagta aattttttgt 3600tgtcataaat atatatgtct
tttttaatgg ggtgtatagt accgctgcgc atagtttttc 3660tgtaatttac
aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta
3720aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg
ccggcatagt 3780acgcagcttc ttctagttca attacaccat tttttagcag
caccggatta acataacttt 3840ccaaaatgtt gtacgaaccg ttaaacaaaa
acagttcacc tcccttttct atactattgt 3900ctgcgagcag ttgtttgttg
ttaaaaataa cagccattgt aatgagacgc acaaactaat 3960atcacaaact
ggaaatgtct atcaatatat agttgctgat atcatggaga taattaaaat
4020gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt
tgtaataaaa 4080aaacctataa atattccgga ttattcatac cgtcccacca
tcgggcgcgg atcagatctg 4140cagcggccgc gggaattcga tccgccatga
cgtatccaag gaggcgttac cgcagaagaa 4200gacaccgccc ccgcagccat
cttggccaga tcctccgccg ccgcccctgg ctcgtccacc 4260cccgccaccg
ctaccgttgg agaaggaaaa atggcatctt caacacccgc ctctcccgca
4320ccttcggata tactgtcaag gctaccacag tcacaacgcc ctcctgggcg
gtggacatga 4380tgagatttaa tattgacgac tttgttcccc cgggaggggg
gaccaacaaa atctctatac 4440cctttgaata ctacagaata agaaaggtta
aggttgaatt ctggccctgc tcccccatca 4500cccagggtga taggggagtg
ggctccactg ctgttattct agatgataac tttgtaacaa 4560aggccacagc
cctaacctat gacccatatg taaactactc ctcccgccat acaatccccc
4620aacccttctc ctaccactcc cgttacttca cacccaaacc tgttcttgac
tccactattg 4680attacttcca accaaataac aaaaggaatc agctttggct
gaggctacaa acctctagaa 4740atgtggacca cgtaggcctc ggcactgcgt
tcgaaaacag taaatacgac caggactaca 4800atatccgtgt aaccatgtat
gtacaattca gagaatttaa tcttaaagac cccccacttg 4860aaccctaaga
attctatcac tagtgaattc gcggccgccg gccgctccag aattctagaa
4920ggtacccggg atcctttcct gggacccggc aagaaccaaa aactcactct
cttcaaggaa 4980atccgtaatg ttaaacccga cacgatgaag cttgtcgttg
gatggaaagg aaaagagttc 5040tacagggaaa cttggacccg cttcatggaa
gacagcttcc ccattgttaa cgaccaagaa 5100gtgatggatg ttttccttgt
tgtcaacatg cgtcccacta gacccaaccg ttgttacaaa 5160ttcctggccc
aacacgctct gcgttgcgac cccgactatg tacctcatga cgtgattagg
5220atcgtcgagc cttcatgggt gggcagcaac aacgagtacc gcatcagcct
ggctaagaag 5280ggcggcggct gcccaataat gaaccttcac tctgagtaca
ccaactcgtt cgaacagttc 5340atcgatcgtg tcatctggga gaacttctac
aagcccatcg tttacatcgg taccgactct 5400gctgaagagg aggaaattct
ccttgaagtt tccctggtgt tcaaagtaaa ggagtttgca 5460ccagacgcac
ctctgttcac tggtccggcg tattaaaaca cgatacattg ttattagtac
5520atttattaag cgctagattc tgtgcgttgt tgatttacag acaattgttg
tacgtatttt 5580aataattcat taaatttata atctttaggg tggtatgtta
gagcgaaaat caaatgattt 5640tcagcgtctt tatatctgaa tttaaatatt
aaatcctcaa tagatttgta aaataggttt 5700cgattagttt caaacaaggg
ttgtttttcc gaaccgatgg ctggactatc taatggattt 5760tcgctcaacg
ccacaaaact tgccaaatct tgtagcagca atctagcttt gtcgatattc
5820gtttgtgttt tgttttgtaa taaaggttcg acgtcgttca aaatattatg
cgcttttgta 5880tttctttcat cactgtcgtt agtgtacaat tgactcgacg
taaacacgtt aaataaagct 5940tggacatatt taacatcggg cgtgttagct
ttattaggcc gattatcgtc gtcgtcccaa 6000ccctcgtcgt tagaagttgc
ttccgaagac gattttgcca tagccacacg acgcctatta 6060attgtgtcgg
ctaacacgtc cgcgatcaaa tttgtagttg agctttttgg aattatttct
6120gattgcgggc gtttttgggc gggtttcaat ctaactgtgc ccgattttaa
ttcagacaac 6180acgttagaaa gcgatggtgc aggcggtggt aacatttcag
acggcaaatc tactaatggc 6240ggcggtggtg gagctgatga taaatctacc
atcggtggag gcgcaggcgg ggctggcggc 6300ggaggcggag gcggaggtgg
