U.S. patent number 5,780,448 [Application Number 08/740,805] was granted by the patent office on 1998-07-14 for dna-based vaccination of fish.
This patent grant is currently assigned to Ottawa Civic Hospital Loeb Research. Invention is credited to Heather L. Davis.
United States Patent |
5,780,448 |
Davis |
July 14, 1998 |
DNA-based vaccination of fish
Abstract
The present invention relates to methods of immunization of
aquaculture species by introducing DNA expression systems into the
aquaculture species. Such DNA expression systems preferably include
DNA sequences encoding polypeptides of pathogens of species of
aquaculture. The present invention also relates to methods of
administration of DNA expression systems into aquaculture. Such
methods include injection, spray, and immersion techniques. The
methods of this invention are useful for prophylactic vaccination
or therapeutic immunization of fin-fish, shellfish, or other
aquatic animals against infectious diseases.
Inventors: |
Davis; Heather L. (Ottawa,
CA) |
Assignee: |
Ottawa Civic Hospital Loeb
Research (Ottawa, CA)
|
Family
ID: |
26675437 |
Appl.
No.: |
08/740,805 |
Filed: |
November 4, 1996 |
Current U.S.
Class: |
514/44R;
435/69.3; 435/69.4; 424/199.1; 424/201.1; 424/227.1; 536/23.72;
536/23.4; 536/23.1; 424/93.1; 424/202.1; 424/204.1; 424/817;
435/69.5; 435/320.1 |
Current CPC
Class: |
A61P
31/04 (20180101); C12N 15/88 (20130101); A61K
47/54 (20170801); C07K 14/28 (20130101); C07K
14/005 (20130101); A61P 37/04 (20180101); A61P
31/12 (20180101); C12N 2760/20022 (20130101); Y02A
50/403 (20180101); A61K 2039/51 (20130101); C12N
2720/10022 (20130101); Y10S 424/817 (20130101); Y02A
50/30 (20180101) |
Current International
Class: |
C12N
15/88 (20060101); A61K 47/48 (20060101); C12N
15/87 (20060101); C07K 14/145 (20060101); C07K
14/195 (20060101); C07K 14/005 (20060101); C07K
14/28 (20060101); C12N 015/00 (); A61K 039/12 ();
A61K 039/29 (); A01N 043/04 () |
Field of
Search: |
;424/93.1,204.1,199.1,201.1,202.1,227.1,817
;435/320.1,69.3,69.4,69.5,240.2 ;536/23.1,23.4,23.72 ;514/44
;800/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
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|
|
|
|
|
WO 91/13987 |
|
Nov 1991 |
|
WO |
|
WO 94/27435 |
|
Dec 1994 |
|
WO |
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Other References
Davis et al., 1994, Vaccine, vol. 12, pp. 1503-1509. .
Hansen et al, 1991, FEBS, vol. 290, pp. 73-76. .
Fynan et al, 1993, PNAS, vol. 90, pp. 11478-11482. .
Lin et al, 1990, Circulation, vol. 82, No. 6, pp. 2217-2221. .
Robinson et al, 1993, Vaccine, vol. 11, pp. 957-960. .
Tang et al, 1992, Nature, vol. 356, pp. 152-154. .
Cheng, L., et al., "In Vivo Promoter Activity And Transgene
Expression In Mammalian Somatic Tissues Evaluated By Using Particle
Bombardment," PNAS 90:4455-4459 (1993). .
Davis, H., et al., "Direct Gene Transfer Into Skeletal Muscle In
Vivo: Factors Affecting Efficiency of Transfer and Stability of
Expression," Human Gene Therapy 4:151-159 (1993). .
Davis, H., et al., "Plasmid DNA Is Superior To Viral Vectors For
Direct Gene Transfer Into Adult Mouse Skeletal Muscle," Human Gene
Therapy 4:733-740 (1993). .
Felgner, P.L. et al. "Improved Cationic Lipid Formulations For In
Vivo Gene Therapy," Annal New York Academy of Sciences pp. 126-139
(1995). .
Leong, J.C, and J.L. Fryer., "Viral Vaccines For Aquaculture,"
Annual Rev. of Fish Diseases 3:225-240 (1993). .
Munn, C.B., "The Use of Recombinant DNA Technology In The
Development of Fish Vaccines," Fish and Shellfish Immunology
4:459-473 (1994). .
Newman, S.G., "Bacterial Vaccines For Fish," Annual Rev. of Fish
Diseases, pp. 145-185 (1993). .
Sato, Y. et al., "Immunostimulatory DNA Sequences Necessary For
Effective Intrademal Gene Immunization," Science 273:352-354. .
Wolff, J.A., et al., "Direct Gene Transfer into Mouse Muscle in
Vivo," Science 247:1465-68 (1990). .
Wolff, J.A., et al., "Conditions Affecting Direct Gene Transfer
Into Rodent Muscle In Vivo," Biotechniques 11(4):474-485 (1991).
.
Zhu, N., et al., "Systemic Gene Expression After Intravenous DNA
Delivery Into Adult Mice," Science 261:209-211 (1993)..
|
Primary Examiner: Mosher; Mary E.
Assistant Examiner: Salimi; Ali R.
Attorney, Agent or Firm: Fish & Richardson, P.C.
Claims
What is claimed is:
1. A composition for inducing an immune response in finfish
comprising:
an expression vector having an expression control sequence capable
of directing expression in finfish of at least one immunogenic
polypeptide and a polypeptide-encoding DNA sequence encoding at
least one immunogenic polypeptide from a fish pathogen.
2. The composition according to claim 1, wherein the vector
additionally comprises an immunostimulatory unmethylated CpG
motif.
3. The composition according to claim 1, wherein the injection of
the vector comprising a fish pathogen induces a protective response
in finfish.
4. The composition for inducing an response according to claim 1 or
3, wherein the vector is selected form the group consisting of
pCMV-G, pCMV-N, pCMV-VP2, pCMV-VP3, pCMV-fstA, and.
5. The composition according to claim 1, wherein the
polypeptide-encoding DNA sequence additionally encodes a different
polypeptide from the same pathogen.
6. The composition according to claim 1, wherein the
polypeptide-encoding DNA sequence additionally encodes a
polypeptide from a different pathogen.
7. The composition according to claim 1, wherein the
polypeptide-encoding DNA sequence additionally encodes a carrier
polypeptide to form a fusion protein with the immunogenic
polypeptide.
8. The composition according to claim 7, wherein the carrier
polypeptide is a surface antigen of the human hepatitis B
virus.
9. The composition according to claim 1, wherein the vector further
comprises a second expression control sequence capable of directing
expression of a polypeptide and a second polypeptide-encoding DNA
sequence under transcriptional control of the second expression
control sequence.
10. The composition according to claim 9, wherein the second
polypeptide-encoding DNA sequence is a cytokine.
11. The composition for inducing an immune response according to
claim 1 further comprising an immune modulator adjuvant.
12. The composition according to claim 11, wherein the adjuvant is
an oligonucleotide having an immunostimulatory unmethylated CpG
motif.
13. The composition according to claim 1 formulated with a
transfection reagent.
14. The composition according to claim 13, wherein the transfection
reagent is selected from the group consisting of liposomes,
fluorocarbon emulsions, cochleates, tubules, gold particles,
biodegradable microspheres, and cationic polymers.
15. The composition according to claim 14, wherein the transfection
reagent is a liposome.
16. The composition according to claim 14, wherein the transfection
agent is a cationic polymer.
17. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded in the genome of a rhabdovirus selected from
the group of viral haemorrhagic septicaemina virus (VHSV),
infectious haematopoietic necrosis virus (IHNV), and spring
viraemia of carp virus (SVCV).
18. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded in the genome of a fish rhabdovirus selected
from the group of viral haemorrhagic septicaemina virus (VHSV),
infectious haematopoietic necrosis virus (IHNV), and spring
viraemia of carp virus (SVCV).
19. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded in the genome of a birnavirus.
20. The composition according to claim 1, wherein the birnavirus is
infectious pancreatic necrosis virus (IPNV).
21. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of VP1, VP2, VP3,
and N structural proteins encoded in the genome of the infectious
pancreatic necrosis virus (IPNV).
22. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of a marine herpesvirus.
23. The composition according to claim 22, wherein the immunogenic
polypeptide is encoded by the genome of the channel catfish virus
(CCV).
24. The composition according to claim 23, wherein the immunogenic
polypeptide is selected from the group consisting of an envelope
protein, a membrane-associated protein) tegumin, a capsid protein,
and a glycoprotein of the channel catfish virus (CCV).
25. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of a marine nodavirus.
26. The composition of claim 25, wherein the nodavirus is selected
from the group consisting of nervous necrosis virus and striped
jack nervous necrosis virus.
27. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of a iridovirus.
28. The composition according to claim 27, wherein the iridovirus
is selected from the group consisting of fish lymphocystis disease
virus (FLDV) and other marine iridoviruses.
29. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of infectious salmon anaemia
virus (ISAV).
30. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of a bacterial pathogen
selected from the group consisting of Aeromonis salmonicida,
Renibacterium salmoninarum, Vibrosis anguillarum and Vibrosis
ordalii, Yersiniosis, Pasteurellosis, Edwardsiellosis ictaluri,
Edwardsiellosis tarda, Cytophaga colummari, and Rickettsia.
31. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of an
iron-regulated outer membrane protein, (IROMP), an outer membrane
protein (OMP), and an A-protein of Aeromonis salmonicida.
32. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of the p57
protein, major surface associated antigen (msa), a surface
expressed cytotoxin (mpr), and a surface expressed hemolysin (ish)
of Renibacterium salmoninarum.
33. The composition according to claim 1, wherein the immunogenic
polypeptide is a flagellar antigen of Yersinosis.
34. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of an
extracellular protein (ECP), an iron-regulated outer membrane
protein (IROMP), and a structural protein of Pasteurellosis.
35. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of an outer
membrane protein (OMP) and a flagellar protein of a member of the
genus Vibrosis, wherein said member is selected from the group
consisting of Vibrosis anguillarum and Vibrosis ordalii.
36. The composition according to claim 1, wherein the immunogenic
polypeptide is selected from the group consisting of a flagellar
protein, an outer membrane protein (OMP) protein, aroA, and purA of
a member of the genus Edwardsiellosis, wherein said member is
selected from the group consisting of Edwardsiellosis ictaluri and
E. tarda.
37. The composition according to claim 1, wherein the immunogenic
polypeptide is a structural or regulatory protein of Cytophaga
columnari.
38. The composition according to claim 1, wherein the immunogenic
polypeptide is a structural or regulatory protein of
Rickettsia.
39. The composition according to claim 1, wherein the immunogenic
polypeptide is encoded by the genome of a marine parasite.
40. The composition according to claim 39, wherein the marine
parasite is a member of the genus Ichthyophthirius.
41. The composition according to claim 39, wherein the immunogenic
polypeptide is surface antigen of the parasite
Ichthyophthirius.
42. A method of inducing immune response in fin-fish against
infection from fish pathogens, comprising administering to the
fin-fish a composition for inducing an immune response comprising
an expression vector having an expression control sequence capable
of directing expression of an immunogenic polypeptide, and a
polypeptide-encoding DNA sequence encoding an immunogenic
polypeptide.
43. The method according to claim 42, wherein the vector
additionally comprises a nucleic acid sequence comprising at least
one unmethylated CpG motif, wherein the nucleic acid sequence is
immunostimulatory.
44. The method according to claim 42, wherein the
polypeptide-encoding DNA sequence additionally encodes a different
polypeptide from the same pathogen.
45. The method according to claim 42, wherein the
polypeptide-encoding DNA sequence additionally encodes a
polypeptide from a different pathogen.
46. The method according to claim 42, wherein the
polypeptide-encoding DNA sequence additionally encodes a carrier
polypeptide to form a fusion protein with the immunogenic
polypeptide.
47. The method according to claim 42, wherein the vector further
comprises a second expression control sequence capable of directing
expression of a polypeptide and a second polypeptide-encoding DNA
sequence under transcriptional control of the second expression
control sequence.
48. The method according to claim 42, wherein the composition is
formulated with a transfection reagent selected from the group
consisting of liposomes, fluorocarbon emulsions, cochleates,
tubules, gold particles, biodegradable microspheres, and cationic
polymers.
49. The method according to claim 42, further comprising
administering to the fin-fish an additional DNA expression vector
comprising an expression control sequence capable of directing
expression of an immunogenic polypeptide and a polypeptide-encoding
DNA sequence capable of inducing an immune response.
50. The method according to claim 42, further comprising
administering to the fin-fish an adjuvant before or after
administration of the composition.
51. The method according to claim 42, further comprising
administering to the fin-fish a recombinant or purified protein,
wherein said protein is administered before, during or after
administration of the composition.
52. The method according to claim 42, wherein the composition is
administered by intramuscular injection or intraperitoneal
injection.
53. The method according to claim 42, wherein the composition is
administered by immersion.
54. The method according to claim 53, wherein the DNA is formulated
with a transfection reagent selected from the group consisting of
cationic liposomes and cationic polymers.
55. The method according to claim 42, wherein the fin-fish is
selected from the group consisting of salmon, salmonid, carp,
catfish, trout (including rainbow trout), yellowtail, seabream, and
seabass.
56. The method according to claim 42, wherein the pathogen is a
viral fish pathogen.
57. The method according to claim 42, wherein said immunogenic
peptide is encoded in the genome of a viral fish pathogen.
58. The method according to claim 42, wherein the immunogenic
polypeptide is encoded in the genome of a rhabdovirus selected from
the group of viral haemorrhagic septicaemina virus (VHSV),
infectious haematopoietic necrosis virus (IHNV), and spring
viraemia of carp virus (SVCV).
59. The method according to claim 42, wherein the immunogenic
polypeptide is encoded in the genome of a birnavirus.
60. The method according to claim 59, wherein the birnavirus is an
infectious pancreatic necrosis virus (IPNV).
61. The method according to claim 42, wherein the immunogenic
polypeptide is selected from the group consisting of VP1, VP2, VP3,
and N structural proteins encoded in the genome of the infectious
pancreatic necrosis virus (IPNV).
62. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of a marine herpesvirus.
63. The method according to claim 62, wherein the immunogenic
polypeptide is encoded by the genome of the channel catfish virus
(CCV) 1.
64. The method according to claim 63, wherein the immunogenic
polypeptide is an envelope protein of the channel catfish virus
(CCV).
65. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of a marine nodavirus.
66. The method according to claim 65, wherein the nodavirus is
selected from the group consisting of nervous necrosis virus and
striped jack nervous necrosis virus.
67. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of a iridovirus.
68. The method according to claim 67, wherein the irridovirus is
selected from the group consisting of fish lymphocystis disease
virus (FLDV) and other marine iridoviruses.
69. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of infectious salmon anaemia
virus (ISAV).
70. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of a bacterial pathogen
selected from the group consisting of Aeromonis salmonicida,
Renibacterium salmoninarum, Vibrosis anguillarum and Vibrosis
ordalii, Yersiniosis, Pasteurellosis, Edwardsiellosis ictuluri,
Edwardsiellosis tarda, Cytophaga columnari, and Rickettsia.
71. The method according to claim 42, wherein the immunogenic
polypeptide is selected from the group consisting of an
iron-regulated outer membrane protein, (IROMP), an outer membrane
protein (OMP), and an A-protein of Aeromonis salmonicida.
72. The method according to claim 42, wherein the immunogenic
polypeptide is selected from the group consisting of the p57
protein, major surface associated antigen (msa), a surface
expressed cytotoxin (mpr), and a surface expressed hemolysin (ish)
of Renibacterium salmoninarum.
73. The method according to claim 42, wherein the immunogenic
polypeptide is a flagellar antigen of Yersinosis.
74. The method according to claim 42, wherein the immunogenic
polypeptide is selected from the group consisting of an
extracellular protein (ECP), an iron-regulated outer membrane
protein (IROMP), and a structural protein of Pasteurellosis.
75. The method according to claim 42, wherein the immumogenic
polypeptide is selected from the group consisting of an outer
membrane protein (OMP) and a flagellar protein of a member of the
genus Vibrosis, wherein said member is selected from the group
consisting of Vibrosis anguillarum and V. ordalii.
76. The method according to claim 42, wherein the immunogenic
polypeptide is selected from the group consisting of a flagellar
protein, an outer membrane protein (OMP) protein, aroA, and purA of
a member of the genus Edwardsiellosis, wherein said member is
selected from the group consisting of Edwardsiellosis ictaluri and
E. tarda.
77. The method according to claim 42, wherein the immunogenic
polypeptide is a structural or regulatory protein of Cytophaga
columnari.
78. The method according to claim 42, wherein the immunogenic
polypeptide is a structural or regulatory protein of
Rickettsia.
79. The method according to claim 42, wherein the immunogenic
polypeptide is encoded by the genome of a marine parasite.
80. The method according to claim 79, wherein the marine parasite
is a member of the genus Ichthyophthirius.
81. The method according to claim 42, wherein the immunogenic
polypeptide is a surface antigen of the parasite Ichthyophthirius
multifiliis.
