U.S. patent number 6,306,625 [Application Number 08/823,578] was granted by the patent office on 2001-10-23 for method for obtaining expression of mixed polypeptide particles in yeast.
This patent grant is currently assigned to SmithKline Beecham Biologicals, Sa. Invention is credited to Eric Jacobs, Apolonia Rutgers.
United States Patent |
6,306,625 |
Jacobs , et al. |
October 23, 2001 |
Method for obtaining expression of mixed polypeptide particles in
yeast
Abstract
The present invention provides a method for obtaining expression
of a mixed hepatitis B surface antigen particle, e.g., a particle
of mixed polypeptide composition, in yeast. Also disclosed is a
method for obtaining multimeric structures presenting two or more
polypeptides by expression in yeast as fusion proteins with the S
antigen of HBsAg.
Inventors: |
Jacobs; Eric (Brussels,
BE), Rutgers; Apolonia (Brussels, BE) |
Assignee: |
SmithKline Beecham Biologicals,
Sa (Rixensart, BE)
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Family
ID: |
27567742 |
Appl.
No.: |
08/823,578 |
Filed: |
March 25, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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498545 |
Jul 5, 1995 |
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329354 |
Oct 26, 1994 |
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170610 |
Dec 21, 1993 |
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028037 |
Mar 8, 1993 |
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846487 |
Feb 28, 1992 |
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368401 |
Jun 19, 1989 |
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292202 |
Dec 30, 1988 |
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Current U.S.
Class: |
435/69.9;
424/227.1; 435/254.11; 435/254.2; 435/254.21; 435/254.23; 435/69.3;
435/71.1 |
Current CPC
Class: |
C07K
14/005 (20130101); C12N 15/81 (20130101); C12N
2730/10122 (20130101) |
Current International
Class: |
C07K
14/005 (20060101); C07K 14/02 (20060101); C12N
15/81 (20060101); C12N 015/09 () |
Field of
Search: |
;435/5,69.3,69.1,69.9,70.1,71.1,254.21,172.3,254.11,320.1,254.2,254.23,255.1 |
References Cited
[Referenced By]
U.S. Patent Documents
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4722840 |
February 1988 |
Valenzuela et al. |
4769238 |
September 1988 |
Rutter et al. |
4818527 |
April 1989 |
Thornton et al. |
4963483 |
October 1990 |
Ellis et al. |
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Foreign Patent Documents
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0 198 474 |
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Oct 1986 |
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EP |
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WO 88/01646 |
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Mar 1988 |
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WO |
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Other References
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Streeck et al., "Expression and Modification of Hepatitis B Surface
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protein in Saccharomyces cerevisiae", Gene, 48, pp. 155-163 (1986).
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Jacobs et al., "Simultaneous Synthesis of the Hepatitis B Surface
Antigens in Saccharomyces Cerevisiae and Assembly as "Mixed
Particles" Similar to those Found in the Sera of Infected Persons",
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(1988). .
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the Complete Viral Surface Protein" Hepatol. 8 No. 1, pp. 82-87
(1988). .
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Hepatitis B Virus (HBsAg)", J. Exp. Med., 168, pp. 293-306 (1988).
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Cregg et al., "Pichia pastoris as a Host System for
Transformations" Mol. Cell. Biol. 5 No. 12, pp. 3376-3385 (1985).
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Schneider et al., "Rosettes from Friend Leukemia Virus Envelope:
Preparation and Physicochemical and Partial Biological
Characterization", J. Virol., 29, pp. 624-632 (1979). .
Schneider et al., "Purification of Murine and Feline Type-C Virus
Envelope Polypeptides as Micellar Protein Complexes", Z.
Naturforsch, 36 c, pp. 353-356 (1981). .
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Friend Leukaemia Virus Glycoprotein", J. Gen. Virol., 64, pp.
559-565 (1983). .
Tamura et al., "Subunit Structure of Islet-Activating Protein,
Pertussis Toxin, in Conformity with the A-B Model", Biochem., 21,
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Jingdong, "Heterologous Gene Expression in Sacharomyces Cerevistae
Using a Dominant Selection and Amplification System", "Thesis from
the University of Ghent", Belgium (1986-87). .
Milich et al., "Immune Response to the Pre-S(1) Region of the
Hepatitis B Surface Antigen (HBsAg): A Pre-S(1)-Specific T Cell
Response can Bypass Nonresponsiveness to the Pre-S(2) and S Regions
of HBsAg", J. of Immunology, 137 No.(1), pp. 315-322 (1986). .
Milich et al., "Antibody production to the nucleocapsid and
envelope of the hepatitis B virus primed by a single synthetic T
cell site" Nature, 329, pp. 547-549 (1987). .
Neurath et al., "Antibodies to a synthetic peptide from the preS
120-145 region of the hepatitis B virus envelope are
virus-neutralizing", Vaccine, 4, pp. 35-37 (1986). .
Neurath et al., "Identification and Chemical Synthesis of a Host
Cell Receptor Binding Site on Hepatitis B Virus", Cell, 46, pp.
429-436 (1986). .
Towler et al., "The Biology and Enzymology of Eukaryotic Protein
Acylation", Ann. Rev. Biochem., 57, pp. 69-99 (1988). .
Itoh et al., "A synthetic peptide vaccine involving the product of
the pre-S(2) region of hepatitis B virus DNA: Protective efficacy
in chimpanzees", Proc. Natl. Acad. Sci. USA, 83, pp. 9174-9178
(1986). .
Itoh et al., "Synthesis in Yeast of Hepatitis B Virus Surface
Antigen Modified P31 Particles by Gene Modification", Biochem. and
Biophy. Res. Comm. 141, No. 3, pp. 942-948 (1986). .
Machida et al., "A Polypeptide Containing 55 Amino Acid Residues
Coded by the Pre-S Region of Hepatitis B Virus Deoxyribonucleic
Acid Bears the Receptor for Polymerized Human as Well as Chimpanzee
Albumins", Gastroenterology86, pp. 910-918 (1984). .
Persing et al., "The preS1 Protein of Hepatitis B Virus Is Acylated
at Its Amino Terminus with Myristic Acid", J. of Virology, 61 No.
5, pp. 1672-1677 (1987). .
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Int. Conf. on Yeast Genetics and Mol. Biol., (1988)..
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Primary Examiner: Scheiner; Laurie
Attorney, Agent or Firm: Kerekes; Zoltan Venetianer; Stephen
Kinzig; Charles M.
Parent Case Text
This is a continuation of application Ser. No. 08/498,545, filed
Jul. 5, 1995, now abandoned which is a continuation of application
Ser. No. 08/329,354, filed Oct. 26, 1994, now abandoned, which is a
continuation of Ser. No. 08/170,6 10, filed Dec. 21, 1993, now
abandoned, which is a continuation of Ser. No.08/028,037 filed Mar.