tggcggtgat gcagacggcg gtttaggctc aaatgtctct 6360ttaggcaaca
cagtcggcac ctcaactatt gtactggttt cgggcgccgt ttttggtttg
6420accggtctga gacgagtgcg atttttttcg tttctaatag cttccaacaa
ttgttgtctg 6480tcgtctaaag gtgcagcggg ttgaggttcc gtcggcattg
gtggagcggg cggcaattca 6540gacatcgatg gtggtggtgg tggtggaggc
gctggaatgt taggcacggg agaaggtggt 6600ggcggcggtg ccgccggtat
aatttgttct ggtttagttt gttcgcgcac gattgtgggc 6660accggcgcag
gcgccgctgg ctgcacaacg gaaggtcgtc tgcttcgagg cagcgcttgg
6720ggtggtggca attcaatatt ataattggaa tacaaatcgt aaaaatctgc
tataagcatt 6780gtaatttcgc tatcgtttac cgtgccgata tttaacaacc
gctcaatgta agcaattgta 6840ttgtaaagag attgtctcaa gctcgccgca
cgccgataac aagccttttc atttttacta 6900cagcattgta gtggcgagac
acttcgctgt cgtcgacgta catgtatgct ttgttgtcaa 6960aaacgtcgtt
ggcaagcttt aaaatattta aaagaacatc tctgttcagc accactgtgt
7020tgtcgtaaat gttgtttttg ataatttgcg cttccgcagt atcgacacgt
tcaaaaaatt 7080gatgcgcatc aattttgttg ttcctattat tgaataaata
agattgtaca gattcatatc 7140tacgattcgt catggccacc acaaatgcta
cgctgcaaac gctggtacaa ttttacgaaa 7200actgcaaaaa cgtcaaaact
cggtataaaa taatcaacgg gcgctttggc aaaatatcta 7260ttttatcgca
caagcccact agcaaattgt atttgcagaa aacaatttcg gcgcacaatt
7320ttaacgctga cgaaataaaa gttcaccagt taatgagcga ccacccaaat
tttataaaaa 7380tctattttaa tcacggttcc atcaacaacc aagtgatcgt
gatggactac attgactgtc 7440ccgatttatt tgaaacacta caaattaaag
gcgagctttc gtaccaactt gttagcaata 7500ttattagaca gctgtgtgaa
gcgctcaacg atttgcacaa gcacaatttc atacacaacg 7560acataaaact
cgaaaatgtc ttatatttcg aagcacttga tcgcgtgtat gtttgcgatt
7620acggattgtg caaacacgaa aactcactta gcgtgcacga cggcacgttg
gagtatttta 7680gtccggaaaa aattcgacac acaactatgc acgtttcgtt
tgactggtac gcggcgtgtt 7740aacatacaag ttgctaacgt aatcatggtc
atagctgttt cctgtgtgaa attgttatcc 7800gctcacaatt ccacacaaca
tacgagccgg aagcataaag tgtaaagcct ggggtgccta 7860atgagtgagc
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
7920cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 7980tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
tcggtcgttc ggctgcggcg 8040agcggtatca gctcactcaa aggcggtaat
acggttatcc acagaatcag gggataacgc 8100aggaaagaac atgtgagcaa
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 8160gctggcgttt
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
8220tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc 8280cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
tacctgtccg cctttctccc 8340ttcgggaagc gtggcgcttt ctcatagctc
acgctgtagg tatctcagtt cggtgtaggt 8400cgttcgctcc aagctgggct
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 8460atccggtaac
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
8520agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa 8580gtggtggcct aactacggct acactagaag gacagtattt
ggtatctgcg ctctgctgaa 8640gccagttacc ttcggaaaaa gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg 8700tagcggtggt ttttttgttt
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 8760agatcctttg
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
8820gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
attaaaaatg 8880aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
tctgacagtt accaatgctt 8940aatcagtgag gcacctatct cagcgatctg