82. A method for expressing a polypeptide in fin-fish comprising
administering to a fin-fish a DNA expression vector comprising an
expression control sequence capable of directing expression of a
polypeptide and a polypeptide-encoding DNA sequence encoding at
least one polypeptide from a fish pathogen.
83. The method according to claim 82, wherein said DNA expression
vector is administered by a technique selected from the group
consisting of intramuscular injection, intraperitoneal injection,
or immersion.
Description
Benefit of the United States Provisional Application No.
60/006,290, filed Nov. 7, 1995 is claimed herefor.
BACKGROUND OF THE INVENTION
Viral and bacterial diseases in fin-fish, shellfish or other
aquatic lifeforms pose a serious problem for the aquaculture
industry. Owing to the high density of animals in the hatchery
tanks or enclosed marine farming areas, infectious diseases may
eradicate a large proportion of the stock in, for example, a
fin-fish, shellfish, or other aquatic lifeforms facility.
Prevention of disease is a more desired remedy to these threats to
fish than intervention once the disease is in progress. Vaccination
of fish is the only preventative method which may offer long-term
protection through immunity.
The fish immune system has many features similar to the mammalian
immune system, such as the presence of B cells, T cells,
lymphokines, complement, and immunoglobulins. Fish have lymphocyte
subclasses with roles that appear similar in many respects to those
of the B and T cells of mammals. Additionally, the efficiency of
the immune response of fish can be affected by outside stresses, as
is true in mammals. However, fish, unlike mammals, display a
temperature-dependent development of protective immunity in
response to antigens.
Most vaccines for fish have been developed against bacteria while
there have been very few fish vaccines made for combating viral or
parasitic diseases. Fish have been immunized by antigen-based
immunization methods using live attenuated pathogens, killed whole
pathogens, or more recently, in laboratory settings, recombinant
proteins. While live attenuated vaccines induce good humoral and
cell-mediated immune responses and can be administered orally or by
immersion or injection, there is the important risk of reversion to
a virulent form. Whole live attenuated vaccines are not preferred
in industrial farming due to the risk of contaminating other
fish--a live attenuated vaccine which may be generally safe for the
target species of fish may be virulent in other species of
fish.
Fish vaccines using whole killed bacteria (i.e. bacterins) or
recombinant proteins from pathogens expressed in cell lines
(subunit vaccines) have the disadvantage of inducing short-lived
immune responses. Injected antigen, including recombinant protein,
is processed solely in an exogenous form usually causing induction
of a humoral response (i.e., production of antibodies) but often a
failure to induce cell-mediated immunity (i.e., cytotoxic
T-cells).
Another disadvantage of whole killed and subunit vaccines is that
they almost always must be injected and they require an adjuvant to
induce an effective immune response. Intramuscular injections of
these adjuvants can cause granuloma formation which scars the flesh
and lowers the market value of the fish. Intraperitoneal injection
of adjuvants may cause adhesions between the viscera which can
affect the health of the fish and retard fish growth.
Recombinant protein vaccines are difficult and expensive to make
especially if the protein must be purified. For example,
bacterially-expressed recombinant proteins may form inclusion
bodies from which recovery of protein in correct configuration may
be low or nonexistent. Induction of an immune response may require
that the antigenic protein be correctly glycosylated and folded,
which may not be accomplished in a cell other than an animal
cell.
Some of the current methodologies for administering vaccines are
not technically or economically practical. For example, direct
injection of recombinant and whole killed pathogen vaccines into
the fish is labor intensive and expensive relative to the future
market value of the fish. Furthermore, injection needles can
cross-infect fish with contaminating pathogenic organisms, and
accidental injection of humans can cause severe or fatal infections
and anaphylactic reactions. Moreover, noninjurious injection of
small fish is very difficult, especially in young fry, which are
particularly susceptible to disease.
A less expensive and easier method which has been used to
administer killed viral or bacterial vaccines is an oral method
wherein the vaccine is added directly to the water or incorporated
into fish food. Oral vaccines have historically shown inconsistent
and relatively low levels of protection suggesting that they may be
best used as a method of revaccination.
Genes have been introduced directly into animals by using live
viral vectors containing particular sequences from an adenovirus,
an adeno-associated virus, or a retrovirus genome. The viral
sequences allow the appropriate processing and packaging of a gene
into a virion, which can be introduced to animals through invasive
or non-invasive infection. Viral vectors have several
disadvantages. Viral vectors being live pathogens, still carry the
risk of inadvertent infection. Furthermore, proteins from viral
vector sequences induce undesirable inflammatory or other immune
responses which may prevent the possibility of using the same
vector for a subsequent vaccine or boost. Viral vectors also limit
the size of the target gene that can be expressed due to viral
packaging constraints.
Naked DNA transfects relatively efficiently if injected into
skeletal muscle but poorly or not at all if injected into other
tissues (Wolff et al., Science 247:1465-1468 (1990), incorporated
herein by reference). Plasmid DNA coated onto the surface of small
gold particles and introduced into the skin by a helium-driven
particle accelerator or "gene-gun" can directly transfect cells of
the epidermis and dermis (Pecorino and Lo, Current Biol., 2:30-32
(1992), which is incorporated herein by reference).
DNA has also been introduced into animal cells by liposome-mediated
gene transfer. DNA-liposome complexes, usually containing a mixture
of cationic and neutral lipids, are injected into various tissues
or instilled into the respiratory passages. Nabel et al., Hum. Gene
Ther., 3:649-656 (1992), which is incorporated herein by reference,
have shown that liposomes may be used to transfect a wide variety
of cell types by intravenous injection in mammals. In addition,
liposome-mediated gene transfer has been used to transfer the
cystic fibrosis transmembrane conductance gene into the nasal
epithelium of mice and humans suffering from cystic fibrosis
(Yoshimura et al., Nucleic Acids Reg., 12:3233-3240 (1992) and
Caplan et al., Nature Med., 1:39-46 (1995), respectively, both of
which are incorporated herein by reference.
Substances may also be administered using biodegradable
microspheres composed of polymers such as polyester
poly(lactide-co-glycolide) (Marx et al., Science, 260:1323-1328
(1993), incorporated herein by reference). It is notable that these
particles can survive the upper digestive system and arrive intact
in cells of gut-associated lymphoid tissue (Eldridge et al., Adv.
Exp. Med. Biol., 251:191-202 (1989), incorporated herein by
reference). Biodegradable microspheres have been used to deliver
recombinant antigens, toxoids or attenuated virus into mammals by
systemic and oral routes (O'Hagan et al., Immunology 73:239-242
(1991); O'Hagen et al., Vaccine 11:149-154 (1993); Eldridge et al.,
Mol. Immunol. 228:287-293 (1991) incorporated herein by reference).
They may also be useful to deliver recombinant plasmid DNA to
gut-associated lymphoid tissue for the purpose of immunization.
While most work has been carried out on mammals, plasmid DNA
encoding reporter genes have been successfully introduced into fish
by intramuscular injection (Hansen et al., FEBS Lett. 290:73-76
(1991), incorporated herein by reference). Thus, cells in fish can
express proteins from a foreign gene with the same types of vector
constructs (i.e., backbones, promoter and enhancer elements) that
are used in mammals.
The induction of an immune response to a protein expressed from an
introduced gene was first suggested by Acsadi et al., New Biologist
3:71-81 (1991), which is incorporated herein by reference, who
found that after plasmid DNA transfer into rat cardiac muscle,
reporter gene expression was transient but could be prolonged by
treatment with an immuno-suppressant. Subsequently, it was shown
that antibodies were induced in rodents against human growth
hormone (Tang et al., Nature, 356:152-154 (1992); Eisenbraun et
al., DNA Cell. Biol., 12:791-797 (1993), both of which are
incorporated herein by reference) or human .alpha.-antitrypsin
(Tang et al., Nature, 356:152-154 (1992), also incorporated herein
by reference) when these proteins were expressed from DNA coated
onto gold particles and introduced into cells of the skin by
bombardment.
DNA-based immunization refers to the induction of an immune
response to an antigen expressed in vivo from a gene introduced
into the animal. This method offers two major advantages over
classical vaccination in which some form of the antigen itself is
administered. First, the synthesis of antigen in a self-cell mimics
in certain respects an infection and thus induces a complete immune
response but carries absolutely no risk of infection. Second,
foreign gene expression may continue for a sufficient length of
time to induce strong and sustained immune responses without
boost.
Several mammalian animal models of DNA-based immunization against
specific viral, bacterial or parasitic diseases have been reported.
These include influenza [(Fynan et al., Proc. Nat'l Acad. Sci. USA,
90:11478-11482 (1993); Montgomery et al., DNA Cell. Biol.,
12:777-783 (1993); Robinson et al., Vaccine, 11:957-960(1993);
Ulmer et al., Science, 259:1745-1749 (1993)], HIV [Wang et al.