8, 1993, now abandoned, which is a continuation of Ser. No.
07/846,487 filed Feb. 28, 1992, now abandoned, which is a
continuation of Ser. No. 07/368,401, filed Jun. 19 , 1989, now
abandoned, which is a continuation-in-part of Ser. No. 07/292,202
filed Dec. 30, 1988, now abandoned.
Claims
What is claimed is:
1. A method for producing a hepatitis B surface antigen particle
comprising coexpressing in a yeast cell a first gene encoding a
first protein S and a second gene encoding a second protein X-S,
wherein S is substantially the major surface protein of hepatitis B
virus and wherein X is all or a portion of the preS1S2 polypeptide
of hepatitis B virus optionally fused to another polypeptide.
2. The method according to claim 1 further comprising coexpressing
a third gene encoding a third protein Y-S, wherein Y is all or a
portion of the preS1S2 polypeptide optionally fused to another
polypeptide, provided Y is not X.
3. The method according to claim 1 further comprising culturing
said yeast cell in an appropriate culture medium and isolating said
particle from a cell lysate or extract of said culture.
4. The method according to claim 1 wherein said particle comprises
the S protein and an M protein of HBsAg or modified versions
thereof.
5. The method according to claim 1 wherein said particle comprises
the S protein and an L protein from HBsAg or modified versions
thereof.
6. The method according to claim 1 wherein said particle comprises
the S protein, an M protein and an L protein from HBsAg or modified
versions thereof.
7. The method according to claim 1 wherein S is the 226 amino acid
mature surface protein of hepatitis B virus.
8. The method according to claim 1 wherein X comprises an epitope
of a pathogenic microorganism, virus, or cell.
9. The method according to claim 1 wherein X is a circumsporozoite
protein of Plasmodium or an immunogenic derivative thereof, an HIV
protein or an immunogenic derivative thereof, an influenza virus
protein or an immunogenic derivative thereof, or epitopes of HBV
core antigen.
10. The method according to claim 1 wherein X is all or part of a
protein which functions to induce an antibody response; or all or
part of a protein which functions as a Tc (cytotoxic T cell) or Th
(helper T cell) epitope.
11. The method according to claim 1 wherein X or Y is a functional
domain of a biologically active enzyme or protein.
12. The method according to the claim 9 wherein X is all of part of
the HIV envelop proteins gp160 and gp120; or proteins derived from
the HA2 subunit of the HA protein of influenza virus.
13. The method according to claim 1 wherein the yeast is selected
from the group consisting of Saccharomyces, Hansenula and
Pichia.
14. The method according to claim 13 wherein said yeast is S.
cerevisiae.
15. The method according to claim 1 comprising integrating by
homologous recombination in said yeast cell a first Ty transposon
expression vector carrying the coding sequence for S and a second
Ty vector carrying the coding sequence for X-S.
16. The method according to claim 1 wherein said coexpression step
comprises cotransformation said yeast with a first vector encoding
a protein comprising the S protein and a second vector encoding a
protein comprising X-S.
17. The method according to claim 14 further comprising
cotransforming said yeast cells by integrating by homologous
recombination a third Ty vector encoding a protein comprising
Y-S.
18. The method according to claim 2 further comprising
cotransforming said yeast cells by integrating by homologous
recombination a first vector encoding a protein comprising the S
protein, a second vector encoding a protein comprising X-S and a
third vector encoding a protein comprising Y-S.
19. The method according to claim 17 wherein said S, X-S and Y-S
coding sequences are carried in compatible vectors.
Description
FIELD OF THE INVENTION
This invention refers generally to the construction of yeast
strains synthesizing simultaneously different heterologous proteins
or polypeptides. More specifically the invention refers to the
production of hepatitis B surface antigen particles by introducing
two or more vectors, each carrying an individual polypeptide
expression cassette into a single yeast cell and allowing assembly
of the mixed (composite) particle.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infection in humans is associated with the
occurrence in the serum of various structures carrying the
hepatitis B surface antigen (HBsAg) [Tiollais et al, Nature, 17:489
(1985)]. In addition to infectious virions, filamentous and
spherical particles of 22 nm in diameter (containing about 100
envelope proteins) are present which are formed by association of
host-derived lipids with the three hepatitis surface proteins: the
major (S), middle (M) and large (L) proteins. These proteins share
the same sequence of 226 amino acids on the HBV genome, known as
the S protein coding sequence. The entire 163 amino acid coding
sequence which immediately precedes the S protein coding sequence
on the HBV genome is referred to herein as the preS coding
sequence. The middle protein includes the additional 55 amino acid
amino-terminal region (preS2 region) which immediately precedes the
S protein (M protein: 55 plus 226 amino acids). The large protein
includes the remaining 108 amino acid region (preS1 region) of the
preS coding sequence (L protein: 108 plus 55 plus 226 amino acids).
See, Heermann et al, J. Virol., 52:396 (1984).
Because the promoter for the S and M specific transcripts is
embedded within the open reading frame of the L protein,
transformation of mammalian cells with DNA encoding the complete
open reading frame for the L protein may result in synthesis of all
three surface proteins. In mammalian cells, HBsAg particles are
secreted [reviewed by Tiollais et al, Nature, 17:489 (1985)].
However, an overproduction of L protein relative to S protein leads
to an inhibition of secretion of HBsAg particles [See, e.g., Ou et
al, J. Virol., 61:782 (1987)].
In yeast, transcription initiation is directed by the 5' yeast
promoter sequence and viral transcription initiation signals within
the HBV genes are not functional. For that reason, expression of
the genes encoding the S, M or L protein in yeast leads in each
case to only one primary translation product. The S protein, the M
protein and the L protein have been expressed independently by
several laboratories [See, e.g., Valenzuela et al, Nature, 298:347
(1982); Valenzuela et al, Biotech., 3:317 (1985); Itoh et al,
Biochem. Biophys. Res. Comm., 138:268 (1986) and others].
Glycosylation of the surface proteins produced in S. cerevisiae
differs from glycosylation observed in mammalian cells. The major
protein is not glycosylated, whereas the middle and the large
protein are produced in N- and O-linked glycosylated and
non-glycosylated forms. N-linked chains of the high mannose type
were identified, as well as O-linked oligosaccharide chain(s) [See,
Itoh, et al, cited above; Langley et al, Gene, 67:229 (1988)]. When
extracted from yeast, both the S and M protein are recovered as
lipoproteic particles closely resembling the 22 nm particle present
in serum of human HBV patients. The L protein was also recovered in
lipoproteic structures, but their exact nature was not elucidated
[See, Rutgers et al, "Viral Hepatitis and Liver Disease", ed. A. J.
Zuckerman, A. R. Liss, New York, pp. 304-308 (1988)].