tctatttcgt tcatccatag ttgcctgact 9000ccccgtcgtg tagataacta
cgatacggga gggcttacca tctggcccca gtgctgcaat 9060gataccgcga
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
9120aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
ctattaattg 9180ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
ttgcgcaacg ttgttgccat 9240tgctacaggc atcgtggtgt cacgctcgtc
gtttggtatg gcttcattca gctccggttc 9300ccaacgatca aggcgagtta
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 9360cggtcctccg
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
9420agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
tgactggtga 9480gtactcaacc aagtcattct gagaatagtg tatgcggcga
ccgagttgct cttgcccggc 9540gtcaatacgg gataataccg cgccacatag
cagaacttta aaagtgctca tcattggaaa 9600acgttcttcg
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
9660acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
tttctgggtg 9720agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
agggcgacac ggaaatgttg 9780aatactcata ctcttccttt ttcaatatta
ttgaagcatt tatcagggtt attgtctcat 9840gagcggatac atatttgaat
gtatttagaa aaataaacaa ataggggttc cgcgcacatt 9900tccccgaaaa
gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
9960aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
gtgaaaacct 10020ctgacacatg cagctcccgg agacggtcac agcttgtctg
taagcggatg ccgggagcag 10080acaagcccgt cagggcgcgt cagcgggtgt
tggcgggtgt cggggctggc ttaactatgc 10140ggcatcagag cagattgtac
tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg 10200cgtaaggaga
aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga
10260agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
gatgtgctgc 10320aaggcgatta agttgggtaa cgccagggtt ttcccagtca
cgacgttgta aaacgacggc 10380cagtgcc 10387920PRTPorcine circovirus
9Ser Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His His Pro Pro Ser1 5
10 15His Leu Gly Gln201019PRTPorcine circovirus 10Pro Arg His His
Tyr Arg Pro Arg Arg Lys Asn Gly Ile Phe Asn Thr1 5 10 15Thr Leu
Ser11233PRTArtificial SequenceThis is an amino acid sequence for
porcine circovirus type 2, open reading frame 2. 11Met Thr Tyr Pro
Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 5 10 15Ser His Leu
Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro20 25 30Arg His
Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg35 40 45Leu
Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Arg Thr50 55
60Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val65
70 75 80Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr
Tyr85 90 95Arg Ile Lys Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro
Ile Thr100 105 110Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile
Leu Asp Asp Asn115 120 125Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr
Asp Pro Tyr Val Asn Tyr130 135 140Ser Ser Arg His Thr Ile Pro Gln
Pro Phe Ser Tyr His Ser Arg Tyr145 150 155 160Phe Thr Pro Lys Pro
Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro165 170 175Asn Asn Lys
Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn180 185 190Val
Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp195 200
205Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu
Phe210 215 220Asn Leu Lys Asp Pro Pro Leu Lys Pro225 230
* * * * *