(1993)], hepatitis B [Davis et al., Hum. Molec. Genet., 2:1847-1851
(1993)], malaria [Sedagah et al., Proc. Nat'l Acad. Sci. USA,
91:9866-9870 (1994)], bovine herpes [Cox et al., J. Virol,
67:5664-5667 (1993)], herpes simplex [Rousse et al., J. Virol.,
68:5685-5689 (1994); Manicken et al. J. Immunol., 155:259-265
(1995)], rabies [Xiang et al., Virology, 199:132-140 (1994)];
lymphocytic choriomeningitis [Yokoyama et al., J. Virol.,
6964:2684-2688 (1995)] and tuberculosis [Lowrie et al., Vaccine,
12:1537-1540 (1994)], all of which are incorporated herein by
reference. In most of these studies a full-range of immune
responses including antibodies, cytotoxic T lymphocytes (CTL),
T-cell help and (where evaluation was possible) protection against
challenge was obtained. In these studies naked DNA was introduced
by intramuscular or intradermal injection with a needle and syringe
or by instillation in the nasal passages, or the naked DNA was
coated onto gold particles which were introduced by a particle
accelerator into the skin.
There is a need for novel systems to vaccinate fin-fish, shellfish,
and other aquatic animals against diseases. These systems should be
inexpensive to produce and administer, avoid the use of live,
attenuated organisms, and induce strong and long-lasting immunity
preferably without boost and with induction of both antibodies and
cell-mediated immunity. More preferably, the system should be
applicable to small fish, be less stressful to fish during
administration, and have the capacity of simultaneously immunizing
many animals for reduced labor-related costs.
SUMMARY OF THE INVENTION
The present invention relates to the immunization of cultured
fin-fish, shellfish, or other aquatic animals ("aquaculture
species") by DNA expression systems to overcome many disadvantages
associated with antigen-based vaccines. The present invention
relates to introduction of DNA plasmids (alone or in a formulation)
containing sequences encoding antigenic components of viral,
bacterial or parasitic diseases by transfection into aquaculture
species. The methods and compositions of this invention are useful
for immunization (i.e., for prophylactic vaccination or therapeutic
immunization) of fin-fish, shellfish or other aquatic animals
against infectious diseases. The DNA sequences according to this
invention are preferably present in vectors capable of inducing
protein expression of these sequences (i.e. expression vectors) and
may be administered alone or in combination with other DNA
sequences in the same or other expression vectors or as
oligonucleotides. These additional DNA sequences may encode
cytokines, costimulatory molecules, or may include
immunostimulatory sequences (e.g., CpG motifs). The DNA sequences
may also be given with other adjuvants, such as alum.
The present invention also relates to methods of administration of
DNA expression vectors to aquaculture species, which may or may not
encode polypeptides from pathogens. DNA vectors of this invention
may be administered to aquaculture species by oral route,
injection, spray, or immersion. In a preferred embodiment, the DNA
expression vectors of this invention are administered by immersion
techniques or automated injection devices.
DESCRIPTION OF THE INVENTION
The present invention provides for methods and compositions for
immunizing cultured fin-fish, shellfish, and other aquatic animals
against infection by viral, bacterial or parasitic pathogens. In
basic outline, DNA encoding a polypeptide component of a pathogen
is introduced into an animal, and the polypeptide is expressed in
cells of the animal, thus inducing an immune response that confers
protection against natural infection by the pathogen or helps
overcome an ongoing and possibly chronic infection.
In a preferred embodiment, the present invention provides a method
for immunizing cultured fin-fish, shellfish, or other aquatic
animals against disease, comprising immersion of the animals in an
aqueous solution containing formulated plasmid DNA encoding one or
more antigenic determinants of an infectious agent (regardless of
codon usage), whereby the DNA enters cells of the animal where it
is expressed leading to induction of immune responses. The
immunization procedure may be prophylactic to prevent infection
from occurring or may be therapeutic to treat pre-existing
infections.
Few anti-viral vaccines have been marketed for fish. This is
largely due to the difficulty of growing virus in culture for the
production of whole killed viral vaccines or safe attenuated
strains of virus. Antigen-based vaccines using purified recombinant
proteins are difficult and expensive to produce in large scale and
may have poor immunogenicity in fish.
DNA-based immunization has several advantages. The antigenic
protein is synthesized in vivo giving rise to both humoral and
cell-mediated (cytotoxic T lymphocytes) immune responses. However,
unlike live attenuated pathogens, which also synthesize protein in
vivo, DNA vaccines carry no risk of inadvertent infection. Unlike
antigen-based immunization, DNA-based vaccination does not require
the use of traditional adjuvants to generate an effective immune
response. Furthermore, DNA used in the methods of this invention is
inexpensive and easy to manufacture and purify.
DNA-based immunization also allows the host animal to produce
foreign antigens within its own tissue thereby resulting in several
advantages. One advantage is the efficient presentation of the
foreign antigen to the immune system due to the expression of a
protein within a self-cell, which could be an antigen-presenting
cell. Another advantage is the correct folding, protein
modification, and disulfide bonding of a protein expressed in a
host cell, especially for viral proteins, which are normally
produced in cells of hosts. Recombinant viral proteins synthesized
in bacterial or yeast cells may be incorrectly post-translationally
modified and are often massed in inclusion bodies, which make the
proteins difficult to purify or ineffective if administered in
unpurified form.
Immune responses in fish are temperature dependent. Antigen-based
vaccines may give rise to sub-optimal immune responses if such
vaccines are given at the wrong temperature. DNA-based immunization
is advantageous because expression of the antigenic protein could
continue over a long period until such time as to stimulate an
immune response when the temperature is optimal.
Another advantage of prolonged synthesis of antigen is the
induction of immune responses as soon as the immune system is
mature. Fish may be unable to induce sufficient immune responses at
a young age. For example, trout and halibut may not produce
lymphoid cells until as late as ten and thirty days after hatching,
respectively, and T-dependent immune responses do not appear until
months after hatching. Using the methods of this invention,
expression of foreign protein in fish can continue at least four
months after transfection indicating that DNA-based immunization
may be preferred for vaccination of young fish.
The term "vaccine" herein refers to a material capable of producing
an immune response. A vaccine according to this invention would
produce immunity against disease in cultured fin-fish, shellfish
and other aquatic species. One of skill in the art would readily
appreciate that activation of CTL activity resulting from in vivo
synthesis of antigen would produce immunity against disease not
only prophylactically but also therapeutically (after development
of disease in culture).
Aquaculture species treated by methods of this invention will
include a diversity of species of cultured fin-fish, shellfish, and
other aquatic animals. Fin-fish include all vertebrate fish, which
may be bony or cartilaginous fish. A preferred embodiment of this
invention is the immunization of fin-fish. These fin-fish include
but are not limited to salmonids, carp, catfish, yellowtail,
seabream, and seabass. Salmonids are a family of fin-fish which
include trout (including rainbow trout), salmon, and Arctic char.
Examples of shellfish include, but are not limited to, clams,
lobster, shrimp, crab, and oysters. Other cultured aquatic animals
include, but are not limited to eels, squid, and octopi.
Purification of DNA on a large scale may be accomplished by anion
exchange chromatography (for example, resins manufactured by
Qiagen, U.S. FDA Drug Master File (DMF-6224)).
DNA which is introduced to aquaculture species will encode foreign
polypeptides (e.g., those derived from viral, bacterial or
parasitic pathogens). Polypeptides of this invention refer to
complete proteins or fragments thereof, including peptides which
are epitopes (e.g., a CTL epitope) associated with an infectious
virus, bacterium or parasite.
DNA sequences encoding a complete or large parts of an antigenic
protein are preferred where humoral immunity is desired rather than
DNA sequences encoding smaller parts, such as only CTL epitopes, as
are preferred where cell-mediated immunity is desired and humoral
immunity may be deleterious. In preferred embodiments, the DNA
sequences encoding polypeptides of viral pathogens may be selected
from the group consisting of glycoprotein (G) or nucleoprotein (N)
of viral hemorrhagic septicemia virus (VHSV); G or N proteins of
infectious hematopoietic necrosis virus (IHNV); VP1, VP2, VP3 or N
structural proteins of infectious pancreatic necrosis virus (IPNV);
G protein of spring viremia of carp (SVC); and a
membrane-associated protein, tegumin or capsid protein or
glycoprotein of channel catfish virus (CCV).