HBsAg particles have been used as carrier matrices for presentation
of foreign epitopes by fusing heterologous sequences within the
preS2 region, leading to particles with the heterologous epitopes
exposed at their surface. In one instance, it was shown that the
presentation resulted in improved immunogenicity. [Valenzuela et
al, Biotech., 3:323 (1985); Rutgers et al, Biotech., 6:1065
(1988)].
S. M. Kingsman et al, Biotech. Gen. Eng. Rev., 3:377 (1985) refers
to expression of heterologous genes in S. cerevisiae, including
HBsAg in unglycosylated form. This document also refers to the S.
cerevisiae Ty element for use in multicopy chromosomal integrative
vectors.
Rutter et al, U.S. Pat. No. 4,769,238 refers to the synthesis of
HBsAg in yeast and the construction of an expression vector
containing the S protein but excluding the 163 amino acid
presequence.
Valenzuela et al, U.S. Pat. No. 4,722,840 refers to hybrid
polypeptides formed of an HBsAg fragment fused to a heterologous
amino acid sequence defining an epitopic site of a pathogen or
toxin. The presurface polypeptide of HBsAg links the particle
forming and heterologous sequences.
A. A. Mohamad et al, Abstracts, Meeting on Hepatitis B. Viruses,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 27
(1987) refers to expression of HBsAg particles in a baculovirus
expression system. The particles are believed to be of the S or M
HBV proteins in glycosylated and unglycosylated form, the L protein
in both forms, or particles having all three proteins.
R. E. Streeck et al, J. Cell Biochem., 12B Suppl., 6 (Abstract
F010) (1988) refers to insertion of sequences, e.g. for poliovirus
type 1, into the S gene of HBV and expression in mammalian cells of
mixed particles containing HBs-Polio-Ag and HBsAg.
European Patent Application 0,198,474 refers to expression in
bacterial cells of plasmids carrying the PreS.sub.1 -PreS.sub.2 -S
protein coding region of HBsAg.
PCT Application WO88/01646 refers to a recombinant DNA containing a
transposase, a control sequence and transposable element enabling
the insertion of exogenous DNA into prokaryotic and eukaryotic
cells.
Although vaccines presently described and in use have great
efficacy, a certain percentage of persons receiving such vaccines,
particularly immunocompromised persons, e.g., hemodialysis
patients, are non- or slow responders [See, e.g., Hadler et al, New
Engl. J. Med., 315:209-215 (1986); and Bruguera et al, PostGrad.
Med. J., 63:155-158 (1987)].
Thus, there remains a need in the art for methods and compositions
useful in preparing additional effective vaccines to HBV.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 and 2 are a schematic diagram of vector pRIT12927 and of
the Ty integrative vector described in Example 1. LTR means Ty1
"long terminal repeat". Restriction sites are identified as
follows: S for SalI, X for XhoI, B for BaglII; E for EcoRI.
Parentheses indicate restriction sites that are destroyed in
constructions. Thin lines represent pBR322 sequences or linkers.
The second schematic shows the pRIT12927 derivative vector with a
selective marker and gene of interest inserted between the SalI
sites of the Ty sequence.
FIG. 2 is a schematic illustrating the expression cassettes of
pRIT13133-L, pRIT13009-L, pRIT13134-L and pRIT13034-L inserted into
the constructs of FIG. 1, in association with appropriate selection
markers. XhoI or BaglII DNA fragments having Ty homology at both
ends are purified for transformation of appropriate S. cerevisiae
host strains. Additional restriction sites are P for PstI, A for
XbaI, T for SacII, C for ClaI. pRIT13034-L is identical to
pRIT13134-L, except that it contains an S expression cassette,
instead of the L cassette. The S expression cassette is a ClaI-SalI
DNA fragment isolated from pRIT12353, which is identical to
pRIT12363 with an additional 547 base pair segment from pBR327
after the ARG3 terminator.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for obtaining
expression of a novel hepatitis B surface antigen (HBsAg) particle
of mixed polypeptide composition in yeast. The method entails
coexpressing in a yeast cell a first protein S, and a second
protein X-S, where S represents substantially the major surface
protein of hepatitis B virus (HBV) and X represents preS1S2 or
preS2 or one or more other polypeptides. Optionally a third protein
Y-S and other additional proteins may also be coexpressed with the
first protein S according to this method, with Y and the other
additional proteins being preS1S2 and preS2 or one or more
other,polypeptides.
In one embodiment, the method involves transforming a selected
yeast cell with a first vector carrying an S protein expression
cassette, and a second vector carrying the X-S expression cassette.
In another embodiment of this method a third vector carrying the
Y-S expression cassette may be cotransformed therewith. The vectors
may be successively integrated and achieve the coexpression and
assembly of the S, X, and optional Y polypeptides by homologous
recombination within the yeast cell. Preferred vectors for use in
such a method are Ty expression vectors described in detail below.
Alternatively, other Ty transposable vectors may be used to
accomplish the coexpression and assembly by the mechanism of
transposition within the yeast cell. Still another alternative
method of the invention employs compatible conventional plasmid or
phage vectors to carry the desired expression cassettes and
transform the yeast host cell.
The method of the invention thus permits the assembly of HBsAg
particles of mixed composition, i.e., an assembly of polypeptides
carrying different heterologous epitopes, or other functional
domains, in a single HBsAg particle. These particles present
several foreign epitopes, or other functional domains, in well
defined relative amounts at the surface of one HBsAg particle for
use as vaccine components.
As another aspect of the present invention there is provided a
mixed HBsAg particle, i.e., a multimeric assembly of two or more
different polypeptides including the S protein of HBsAg, or a
multimeric assembly of the S protein with one or more hybrid
polypeptides into a particulate structure of mixed polypeptide
composition (e.g., S and X-S, Y-S and the like). For example, the
mixed particle can contain the S and the M protein of HBsAg, the S
and the L protein from HBsAg, the S, M and L proteins from HBsAg,
or can also contain modified S, M and L proteins. Alternatively, X
or Y can represent an immunoprotective epitope, a B cell epitope,
or a T cell helper cell epitope or cytotoxic T cell epitope of a
pathogenic microorganism, virus, or other cell.
Still a further aspect of this invention are vaccines comprising a
mixed, composite particle of the present invention, alone or in
combination with other vaccinal agents and pharmaceutically
acceptable vaccinal carriers and adjuvants. These mixed particles
of the present invention thus broaden the spectrum of antibodies
against HBV, and may provide an earlier response to viral challenge
by the person vaccinated with the vaccines containing these
proteins. Additionally the vaccines containing these particles may
enable persons who do not respond to S protein to mount a preS
response, or may augment or enhance the S response.
Other aspects and advantages of the present invention are described
in the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for obtaining expression of a mixed
HBsAg particle of mixed polypeptide composition.