In other preferred embodiments, the DNA sequences encoding
polypeptides of bacterial pathogens may be selected from the group
consisting of an iron-regulated outer membrane protein, (IROMP), an
outer membrane protein (OMP), and an A-protein of Aeromonis
salmonicida which causes furunculosis, p57 protein of Renibacterium
salmoninarum which causes bacterial kidney disease (BKD), major
surface associated antigen (msa), a surface expressed cytotoxin
(mpr), a surface expressed hemolysin (ish), and a flagellar antigen
of Yersiniosis; an extracellular protein (ECP), an iron-regulated
outer membrane protein (IROMP), and a structural protein of
Pasteurellosis; an OMP and a flagellar protein of Vibrosis
anguillarum and V. ordalii; a flagellar protein, an OMP protein,
aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and
surface antigen of Ichthyophthirius; and a structural and
regulatory protein of Cytophaga columnari; and a structural and
regulatory protein of Rickettsia.
In yet another preferred embodiment, the DNA sequences encoding
polypeptides of a parasitic pathogen may be selected from one of
the surface antigens of Ichthyophthirius.
The methods of this invention could also be used to introduce
plasmid vectors encoding polypeptides endogenous to the animal, but
which might be normally present in low concentrations (e.g., growth
hormones). In this case the expression proteins would serve a
physiological role (i.e. enhanced growth) rather than induce an
immune response.
Vectors useful in the making of expression plasmids include, but
are not limited to, vectors containing constitutive promoters,
inducible promoters, tissue-specific promoters, or promoters from
the gene of the antigen being expressed. Constitutive promoters may
include strong viral promoters, for example, promoter sequences
from cytomegalovirus (CMV), Rous sarcoma virus (RSV), simian
virus-40 (SV40), or herpes simplex virus (HSV). Tissue-specific
promoters may include the muscle beta-actin promoter or the
thymidine kinase promoter. An inducible or regulatable promoter,
for example, may include a growth hormone regulatable promoter, a
promoter under the control of lac operon sequences or an antibiotic
inducible promoter or a Zinc-inducible metallothionein
promoter.
The vector should include an expression control sequence comprising
a promoter (e.g., inducible or constitutive promoters described
above) DNA sequence, and may include, but is not limited to, an
enhancer element, an RNA processing sequence such as an intronic
sequence for splicing of a transcript or a polyadenylation signal
(e.g., from simian virus-40 (SV40) or bovine growth hormone (BGH)),
a signal sequence for secretion of the expressed protein, or one or
more copies of immunostimulatory DNA sequences known as CpG motifs.
The vector should also include one or more of the following DNA
sequences: bacterial origin of replication sequences, a selectable
marker, which may be for antibiotic resistance (e.g., kanamycin) or
for non-antibiotic resistance (e.g., .beta.-galactosidase
gene).
Oligonucleotides having unmethylated CpG dinucleotides have been
shown to activate the immune system (A. Krieg, et al., "CpG motifs
in Bacterial DNA Trigger Directed B Cell Activation" Nature
374:546-549 (1995)). Depending on the flanking sequences, certain
CpG motifs may be more immunostimulatory for B cell or T cell
responses, and preferentially stimulate certain species. Copies of
CpG motifs in DNA expression vectors act as adjuvants facilitating
the induction of an immune response against an expressed protein. A
CpG motif, a stretch of DNA containing CpG dinucleotides within a
specified sequence, may be as short as 5-40 base pairs in length.
Multiple CpG motifs may be inserted into the non-coding region of
the expression vector. When a humoral response is desired,
preferred CpG motifs will be those that preferentially stimulate a
B cell response. When cell-mediated immunity is desired, preferred
CpG motifs will be those that stimulate secretion of cytokines
known to facilitate a CD8+ T cell response.
Other CpG motifs have be found to inhibit immune responses. In a
preferred embodiment of the application, these immunoinhibitory CpG
motifs would be removed or mutated in a DNA expression vector used
by the methods of this invention, without disrupting the expression
of polypeptides therefrom.
An additional preferred embodiment of this invention relates to the
administration of a vector containing one or more different DNA
sequences, one sequence encoding an antigen and the others encoding
polypeptides which may or may not be antigenic. For example, the
vector may encode two antigens from the same pathogen.
Alternatively, the different antigen(s) may induce an immune
response against a different pathogen and thus serve as a
multivalent vaccine. Alternatively, the other polypeptides may
serve to enhance an immune response against a targeted pathogen
(e.g., helper epitopes, cytokines, carrier polypeptides, cholera
toxin subunits, or other immunostimulants).
When two or more polypeptide-encoding DNA sequences are present in
one vector, the transcription of each antigen-encoding DNA sequence
may be directed from its own promoter. Alternatively, one promoter
may drive the expression of two or more antigen-encoding DNA
sequences joined in frame to each other to express a fusion
protein. For example, VP2 and VP3 proteins of infectious pancreatic
necrosis virus (IPNV) may be fused. In another embodiment, DNA
sequences encoding two or more antigens from different diseases may
be joined to form a multivalent vaccine when expressed.
Alternatively, a DNA sequence encoding an antigenic polypeptide may
be fused to a DNA sequence encoding a carrier polypeptide. In a
preferred embodiment, the carrier polypeptide may contain one or
more envelope proteins of the hepatitis B virus, preferably from
the human hepatitis B virus. In a more preferred embodiment, the
envelope proteins of hepatitis B virus will be the small and major
protein (also referred to as surface antigen).
In another embodiment, each polypeptide-encoding DNA sequence in
the vector may be under the control of its own promoter for
expression of two or more non-fused polypeptides.
Alternatively, the DNA sequences encoding additional antigens may
be administered by using a second vector containing such sequences.
Such sequences may encode antigens from the same pathogen or
different pathogens, or cytokines, cholera toxin subunits, or other
immunostimulants. Such a vector may be administered concurrently or
sequentially with the first expression vector. A preferred
embodiment of this invention is the concurrent administration of
expression vectors. One vector may be induced to express protein
simultaneously with or after expression of protein from the other
vector.
In yet another embodiment of this invention, antigen-expressing
vectors may be administered concurrently with an antigen-based
vaccine such as a recombinant protein or whole-killed vaccine. In a
preferred embodiment, the antigen-expressing vector is administered
simultaneously with a protein antigen (i.e. recombinant protein or
whole killed pathogen). Another preferred embodiment would be to
first administer a DNA vaccine to prime the immune response
followed by administration of the protein antigen two to eight
weeks later, preferably orally or by immersion, to boost the immune
response.
The DNA used in the method of this invention is preferably purified
plasmid DNA(s) simply dissolved in an aqueous solution or in a
formulation. One of skill in the art would readily appreciate how
to formulate DNA used in the methods of this invention with known
transfection reagents such as cationic liposomes, fluorocarbon
emulsions, cochleates, tubules, gold particles, biodegradable
microspheres, or cationic polymers.
Liposomes useful for transfection of DNA of this invention include
commercially available liposomes and liposomes containing either
cationic lipids or cationic polymers. In a preferred embodiment of
this invention, liposomes would include a mixture of a neutral
lipid such as dioleoylphosphatidylethanolamine (DOPE) or
cholesterol and a cationic lipid.
In a more preferred aspect of the invention, liposomes would
include a mixture of cationic polymers and neutral lipids such as
DOPE or cholesterol. Such liposomes may be prepared as described
herein and in United States Provisional Patent Application
entitled, "A Novel Class of Cationic Reagents for High Efficient
and Cell-Type-Specific Introduction of Nucleic Acids into
Eukaryotic Cells", incorporated by reference herein. Unlike
cationic lipids, cationic polymers do not have ester-linkages and
have greater stability in vivo as a result. Cationic polymers (also
referred to as dendrimers) may be dimeric, cyclic, oligomeric, or
polymeric in structure.
Cationic polymers in an aqueous solution without neutral lipids are
also preferred transfection reagents according to the preferred
embodiments of this invention. Cationic polymers have been shown to
work well for transfecting fish cells in vitro with plasmids
expressing fish pathogen antigens (see Table 1, Example 1).
Cochleates, which are stable phospholipid-calcium precipitates
composed of phosphatidylserine, cholesterol and calcium are
desirable non-toxic and non-inflammatory transfection reagents that
can survive the digestive system. Biodegradable microspheres
composed of polymers such as polyester poly(lactide-co-glycolide)
have been used to microencapsulate DNA for transfection.
Tubules have been previously described in the literature as
lipid-based microcylinders consisting of helically wrapped bilayers
of lipid, the edges of which are packed together. DNA may be placed
in the hollow center for delivery and controlled release in
animals.
With immersion, DNA may enter cells of the epithelium of the skin,
the gills or the gut wall. With injection, DNA may enter muscle
cells or other cells in muscle tissue (e.g. fibroblasts, immune
cells) or cells of viscera within the intraperitoneal cavity. DNA
may then be expressed in these transfected cells leading to
induction of appropriate immune responses in regional or systemic
lymphoid tissue.
The invention provides for pharmaceutical compositions comprising
DNA vaccines in an amount effective for the treatment and
prevention of diseases caused by pathogens of aquaculture species.
According to another embodiment, the pharmaceutical compositions of
this invention further comprise a second DNA vaccine, an adjuvant,
a recombinant protein, a transfection reagent, or some combination
thereof.
Methods of this invention may be useful in the immunization of
aquaculture species against many pathogens. Such pathogens include
but are not limited to hemmorrhagic septicemia virus, infectious
hematopoietic necrosis virus, infectious pancreatic necrosis virus,
virus causing spring viremia of carp, channel catfish virus
(Herpesvirus ictaluri), grass carp hemorrhagic virus, nodaviridae
such as nervous necrosis virus or striped jack nervous necrosis
virus, infectious salmon anaemia virus, Aeromonis salmonicida,
Renibacterium salmoninarum, Yersinia, Pasteurella (including
piscicida), Vibrosis (including anguillarum and ordalii),
Edwardsiella (including ictaluri and tarda), Streptococci, and
Ichthyophthirius.
In one embodiment of this invention, recombinant plasmid DNA is
introduced into animals orally. DNA for oral use may be formulated
with biodegradable microspheres, fluorocarbon emulsions,
cochleates, or tubules. This is a non-stressful method of
immunizing aquaculture species by which DNA may be coated onto or
milled into feed in the form of a paste or liquid suspension or
incorporated into gelatin capsules and introduced into the
environment of the aquaculture species. Preparations of DNA for
oral use may include lactose and corn starch. The DNA can be used
with or without products to enhance entry into cells of the gut
epithelium or more deeply situated cells.
In another embodiment, pure recombinant plasmid DNA is introduced
into animals by injection with a needle or a jet-injection system,
which does not have a needle. Injection areas of the fin-fish
include but are not limited to intraperitoneal, intramuscular, and
subcutaneous areas of the fish. In a preferred embodiment, large
fin-fish are immunized by injection methods of this invention.
Typically, fish are injected with 0.1-0.5 ml of a solution
containing DNA. DNA may be injected in a pure form or may be
formulated with liposomes, cationic polymers, fluorocarbon
emulsions, cochleates, or tubules.
In yet another embodiment of this invention, pure DNA is introduced
into a fin-fish by particle bombardment. This method introduces
DNA-coated gold particles into the epidermis of a fin-fish using a
"gene-gun", which uses compressed helium to shoot the gold
particles at high speed into the skin. This method has been shown
to be particularly efficient for induction of cell-mediated immune
responses with small quantities of DNA in mice.
In another embodiment of this invention, plasmid DNA is introduced
to fish by spray. Typically, fish are exposed to spray for at least
2 seconds. Fish may pass through a mist of DNA solution by forcing
the vaccine through high-pressure paint-sprayer-type nozzles.
Typically, any pressure up to 90 psi is satisfactory. Due to the
number of pounds of fish per unit volume that can be vaccinated by
spray, it may be more economical to immunize larger fish by this
method than by immersion. The DNA can be used with or without
products to enhance entry into cells of the skin. For example, the
DNA may be associated with liposomes or cationic polymers.
In a more preferred embodiment of this invention, a large number of
animals can be immunized simultaneously by immersion in a solution
containing DNA. In one embodiment, fish are dip-netted into
suspensions containing DNA formulations (e.g., DNA formulated with
cationic polymers or liposomes) for at least several seconds. The
fish are then returned to the holding tanks in which they develop
immunity. In another embodiment, fin-fish, shellfish, or other
aquatic animals are placed into tanks containing a relatively small
volume of water. Concentrated DNA formulations (e.g., DNA
formulated with cationic polymers or liposomes) is added to the
tank, and animals are left for a period of time up to several hours
before the tank is refilled with water to restore the normal
aquatic environment. This method of immersion is preferred for the
immunization of small fry, which cannot be immunized by direct
injection.
The amount of the expression plasmid DNA that may be combined with
a carrier material to produce a single dosage form will vary
depending upon the host treated, and the particular mode of
administration. It should be understood, however, that a specific
dosage and treatment regimen for any particular fish will depend
upon a variety of factors, including the expression of the
particular plasmid DNA employed, the stability and activity of the
particular protein or peptide expressed, age, body weight, general
health, species of fish, the progress of the disease being treated,
and nature of the disease being immunized against or dreaded. The
amount of expression plasmid DNA may also depend upon whether other
therapeutic or prophylactic agents including additional expression
plasmid DNAs and adjuvants, if any, are co-administered with the
expression plasmid.
Without being bound by the values listed below, dose ranges for the
administration of DNA used in the methods of this invention may be
generalized as follows. For immunization of fish via oral routes,
0.1 to 50 .mu.g DNA per fish administered over several consecutive
days may be used. For DNA-based immunization by intramuscular or
intraperitoneal injection, 0.1 to 10 .mu.g of DNA may be used. For
spray immunization, a volume of 1 ml per fish of 0.1 to 10 mg/ml
DNA solution may be useful. Fish immunized by immersion methods of
this invention may be incubated in a 1 to 100 .mu.g/ml DNA solution
at a volume sufficient for fish to survive for a time period
necessary for uptake of DNA to produce an immune response by the
fish. An effective dosage range for immunization of fish via
gene-gun route may be 10 ng to 1 .mu.g.
Adjuvants for immunization are well known in the art and suitable
adjuvants can be combined with the DNA sequences described herein
by a person skilled in the art to form a pharmaceutical
composition. Oil adjuvants are least desirable for the methods of
this invention because they create undesirable side-effects such as
visceral adhesions (which can restrict growth) and melanized
granuloma formations (which can lower the grade of the fish at
market) and because they cannot form a homogeneous mixture with DNA
preparations. DNA-based immunization does not require oil adjuvants
and thus avoids these undesirable effects.
Adjuvants used in immunization with DNA expression plasmids of this
invention may include alum or a DNA molecule having unmethylated
CpG dinucleotides therein (also referred to as CpG adjuvant).
Oligonucleotides having unmethylated CpG dinucleotides have been
shown to activate the immune system (A. Krieg, et al., "CpG motifs
in Bacterial DNA Trigger Directed B Cell Activation" Nature
374:546-549 (1995)). CpG motifs may be inserted into a plasmid DNA
vaccine vector, and replicated in bacteria thereby allowing the CpG
motifs to retain their unmethylated form. As such, administration
of a CpG adjuvant cloned into plasmid vectors would be simultaneous
with the administration of a plasmid DNA vaccine. Alternatively, a
CpG adjuvant in the form of free oligonucleotides may be
administered before, during or after the administration of a
plasmid DNA vaccine.
Oligonucleotides having CpG motifs may be optionally modified at
their phosphodiester linkages for stability purposes. Such
modifications are well known by those of skill in the art. For
example, phosphodiester bonds in an oligonucleotide may be replaced
by phosphorothioate linkages.
The present invention also includes pharmaceutical products for all
of the uses contemplated in the methods described herein. For
example, a pharmaceutical product comprising pure plasmid DNA
vector or formulations thereof, operatively coding for an
immunogenic polypeptide or peptide, may be prepared in
physiologically acceptable administrable form (e.g., saline). The
pharmaceutical product may be placed in a container, with a notice
associated with the container in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the DNA for veterinary administration. Such
notice, for example, may be labeling approved by the Biologics
Division of Agriculture and Agri-Food Canada or the United States
Department of Agriculture (USDA) or the approved product
insert.
In order that this invention may be more fully understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in anyway.
EXAMPLES
Example 1
Cloning of DNA encoding Antigenic Proteins Into Plasmid DNA
Vectors
DNA encoding proteins of fish pathogens is useful in developing DNA
fish vaccines. Table 1 below recites fish pathogen protein
expression plasmids. Table 1 describes nucleotide sequences
encoding proteins from pathogens cloned into vectors having the
cytomegalovirus promoter (CMV), i.e., pcDNA3 (from Invitrogen) or a
vector containing the CMV promoter and intron A of CMV to promote
better expression of protein (pCMV.sub.A vector). For example,
genetic sequences coding for the major glycoprotein (G) or
nucleoprotein (N) of the viral hemorrhagic septicemia virus (VHSV)
were cloned into the EcoRI site of either the pcDNA3 or pCMV.sub.A
vector. Nucleotide sequences encoding the VP2 and VP3 structural
proteins of the infectious pancreatic necrosis virus (IPNV) were
cloned into same vectors. The gene encoding the ferric siderophore
receptor (fstA) of Aeromonas salmonicida has also been cloned into
exp ression vectors. The fstA protein is one of several possible
iron-regulated outer membrane proteins that could be expressed as
an antigen from a DNA vaccine.