For ease in reference, the following Table I sets out the DNA
coding sequences and amino acid sequences for the preS1, preS2 and
S regions of the HBV proteins discussed herein; amino acid numbers
are above the codons in parentheses. These coding sequences are
from an HBV adw subtype, which was isolated from plasmid pRIT10616.
This plasmid, harbored in E. coli K12 strain 600, is on deposit at
the American Type Culture Collection, Rockville, Md., U.S.A. under
accession number ATCC# 38131.
TABLE I PRE-S1 REGION NcoI (12) ATG GGG ACG AAT CTT TCT GTT CCC AAC
CCT CTG GGA TTC TTT Met Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly
Phe Phe (26) CCC GAT CAT CAG TTG GAC CCT GCA TTC GGA GCC AAC TCA
AAC Pro Asp His Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn (40)
AAT CCA GAT TGG GAC TTC AAC CCC ATC AAG GAC CAC TGG CCA Asn Pro Asp
Trp Asp Phe Asn Pro Ile Lys Asp His Trp Pro (54) GCA GCC AAC CAG
GTA GGA GTG GGA GCA TTC GGG CCA GGG CTC Ala Ala Asn Gln Val Gly Val
Gly A1a Phe G1y Pro Gly Leu (68) ACC CCT CCA CAC GGC GGT ATT TTG
GGG TGG AGC CCT CAG GCT Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser
Pro Gln Ala (82) CAG GGC ATA TTG ACC ACA GTG TCA ACA ATT CCT CCT
CCT GCC Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala
(96) TCC ACC AAT CGG CAG TCA GGA AGG CAG CCT ACT CCC ATC TCT Ser
Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser (110) (119) CCA
CCT CTA AGA GAC AGT CAT CCT CAG GCC Pro Pro Leu Arg Asp Ser His Pro
Gln Ala PRE-S2 REGI0N (120) ATG CAG TGG AAT TCC ACT GCC TTC CAC CAA
GCT CTG CAG GAT Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln
Asp (134) CCC AGA GTC AGG GGT CTG TAT TTT CCT GCT GGT GGC TCC AGT
Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser (148) TCA
GGA ACA GTA AAC CCT GCT CCG AAT ATT GCC TCT CAC ATA Ser Gly Thr Val
Asn Pro Ala Pro Asn Ile Ala Ser His Ile (162) (174) TCG TCA AGC TCC
GCG AGG ACT GGG GAC CCT GTG ACG AAC Ser Ser Ser Ser Ala Arg Thr G1y
Asp Pro Val Thr Asn S PROTEIN ATG GAG AAC ATC ACA TCA GGA TTC CTA
GGA CCC CTG CTC GTG Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu
Leu Val TTA CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC ACA ATA Leu
Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile CCG CAG AGT CTA
GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA Pro Gln Ser Leu Asp Ser Trp
Trp Thr Ser Leu Asn Phe Leu GGG GGA TCA CCC GTG TGT CTT GGC CAA AAT
TCG CAG TCC CCA Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser
Pro ACC TCC AAT CAC TCA CCA ACC TCC TGT CCT CCA ATT TGT CCT Thr Ser
Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro GGT TAT CGC TGG ATG
TGT CTG CGG CGT TTT ATC ATA TTC CTC Gly Tyr Arg Trp Met Cys Leu Arg
Arg Phe Ile Ile Phe Leu TTC ATC CTG CTG CTA TGC CTC ATC TTC TTA TTG
GTT CTT CTG Phe I1e Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu
GAT TAT CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA Asp Tyr Gln
Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly TCA ACA ACA ACC AAT ACG
GGA CCA TGC AAA ACC TGC ACG ACT Ser Thr Thr Thr Asn Thr Gly Pro Cys
Lys Thr Cys Thr Thr CCT GCT CAA GGC AAC TCT ATG TTT CCC TCA TGT TGC
TGT ACA Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr AAA
CCT ACG GAT GGA AAT TGC ACC TGT ATT CCC ATC CCA TCG Lys Pro Thr Asp
Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser TCC TGG GCT TTC GCA AAA TAC
CTA TGG GAG TGG GCC TCA GTC Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu
Trp Ala Ser Val CGT TTC TCT TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG
TGG Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp TTC GTA
GGG CTT TCC CCC ACT GTT TGG CTT TCA GCT ATA TGG Phe Val Gly Leu Ser
Pro Thr Val Trp Leu Ser Ala Ile Trp ATG ATG TGG TAT TGG GGG CCA AGT
CTG TAC AGC ATC GTG AGT Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser
Ile Val Ser CCC TTT ATA CCG CTG TTA CCA ATT TTC TTT TGT CTC TGG GTA
Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val TAC ATT TAA
Tyr Ile
Additionally the following terms used throughout the specification
are defined as follows:
An "expression cassette" is defined as any discrete region of DNA
which functions in yeast as a complete gene expression unit.
A "complete gene expression unit" is a structural gene and the
promoter and regulatory regions required for its transcription and
translation.
A "functional DNA coding region" means a DNA coding sequence which,
when fused in phase to the S protein coding sequence, does not
interfere with the assembly of the HBsAg mixed particle.
A "functional derivative" means a coding sequence having amino acid
alterations which do not interfere with particle formation and
which retain immunogenicity or other functionality. Such functional
derivatives can be prepared by conventional site specific
mutagenesis [See, e.g., Botstein et al, Science, 229:1193 (1985)]
or by other standard techniques.
A "vector" is defined as DNA which can carry and maintain the DNA
fragment of the invention, including, for example, phages and
plasmids. These terms are understood by those of skill in the art
of genetic engineering.
The method of this invention involves coexpressing in a yeast cell
a first protein S and a second protein X-S, where S is
substantially the major surface protein of HBV and where X is
encoded by a functional DNA coding region fused in phase to the S
coding sequence and X is either preS1S2 or preS2, or one or more
other immunologically important polypeptides, or a functional
derivative thereof. The method of this invention may also involve
optional third, fourth and other proteins of the formula Y-S, where
Y is defined identically to X but is different from X.
According to the invention, the HBsAg antigen S protein is
coexpressed with the X or Y amino acid sequence, with the X or Y
sequence being in the form of a fusion protein with S. The coding
regions of the selected X or Y, as well as the sequence for the S
protein may be selected from published sequences and constructed by
conventional chemical or recombinant DNA techniques.
Preferred functional DNA coding regions for X and/or Y include, but
are not limited to, the entire HBV preS protein coding sequence, or
an immunogenic derivative thereof, e.g., the preS2, preS or preS1S2
protein coding sequence, or immunogenic derivatives thereof. L
proteins when incorporated into the mixed particles of this
invention, produce a particle comprising S proteins and proteins
having the important CTL epitopes from the preS region of the L
protein.
Certain S, or preS sequences are published and may include those
sequences disclosed in copending U.S. patent application Ser. No.
009,325 incorporated herein by reference, as well as the sequences
provided in Table I.