TABLE 1 ______________________________________ Plasmid Vector
Antigen Pathogen ______________________________________ pCMV-G
pcDNA3 G glycoprotein viral hemorrhagic (EcoRI site) (#1-1565)*
septicemia virus pCMV.sub.A -G pCMV.sub.A vector G glycoprotein
viral hemorrhagic (EcoRI site) (#1-1565)* septicemia virus pCMV-N
pcDNA3 N nucleoprotein viral hemorrhagic (EcoRI site) (#92-1306)*
septicemia virus pCMV.sub.A -N pCMV.sub.A vector N nucleoprotein
viral hemorrhagic (EcoRI site) (#92-1306)* septicemia virus
pCMV-VP2 pcDNA3 VP2 infectious pan- creatic (HindIII/XbaI site)
(#117-1760)* necrosis virus pCMV.sub.A - pCMV.sub.A vector VP2
infectious pan- VP2 creatic (Sall/XbaI) (#117-1760)* necrosis virus
pCMV-VP3 pcDNA3 VP3 infectious pan- creatic (EcoRI/XbaI site)
(#2325-3011)* necrosis virus pCMV.sub.A - pCMV.sub.A vector VP3
infectious pan- VP3 creatic (EcoRI/XbaI site) (#2325-3011)*
necrosis virus pCMV-fstA pcDNA3 IROMP fstA Aeromonis salmonicida
(EcoRI/XbaI site) (#76-2630)* pCMV.sub.A - pCMV.sub.A vector IROMP
fstA Aeromonis fstA salmonicida (EcoRI/XbaI slte) (#76-2630)*
______________________________________ *# indicates the nucleotide
sequences within the genome of the pathogen which have been cloned
to code for the antigen
Example 2
Expression of Foreian Protein In Fish Injected with Pure Plasmid
DNA Vector
The pCMV-luc plasmid used in the following experiments contains the
luciferase reporter gene (luc) under the control of the
cytomegalovirus promoter. Purified plasmid DNA was prepared by
using commercially available Qiagen DNA purification columns. The
purified plasmid DNA was then dissolved in endotoxin-free
Dulbecco's phosphate buffered saline (DPBS) without calcium
chloride or magnesium chloride, or in 0.15M NaCl dissolved in
deionized distilled water for a final concentration of 0.001 mg/ml
to 5 mg/ml DNA. Fish were anaesthetized with 0.168 mg/ml tricaine
(3-amino benzoic acid ethylester) in water or by placing the fish
on ice for 30-60 seconds before injection. Trout and zebra fish
were injected intramuscularly between the dorsal fin and the
lateral line with 10 .mu.l of the DNA solution.
Luciferase activity was measured in the muscle and gills 2.5 days
after injection. Rainbow trout were euthanized by an overdose of
tricaine (0.1% w/v). Zebra fish were killed by immersion in ice.
The muscle or gills of the fish were removed on ice, homogenized,
centrifuged to pellet cellular debris, and the supernatants
containing soluble proteins were assayed for luciferase activity.
Luciferase assays were carried out using a kit commercially
available from Promega Corporation. Light emission in relative
light units (RLU) was quantitated by a luminometer (Analytical
Luminescence Laboratory) over a ten second interval and background
values from control samples were subtracted. The concentration of
protein in the supernatants was determined and luciferase activity
was expressed as RLU/sec/mg protein.
The results summarized in Table 2 (below) indicate that purified
plasmid DNA can efficiently transfect fish cells after
intramuscular injection. Nanogram amounts of plasmid DNA were able
to induce detectable protein expression in both the injected muscle
as well as more distant cells (e.g., in gills), showing that
different types of cells, possibly including antigen presenting
cells (APC), are transfectable by plasmid DNA and are capable of
synthesis of foreign protein. Cells distant to the site of
injection (i.e. gills) expressed lower amounts of protein than the
injected muscle cells. Zebra fish and trout are not closely related
species of fish. Therefore, the results in Table 2 indicate that
most species of fish could take up and express foreign proteins
from injected plasmids.
TABLE 2 ______________________________________ Dose Luciferase
Activity (RLU/sec/mg of protein)* of DNA Trout Zebra Fish (.mu.g)
Muscle Gills Muscle Gills ______________________________________
0.01 3,449 4 502 18 (.+-.1548) (.+-.3) (.+-.307) (.+-.8) 0.1 22,768
36 11,665 94 (.+-.12,708) (.+-.14) (.+-.2,989) (.+-.66) 1 78,408
618 826,486 228 (.+-.51,523) (.+-.567) (.+-.368,790) (.+-.115) 10
280,051 982 199,285 833 (.+-.172,749) (.+-.743) (.+-.97,134)
(.+-.621) 50 417,226 980 145,891 5,519 (.+-.165,164) (.+-.393)
(.+-.85,645) (.+-.4791) ______________________________________
*mean .+-. standard error of mean (n = 10 fish per group)
Example 3
Kinetics And Longevity of Foreign Gene Expression in Fish
One microgram of pCMV-luc plasmid in 10 .mu.l of saline was
injected intramuscularly into adult zebra fish and 3-4 month old
rainbow trout as previously described in Example 2. Luciferase
activity in the muscle and gills of the injected fish was
determined at various times between 2.5 days and 8 weeks using the
methods described in Example 2. In Table 3 (below), the plasmid DNA
directs protein expression within days of injection and protein
expression in post-mitotic muscle remains stable for at least eight
weeks. Luciferase expression in the gills falls off over time,
possibly due to cell turnover.
TABLE 3 ______________________________________ Luciferase Activity
(RLU/sec/mg of Protein)* Time Trout Zebra Fish (days) Muscle Gills
Muscle Gills ______________________________________ 0 0 0 0 0 2.5
78,408 618 2,107,048 8,705 (.+-.51,523) (.+-.567) (.+-.1,281,284)
(.+-.6853) 14 54,004 211 4,160,080 6,965 (.+-.19,411) (.+-.133)
(.+-.2,553,955) (.+-.3,672) 28 90,686 39 5,236,613 6,056
(.+-.46,044) (.+-.20) (.+-.4,536,744) (.+-.4048) 56 18,219 30
6,395,781 3,246 (.+-.11,785) (.+-.23) (.+-.1,764,195) (.+-.1,040)
______________________________________ *mean .+-. standard error of
mean (n = 10 fish per group)
Example 4
Kinetics and Longevity of Foreign Gene Expression in Zebra Fish
Zebra fish were injected intramuscularly with 0.1 .mu.g of purified
plasmid pCMV-luc DNA in 10 .mu.l of saline as previously described
in Example 2. The results from Table 4 (below) indicate that even
ten-fold less DNA than used in Example 3 is capable of producing
detectable levels of protein for at least sixteen weeks. The
results also indicate that protein expression begins within hours
after injection.
TABLE 4 ______________________________________ Luciferase Activity
(RLU/sec/mg Time of Protein)* (days) Muscle Gills
______________________________________ 0.16 64 (.+-.35) 1 (.+-.1)
0.33 1,620 (.+-.1418) 3 (.+-.2) 0.5 2,739 (.+-.1359) 30 (.+-.19) 1
2,629 (.+-.1,129) 15 (.+-.2) 2.5 11,665 (.+-.2,989) 94 (.+-.66) 112
82,424 (.+-.49,208) 103 (.+-.50)
______________________________________ *mean .+-. standard error of
mean (n = 10 fish per group)
Example 5
Transfection of Fish Cells by Injection of Plasmid DNA Formulated
With a Cationic Lipid
Zebra fish were injected intraperitoneally (IP) (i.e., in the
abdomen) with 0.1 .mu.g of pCMV-luc alone or associated with 0.5
.mu.g of a cationic lipid, G304 (obtained from Gibco BRL, New York,
USA) in 10 .mu.l.
Luciferase activity in muscle, gills, and viscera (liver, spleen,
intestine, stomach, swim bladder, pyloric caecae, and ovary or
testis) was measured 2.5 days after DNA injection. The fish tissues
were prepared as described previously (Example 2).
Table 5 (below) shows that protein is expressed from DNA that is
injected intraperitoneally into fish. Injection of plasmid DNA
formulated with cationic lipid resulted in higher foreign protein
expression in the viscera than injection of DNA alone. Muscle
tissue, on the other hand, expressed greater levels of luciferase
enzyme when the plasmid pCMV-luc DNA was injected without the
cationic lipid. Therefore, a cationic lipid may increase
transfection efficiency depending the target tissue.