Other X and/or Y fusion components include other immunogenic coding
sequences such as coding sequences from the HBV core protein or the
coding sequence of the repeat region of the circumsporozoite
protein of Plasmodium, or an immunogenic derivative thereof [see,
e.g., published application PCT/BE88/00002]; an HIV protein or an
immunogenic derivative thereof, such as, all or part of the coding
sequences for the HIV envelope proteins gp160 and gp120; an
influenza virus protein or an immunogenic derivative thereof, such
as proteins derived from the HA2 subunit of the HA protein, such as
the "C13" and "D" proteins disclosed in copending application U.S.
Ser. No. 07/238,801, which is incorporated herein by reference..
Such sequences are published and known to those of skill in the
art.
Alternatively, X or Y or additional fusion coding sequences used in
this method may each be an immunologically important epitope(s), or
part of a protein selected from any pathogenic microorganism,
virus, or cell of interest. Such epitope or part of a protein may
function to induce an antibody response, a T.sub.h (helper T cell)
response, or may function as a T.sub.c (cytotoxic T cell) epitope
and thereby function to induce a CTL response which has been shown
to be important in immunoprotection against several infectious
disease states.
Alternatively, X or Y can comprise a functional domain of a
biologically active- enzyme or other protein. The method of the
present invention is not limited to the particular component of X
or Y, nor to the number of X-S and Y-S type proteins employed in
the co-expression.
According to this method S is preferably the entire mature surface
protein (about 226 amino acids) of HBV (see Table I). In the
protein fusions used in the method of this invention, e.g., X-S, S
means a protein encoded by an S protein coding sequence of the HBV
genome, wherein S is substantially the same as an authentic HBsAg S
protein. Truncates of S can also be employed in this method as long
as particle assembly is not adversely affected.
It now appears that there are epitopes within the pre-S region of
the L protein which are important in inducing a cytotoxic T cell
(CTL) response. However, particles consisting of L protein are
difficult to produce. However the L proteins may be employed in the
mixed particles of this invention. This invention thereby provides
a means to produce a particle comprising S proteins and L proteins
having the important CTL epitopes from the pre-S region.
The HBsAg S protein coding sequence can be isolated from DNA
extracted from Dane particles in infected human serum by filling in
the single strand region with a DNA polymerase, preferably the
endogenous polymerase, followed by digestion with a restriction
endonuclease. The choice of endonuclease will depend, in part, on
the particular Dane particles. For example, the HBsAg coding
sequence of HBV DNA of certain Dane particles of the adw serotype
can be isolated on a single BamHI fragment; the HBsAg coding
sequence of HBV DNA of certain Dane particles of the ayw serotype
can be isolated on a HhaI fragment. HBV DNA of Dane particles of
the same serotype may also exhibit different patterns of
restriction sites.
Once isolated as described above, the S protein coding sequence is
employed in the coexpression method of the present invention in two
ways. First the S protein coding sequence is placed into a vector
or an expression cassette employing conventional techniques to
allow expression of the S protein in the yeast host. Secondly, the
S protein coding sequence or a substantial portion thereof is
employed in the fusion protein with X or Y, or any other additional
epitope.
Fusing of the HBsAg S sequence to the polypeptides X or Y can be
accomplished by use of intermediate vectors. Alternatively, the
HBsAg S sequence can be inserted directly into a vector which
contains the X or Y polypeptide coding region. Techniques for
cloning DNA fragments in phages are disclosed, for example, by
Charnay et al, Nature, 286:893-895 (1980) and Tiollais, United
Kingdom Patent Application 2,034,323.
Thus, the protein X-S, for example, is constructed in a vector
comprising a DNA molecule operatively linked to a regulatory
region. The DNA molecule contains the functional DNA coding
sequence X fused in the proper reading frame to the HBsAg protein
S. This DNA molecule or an expression cassette containing the X
coding sequence, which may be any functional DNA coding sequence of
interest, fused in frame to a portion of the S protein coding
sequence can be constructed by conventional techniques, such as by
ligating the X coding sequence in the appropriate reading frame to
any codon within the S protein coding region.
Such fusion may occur at the N-terminus of the S protein coding
sequence or at some-point within the S protein. The fusion may
occur at the 5' terminus of the preS2 region, or at some point
within the preS2 region so that preS2 DNA flanks both sides of the
functional DNA coding sequence, or at the 5' terminus of a
truncated portion of the preS2 region, or at the 5' terminus of the
S protein coding region. Preferably, enough of the S coding
sequence remains after ligation of the X coding sequence,
especially where X represents a B epitope-of interest, to insure
optimal presentation of the X protein on the resulting HBsAg
particle surface. Where X represents a T cell coding sequence,
enough of the S coding sequence should remain after,ligation of the
X coding sequence to ensure particle formation. The construction of
the fusion proteins may also be prepared according to methods
described in copending U.S. patent application Ser. No. 009,325,
incorporated herein by reference.
Restriction of DNA to prepare DNA fragments used in the invention,
ligation of such fragments to prepare recombinant DNA molecules
used in the invention and insertion into microorganisms are carried
out by known techniques such as techniques disclosed in the
previously and subsequently cited references. Conditions are
selected to avoid denaturation of the DNA and enzymes. Restriction
enzymes and ligases used in carrying out this invention are
commercially available and should be used in accordance with
instructions included therewith. T4 DNA ligase is the preferred
ligase.
The various fragments and final constructions used for the
coexpression of the S protein, the X-S and, Y-S fusion proteins,
and the components of the vectors carrying these coding sequences
for coexpression into yeast cells in the method of the present
invention may be joined together using with conventional methods
known to those of skill in the art. In many cases, genes have been
isolated and restriction mapped, as well as sequenced. To that
extent, one can select the sequence of interest, such as the HBsAg
sequence, by restriction of the gene, employing further
manipulation as necessary such as resection with Bal31 in vitro
mutagenesis, primer repair, or the like, to provide a fragment of a
desired size, including the desired sequence, and having the
appropriate termini. Linkers and adapters can be used for joining
sequences, as well as replacing lost sequences, where a restriction
site employed was internal to the region of interest. The various
fragments which are isolated, may be purified by electrophoresis,
electroeluted, ligated to other sequences, cloned, reisolated and
further manipulated.
The use of regulatory regions for controlling transcription of the
structural gene of interest, such as the HBsAg sequence S and the
X-S sequence, e.g., the M protein of HBsAg, may allow for growing
the host cells to high density with no or low levels of expression
of the structural gene, and then inducing expression by changing
the environmental conditions, such as nutrient, temperature, and
the like.
The vector carrying the S protein coding sequence and the one or
more vectors carrying the X-S, Y-S or other fusion proteins of that
formula are coexpressed into a yeast host cell by conventional
techniques. The cell is then cultured in appropriate culture media,
i.e., media which enables the host to express the S protein and X-S
and Y-S fusion proteins in mixed particles in recoverable quantity.
One exemplary medium for such use is Yeast Nitrogen Base (YNB)
minimal medium. The appropriate culture media may be selected by
one of skill in the art depending upon the particular host employed
in the method.
Resulting hybrid X-S and Y-S proteins will be produced
simultaneously in yeast cells and assembled as mixed particles
together with the S protein. Such mixed particles are herein
defined as a multimeric assembly of two or more polypeptides into a
particulate structure, which is therefore characterized by a
composite nature. At the surface of the mixed particles the
different peptides are disposed in different spatial association.
The method of the present invention thus allows the formation of
conformational epitopes of pathogenic microorganisms at the surface
of an HBsAg mixed particle.
Where X and Y are components of a conformational determinant, the
method enables spatial assembly of the amino acid sequences, e.g.
X-S and Y-S, in such a manner that the presence of X and Y on the
HBsAg particle will elicit an immune response similar to that
associated with recognition of the native determinant. For example,
the components X and Y may comprise the preS2 and/or preS1 protein
of HBsAg. When employed as fusions to S, mixed particles are
produced. The present invention thereby enables the production of
HBsAg particles in yeast which mimic natural particles based on
their content of the S, M and/or L proteins of HBV.
The isolation of the mixed particle of the invention from a cell
lysate or extract of the culture medium is performed by
conventional protein isolation techniques.
A preferred embodiment of the method according to the present
invention employs Ty integrative vectors for co-expression of the
fused proteins in the yeast cell. Ty elements are a family of
naturally occurring transposable elements from the yeast
Saccharomyces cerevisiae that are structurally similar to
retroviral proviruses and the copia element family of Drosophila.
Such Ty elements and examples of vector systems are described in
detail in Mellor et al, Yeast, 2:145 (1986); Boeke et al, Cell,
40:491 (1985); Boeke et al, Cell, 42:507 (1985) and copending U.S.
patent applications Ser. Nos. 101,463 and 233,631 [SKB 12083 and
12083-1], which applications and references are incorporated herein
by reference. Preferably the Ty based plasmids and linear vectors
are derivatives of the Ty1 element isolated from the CAR1 locus of
the strain cargA.sup.+ O.sup.h -1 [Jauniaux et al, EMBO J., 1:1125
(1982)] and plasmid YEP URA3.sup.+ cargA.sup.+ O.sup.h -1.
Ty elements may be isolated from strains of S. cerevisiae by
standard techniques known to those of skill in the art. Numerous
strains of S. cerevisiae are available from public depositories and
laboratories, e.g., American Type Culture Collection, Rockville,
Md. (ATCC). The Ty elements are approximately 5.9 kb in length with
terminally repeated delta sequences or direct LTRs of approximately
335 bp surrounding the internal region of 5.3 kb. Two classes of Ty
elements may be employed in the present invention, Ty1 and Ty2,
which differ essentially by two large internal substitutions.
Construction of Ty vectors for use in carrying the S protein and
the X-S and/or Y-S fusions of the present invention allow these
sequences to be stably integrated into the chromosomes of the yeast
host cell. Such integrations are stable due to their low frequency
of excision. The vectors are thus homogeneously distributed in the
cell population, allowing a similar rate of protein synthesis in
each cell. The Ty vectors ensure that the relative copy number of
the integrated copies can be controlled and can remain the same in
each cell. Use of the Ty vectors permits the production of
substantially homogenous mixed particles according to this
embodiment of the invention. The method thus allows the yeast cell
to produce two or more gene products in the same strain at well
defined levels and with the same efficiency in each cell of the
population.
The use of this Ty vector expression system according to the method
of the invention is exemplified as follows. Two independently
different expression cassettes may be introduced into the same
yeast host strain and stably integrated into the chromosomes. One
Ty1 vector contains, e.g. the URA3 marker and contains the
TDH3-HBsAg (S antigen)-ARG3 cassette described in Example 1,
containing a strong glycolytic promoter allowing constitutive
expression of the S antigen. The other vector is introduced into
the same yeast strain carrying the LEU2 or TRP1 marker with a
preS-S expression cassette (preS2S or preS1S2S), e.g., as described
in U.S. patent application Ser. No. 009,325.
Promoters useful for expressing the preS-S cassettes include ARG3
or other promoters which allow modulation of the level of the
preS-S antigen relative to the S antigen depending on the
composition of the growth medium. In the case of ARG3, media
containing arginine or no arginine allow different expression
levels. Transformants, growing on different media, can synthesize
and assemble the mixed particles of the invention having various
compositions.
Ty sequences can be used to direct integration of vectors into the
chromosomes of a yeast host cell by standard homologous
recombination with resident Ty1 elements. More than ten Ty1 copies
are present in the chromosomes of most conventionally used S.
cerevisiae laboratory strains. According to the method employing Ty
vectors for homologous recombination, different haploid yeast
strains are transformed independently with the different vectors.
These strains can have the same or different mating type. The
transformants are easily characterized by stability of the vector,
copy numbers and expression levels. Haploid transformants having
different vector copy numbers and different expression levels can
be chosen and different diploid strains can be constructed. In such
diploid strains different relative copy numbers of the two or more
vectors can be obtained.
Copy numbers can also be increased by classical. genetics using
series of haploid transformants carrying integrated vectors at
different Ty loci. Also, the S. cerevisiae CUP1 metallothionein
structural gene which allows resistance to copper can be used in Ty
vectors with CUP.sub.S or cup1.tangle-solidup. recipient strains
(to avoid amplification of CUP1 genes at the CUP1 locus) and allow
the monitoring of vector copy numbers. Such cup1.tangle-solidup.
strains have been obtained previously, as described, for example in
D. H. Hamen et al, Science, 228:685 (1985).
Other S. cerevisiae genes which may be used as marker genes for
selection of Ty vector integrations in the same host strain are the
following: URA3, TRP1, LEU2, and ARG3. Still other marker genes in
other yeast species may also be used in an analogous method to
construct integrative vectors according to the present invention.
These two Ty1 based vector systems can be used to obtain strains of
S. cerevisiae synthesizing two or more different polypeptides
susceptible to association in vivo or during extraction to form the
hybrid particles.
Other integratable vectors may also be employed in this method,
including, for example, the Ty2 element, in a manner analogous to
the use of the Ty1 element described above and in the examples.
Additionally, retrotransposition vectors described in U.S. patent
application Ser. No. 07/233,631, incorporated herein by reference,
may also be designed. Additionally, other genomic repetitive DNA
sequences may be employed in a manner analogous to the employment
of the Ty sequences. For example, single Ty LTR sequences may also
be used to assist in the stable integration of the S, X-S and Y-S
fusion protein coding sequences into the chromosome of a yeast
strain, as well as any other repetitive genomic DNA sequences,
e.g., the Ty delta sequence. [See, e.g., Z. Jingdong, "Heterologous
Gene Expression in Saccharomyces cerevisiae Using a Dominant
Selection and Amplification System", thesis from the University of
Ghent, Belgium (1986-87)].