TABLE 5 ______________________________________ Luciferase Activity
(RLU/sec/mg Zebra Fish of protein)* Tissue DNA alone DNA + lipid
______________________________________ Muscle 151 (.+-.127) 32
(.+-.10) Gills 15 (.+-.15) 8 (.+-.8) Viscera 3 (.+-.2) 85 (.+-.40)
______________________________________ *mean .+-. standard error of
mean (n = 10 fish per group)
Example 6
Comparison of Foreign Gene Expression after Injection of DNA in
Fish and in Mice
Fish and mice were injected intramuscularly with a range of 0.1
.mu.g to 50.0 .mu.g of pCMV-luc plasmid DNA in 50 .mu.l . Total
luciferase activity for the whole muscle of mouse or fish was
assayed 2.5 days after injection. The fish and mouse tissues were
prepared as described previously (Example 2).
For each dose of DNA, injected trout demonstrated higher levels of
luciferase activity than injected mice (Table 6 below). In general,
for a given dose of DNA, luciferase activity was approximately 100
times higher in fish than in mouse. Therefore, the knowledge that
mice can be immunized against numerous diseases using doses of DNA
within the range tested here, and the finding that fish muscle is
more easily transfected and/or that fish muscle expresses
transgenes more efficiently, indicate that fish should be good
candidates for DNA-based immunization.
TABLE 6 ______________________________________ Luciferase Activity
(RLU/sec Dose of DNA total)* (.mu.g) mouse trout
______________________________________ 0.01 N/A 65,898 (.+-.30,774)
0.1 1,649 327,724 (.+-.542) (.+-.177,583) 1.0 5,466 1,100,347
(.+-.1536) (.+-.669,634) 10 43,082 3,225,068 (.+-.5,419)
(.+-.1,869,474) 50 70,713 4,520,741 (.+-.15,921) (.+-.1,609,457)
______________________________________ *mean .+-. standard error of
mean (n = 5 for trout except for the 50 .mu. dose group for which n
= 12; n = 10 for mouse groups)
Example 7
Expression of Plasmid DNA after Immersion of Fish in DNA-containing
Solutions
Cationic lipid, G304, was obtained from Gibco BRL, New York, USA.
Cationic polymer liposomes designated Q203, Q205, Q206, Q208, Q250,
and QX were obtained from Qiagen GmbH, Hilden, Germany. Cationic
polymer liposomes are composed of a mixture of cationic polymers
and neutral lipids. Such transfection reagents were prepared as
described in U.S. Provisional Patent Application entitled, "A Novel
Class of Cationic Reagents for High Efficient and
Cell-Type-Specific Introduction of Nucleic Acids into Eukaryotic
Cells", incorporated by reference herein.
For example, a cationic polymer (either Q203, Q205, Q206, Q208,
Q250 or QX, described below in Table 7) and a neutral lipid,
dioleoyloxiphosphatidylethanolamine (DOPE) were mixed together for
a final concentration of 2 mM in chloroform, which was then
evaporated off in a rotary evaporator at 60.degree. C. The mixture
was dried for 10 minutes under a reduced pressure of 10 to 15 mbar.
Under sterile conditions, endotoxin free deionized water was added
to the mixture, which was then heated while stirring at 60.degree.
C.
Next, Q203, Q205, Q250, and QX were sonicated once for 300 seconds
at 60.degree. C. In the case of Q250, trans .beta. carotene was
added to a final concentration of 0.37 mM before sonication. Q206
and Q208 were not sonicated but were stirred at 60.degree. C. until
the solutions became transparent or slightly opalescent. The total
concentration of DOPE+cationic polymer for all liposomes was 2 mM.
The concentration of DOPE in each liposome can be calculated by
multiplying the X(DOPE) value in Table 7 by 2 mM so that, for
example, Q203-containing liposomes are 1.7 mM DOPE and 0.3 mM Q203.
Table 7 (below) summarizes the cationic polymer liposomes used in
the methods of this invention.
TABLE 7 ______________________________________ Cationic Polymer
Liposome Method of Reagent Cationic Polymers X(DOPE) Preparation
______________________________________ Q203
butandiyl-1,4-bis(octadecyl with sonication. dimethylammonium
bromide) 0.85 Q205 butandiyl-1,4-bis(octadecyl 0.82 with
sonication. dimethylammonium bromide) Q206
butandiyl-1,4-bis(octadecyl 0.78 without soni- dimethylammonium
bromide) cation. Q208 butandiyl-1,4-bis(octadecyl 0.75 without
soni- dimethylammonium bromide) cation. Q250 didodecyldimethyl
ammonium 0.571 with sonication. bromide Add trans .beta. carotene
to final concentration of 0.37 mM. QX didodecyldimethyl ammonium
0.375 with sonication. bromide
______________________________________
DNA:liposome complexes were prepared by independently diluting DNA
and liposome solutions in 0.15M NaCl, then mixing the two solutions
and vortexing, and then incubating the mixture at room temperature
for 30-45 minutes. The solutions were diluted further with water
and incubated for an additional 10-15 minutes at room temperature
prior to use with fish.
Each fish was immersed in the solution of liposome formulated DNA
(2.5 ml or 5 ml per fish) for 90 minutes and then returned to its
normal holding tank. After 2.5 days, the fish were homogenized or
gills and muscle were homogenized separately and assayed for
luciferase activity.
Table 8 (below) shows luciferase activity above background in
individual zebra fish after immersion. Thus, the results of Table 8
indicate that the majority of fish were successfully transfected
and able to express foreign protein after immersion in DNA:liposome
solutions. No fish expressed luciferase activity after immersion in
pCMV-luc DNA without liposomes. Therefore, lipid-containing
transfection reagents appear to significantly contribute to the
transfection efficiency of DNA into fish with the immersion
technique.
TABLE 8 ______________________________________ Transfection Total
Luciferase Activity Reagent (RLU/ second)
______________________________________ G304 330, 65, 0, 1643, 1581,
143, 5, 165, 0, 0, 257 Q203 268, 82, 106, 264 Q205 188, 268, 166,
136 Q206 208, 286, 170, 108, 174 Q208 668, 204, 1060, 0, 0, 180,
842, 242, 90, 36 Q250 358, 398, 60, 10, 134, 1742, 54, 136, 84, 136
QX 166, 80, 302, 74, 432, 630, 28, 28, 260, 260
______________________________________
Example 8
Induction of an Immune Response against a Protein Derived from a
Fish Pathogen in Fish and Mice by Injection of Antigen-Encoding
Plasmid DNA
Purified pCMV.sub.A -VP3 DNA (encoding the VP3 protein of
infectious pancreatic necrosis virus) was prepared as described
previously for pCMV-luc DNA and injected intramuscularly in mice or
trout. Each of three adult female BALB/C mice received 100 .mu.g in
tibialis anterior muscle while a single one-year old female trout
received 200 .mu.g in the tail muscle. Two weeks later, the humoral
immune response against the expressed antigen was determined by
ELISA assay of plasma taken from the mice and fish to detect
anti-VP3 antibody.
The ELISA assay was performed using standard techniques. In
particular, 96-well plates were coated with infectious pancreatic
necrosis virus (IPNV) particles, blocked with a non-specific
protein, and then washed. Ten-fold serial dilutions of trout or
mice plasma and control plasma (obtained from non-injected mice and
fish or from animals injected with control DNA not encoding VP3)
were put in the appropriate wells (100 .mu.l/well) were incubated
for 2 hours. After washing, bound anti-VP3 antibodies in trout and
mouse plasma were detected by addition of horse-radish
peroxidase-labeled (HRP) mouse anti-trout or goat anti-mouse IgG
monoclonal antibodies, respectively. Amounts of bound antibody were
quantitated by reaction with O-phenylenediamine dihydrochloride,
which is cleaved by HRP producing a color measurable by a
spectrophotometer at OD.sub.450.
ELISA titer values in Table 9 (below) indicate the dilution factor
which gave an OD.sub.450 value twice that of background. Table 9
shows that DNA-based immunization of fish or mice by intramuscular
injection of plasmid DNA can induce an immune response against an
antigenic protein of a fish pathogen such as the VP3 protein of
IPNV.
TABLE 9 ______________________________________ Species anti-VP3
ELISA titers ______________________________________ Mouse 527.2
Trout 14.1 ______________________________________
The collective results of the examples show the expression of
foreign proteins in cells of fish after administration of pure
plasmid DNA, either by intramuscular or intraperitoneal injection
of pure or formulated plasmid DNA, or by injection of or immersion
in DNA formulated with cationic liposomes. Furthermore, the
collective results show that an immune response can be induced if
the protein is antigenic, for example a protein of a fish pathogen.
This should lead to protection against natural infection by
virulent pathogen.
While we have hereinbefore presented a number of embodiments of
this invention, it is apparent that our basic construction can be
altered to provide other embodiments which utilize the methods of
this invention. Therefore, it will be appreciated that the scope of
this invention is to be defined by the claims appended hereto
rather than the specific embodiments which have been presented
hereinbefore by way of example.
* * * * *