In other yeast genera no Ty sequences are present and therefore
vector systems must be designed to provide coexpression of the S,
X-S and Y-S protein coding sequences in yeast to create the mixed
particles described. Therefore, expression cassettes may be
designed for easy integration into the yeast chromosomes employing
other repetitive DNA sequences or plasmids such as ARS based
plasmids in yeast such as Pichia, allowing integration into the
chromosomes. An example of such applicable ARS plasmids is
described in Cregg et al, Mol. Cell. Biol., 5(12):3376 (1985). In
the expression systems described above, e.g. both the Ty expression
systems and others, the regulatory element comprises a promoter
which effects RNA polymerase binding and transcription.
Regulatable, i.e., inducible or derepressible, promoters, can also
be used. A variety of useful promoters are available for expression
of heterologous polypeptides in yeast. These include the copper
inducible metallothionein gene (CUP1) promoter and the constitutive
promoter of the glycolytic genes, glyceraldehyde-3-phosphate
dehydrogenase (TDH3) and alcohol dehydrogenase (ADH). Regions for
transcription termination in yeast are derived from the 3' end of
any of several yeast genes, for example, the gene for
iso-1-cytochrome C (CYC1). Expression systems for use in
Kluyveromyces are disclosed, e.g., in PCT WO83/04050, Hollenberg et
al; in Schizosaccharomyces, e.g., in EP-A-107170, Yamomoto; in
Pichia, e.g., in Cregg et al, Bio/Technology, 5:479 (1987); and in
Hansenula, e.g., in EP-A-299108, Hollenberg et al.
Thus, any known promoter capable of controlling the expression of
the fusion protein coding sequences in yeast may be selected. For
example, PGK, TDH3, ARG3, and APH1 are illustrative promoters
useful in the vectors of the present invention. However, if
regulatable promoters are chosen, for example, PHO5, GAL1, and
CUP1, protein synthesis must be induced in such a way that the
different constituents of the particles will be produced in well
defined relative amounts. One way to control these relative
expression levels of the different proteins is to use two or more
promoters having different strengths. For example, strong promoters
such as TDH3 could be used to control the expression for abundant
proteins, and less efficient promoters such as ARG3 could be used
to control the expression of minor proteins when coexpressed in
yeast to produce the particles of the invention.
In the practice of the methods of this invention, any species of
yeast host for which transformation, cloning and expression systems
are available may be employed in developing "mixed particles" of
HBsAg. The examples below employ Saccharomyces, specifically S.
cerevisiae. However, other yeast species may be employed including,
without limitation, Hansenula, Pichia, Candida, Kluyveromyces and
Schizosaccharomyces.
Specifically provided by the examples of this invention is an S.
cerevisiae strain containing two or more Ty vectors capable of
synthesizing simultaneously different HBsAg molecules and forming
mixed HBsAg particles in vivo in such strains or during extraction.
For example, the method of the invention using Ty1 based
integrative vectors in Examples 1-4, allows variation of the copy
numbers of three expression cassettes and, consequently, the
relative expression levels of the three envelope proteins of HBsAg.
Individual expression of S or M protein in yeast leads to recovery
of these proteins in particles similar to the 22 nm particles
present in serum of HBV patients. Thus, the mixed (S,M) lipoprotein
structures detected upon simultaneous expression of the S and M
protein are likely true particles. Mixed particles were only found
when the different surface proteins are expressed in the same cell.
Simultaneous cell rupture and subsequent processing of two strains
producing different surface proteins (mixture of cells from
independent cultures) does not give rise to mixed structures.
Preferred yeast strains are devoid of functional chromosomal CUP1
genes, which are sensitive to copper toxicity, in order to readily
select transformants having integrated several copies of vectors.
Two preferred cup1.tangle-solidup. yeast strains are described
below in Example 9.
The method of the present invention for producing such mixed
particles has advantageous utility in the production of vaccines to
pathogenic microorganisms. For example, the mixed HBsAg particle
described in the examples may be constructed to resemble particles
or filaments or virions present in the blood of a patient that
contain preS2 and preS1 epitopes exposed at their surface in
addition to epitopes from the major proteins. These particles
therefore may be useful in various formulations for an HBV vaccine.
In the formation of these vaccines only a fraction of the envelope
proteins present in the particles must be M or L proteins in order
to avoid the masquage of epitopes present on the major proteins.
Mixed particles may have immunogenic properties that are not
obtained with individual mixtures of S, M and L proteins.
Further, because purification of L particles is difficult and has
not been reported with accuracy, the method of the present
invention provides a method for producing particles carrying the
entire preS1S2 region.
The method of the present invention also allows the formation of
conformational epitopes using mixed hybrid particles of other than
HBsAg origin. For example, the malarial circumsporozoite protein,
particularly the repeat region, or various proteins of HIV virus
such as fragments of gp160 or gp120 and other proteins from various
microorganisms, viruses or cells may be employed in the method
according to the present invention. These multimeric structures may
also be employed as vaccine components for the appropriate
diseases.
For example, the method of the invention allows the formation of
mixed hybrid particles carrying both B-cell epitopes and the
relevant T-helper epitopes to enhance the B-cell response against a
pathogen of interest. [See, e.g., Milich et al, Nature, 329:547
(1987).] The method of the invention also allows the formation of
mixed hybrid particles carrying cytotoxic T cell epitopes to induce
a protective cellular immune response against a pathogen of
interest. Mixed hybrid particles may be an efficient delivery
system to elicit T-cell responses [See, e.g., Yiu et al, J. Exp.
Med., 168:293 (1988)]. The method of the invention also allows the
formation of mixed hybrid particles carrying any combination of B,
T.sub.H and T.sub.C epitopes to induce both a humoral and cellular
immune response to the pathogen of interest.
Thus this invention encompasses vaccines containing the mixed
particles of the invention. Such vaccine will contain an
immunoprotective quantity of the mixed particles, i.e., enough of
the particles are administered to elicit sufficient protective
antibody response against the agent desired to be protected against
without serious side effects. Such vaccines may be prepared by
conventional techniques. For example, a vaccine for stimulating
protection against HBV infection in humans may contain the mixed
particle described above and a suitable conventional carrier. The
mixed particle may be in an aqueous solution buffered at
physiological pH for direct use. Alternatively, the particle can be
admixed or adsorbed with a conventional adjuvant, such as; aluminum
hydroxide or aluminum phosphate. Such a mixed particle may also be
combined with other immunogens to prepare combination vaccines.
See, e.g., New Trends and Developments in Vaccines, eds. Voller et
al, University Park Press, Baltimore, Md. (1978).
Such vaccines can be administered by an appropriate route, e.g., by
the subcutaneous, intravenous or intramuscular routes. The amount
of the mixed particle of the invention present in each vaccine dose
is selected by the attending physician with regard to consideration
of the patient's age, weight, sex, general physical condition and
the like. The amount to induce an immunoprotective response in the
patient without significant adverse side effects may vary depending
upon the immunogen employed and the optional presence of an
adjuvant. Generally, it is expected that each dose will comprise
1-1000 micrograms of protein, preferably 1-200 micrograms. Initial
doses may be optionally followed by repeated boosts, where
desirable.
Another use for the mixed particles of this invention may be as
probes or reagents for detection of HBV or other pathogenic
organisms in biological samples by various conventional
immunoassays and the like.
The following Examples 1-4 illustrate the method of the invention
in the construction of a yeast strain carrying HBsAg expression
cassettes encoding respectively the S and M, the S and L, or the
three surface proteins integrated in their genomes. Such strains
synthesize simultaneously different surface proteins. The presence
of (S,M), (S,L) or (S,M,L) mixed lipoprotein structures in yeast
extracts is also demonstrated by immuneprecipitation of the major
protein with monoclonal antibodies that are specific for the middle
and the large proteins. Examples 5-8 demonstrate immunogenicity
testing of the particles and other applications of the methods of
the present invention. Example 9 describes certain preferred yeast
strains for production of the particles of this invention.
EXAMPLE 1
Ty Integrative Vectors Carrying HBsAg Expression Cassettes
The Ty vectors containing expression cassettes and selective
markers are constructed as follows. Expression cassettes for HBsAg
proteins were designed by linking DNA sequences encoding the major
(S), middle (M) or large (L) envelope proteins to the TDH3
(glyceraldehyde 3 phosphate dehydrogenase) sequences upstream from
the ATG (including the TDH3 promoter sequences) and to the ARG3
transcriptional terminator forming vectors pRIT12363(S),
pRIT12660(M) and pRIT12845(L), substantially as described in
Rutgers et al, (1988) cited above, which is incorporated herein by
reference.
pRIT12363 contains a 2900 base pair (bp) expression cassette
consisting of a HBV DNA derived coding sequence for the 226 amino
acids of the S protein flanked 5' by, and under the control of, a
1050 bp TDH3 promoter fragment and 3' by a 1150 bp fragment
carrying the ARG3 transcription terminator [Harford et al, Post.
Grad. Med. J., 63:Suppl. 2, 65-70 (1987)].
pRIT12660 contains a 3050 bp expression cassette consisting of a
HBV DNA derived coding sequence for the 281 amino acids of the M
protein flanked 5' by, and under the control of, a 1050 bp TDH3
promoter fragment and 3' by a 1150 bp fragment carrying the ARG3
transcription terminator.
pRIT12845 contains a 3370 bp expression cassette consisting of a
HBV DNA derived coding sequence for the 389 amino acids of the L
protein flanked 5' by, and under the control of, a 1050 bp TDH3
promoter fragment and 3' by a 1150 bp fragment carrying the ARG3
transcription terminator.
Co-owned copending U.S. patent application Ser. No. 009,325 is also
incorporated by reference for description of these vectors, and
sequences of preS1 and preS2. However, these particular vectors and
expression cassettes are merely exemplary and not essential to the
practice of the present invention.
The Ty based plasmids and linear vectors are derivatives of the Ty1
element isolated from the CAR1 locus of the plasmid strain YEP
URA3.sup.+ cargA.sup.+ O.sup.h -1 [Jauniaux et al, (1982) cited
above]. The HindIII fragment containing this Ty1 element and
adjacent CAR1 sequences was cloned in the SalI site of pUC9
[Amersham, U.K.; Pharmacea, Sweden] after T4 polymerase treatments,
giving rise to plasmid pRIT12927 (see FIG. 1A). All Ty1 based
vectors described herein are derivatives of pRIT12927 in which the
heterologous DNA sequences of interest replace the Ty1 internal
SalI fragment (see FIG. 1B). Linear vectors are either BglII or
XhoI DNA fragments purified from the Ty plasmids after
electrophoresis on 0.8% agarose gels. These vectors can be
integrated into the genome by homologous recombinations with
resident Ty1 elements. URA3 or LEU2 genes were used as markers for
selection of the transformants through complementation of the ura3
or leu2 mutation.
A. Vector pRIT13133-L
To form this vector, the SalI fragment inserted in Ty1 is a
derivative of pBR322 containing the S. cerevisiae URA3 gene, the
APH1 (Km.sup.r) gene from Tn903 and the HBsAg cassette
pTDH3-S-tARG3 [See FIG. 1B]. The APH1 gene from Tn903 which is
present on the vector pRIT13133-L is expressed in S. cerevisiae,
conferring G418 resistance [Jimenez et al, Nature, 287:869 (1980)],
and can be used as an additional selective marker. The APH1 gene
was inserted as a 1.7 PvuII fragment [Grindley et al, Proc. Natl.
Acad. Sci. USA, 77:7176 (1980)] in the HINDIII site of pBR322 after
T4 polymerase treatments. The URA3 gene from vector YIP5 [Struhl et
al, Mol. Gen. Genet., 176:335 (1979)] is inserted in pBR322 by
replacement of the BglI fragment (pBR322, 1169-3488) after T4
polymerase treatments. The expression cassette for the S protein
from plasmid pRIT12363, described above, was inserted in the PstI
site of pBR322 as a HindIII fragment after T4 polymerase treatments
to produce pRIT13133L.
B. Plasmid pRIT13133
In the plasmid pRIT13133, the DNA sequences inserted in Ty1 are the
same as in the vector pRIT13133-L. Ty sequences extend to the LTR
XhoI sites and were ligated at XhoI.
C. Vector pRIT13009-L
The LEU2 gene was inserted in the SalI site of Ty1 as a SalI-XhoI
fragment isolated from the vector pCV9 [Petes, Cell, 19:765
(1980)]. The expression cassette for the M protein from plasmid
pRIT12660, described above, was subsequently inserted in the
remaining SalI site as a HindIII fragment after T4 polymerase
treatments.
D. Vector pRIT13134-L
URA3 gene [Rose et al, Gene, 29:113 (1984)] is carried on a BglII
fragment from plasmid pFL44 [Laboratoire de Genetique
Physiologique--C.N.R.S. Strasbourg]. The CUP1 gene is a XbaI-KpnI
DNA fragment [Butt et al, Proc. Natl. Acad. Sci., 81:3332 (1984);
Fogel et al, Curr. Genet., 7:347 (1983)]. The CUP1 gene present on
the vector pRIT13136-L is an additional marker that can be used in
CUP1.sup.s or cup1.tangle-solidup. recipient strains. The
expression cassette for the L protein is a HindIII fragment from
plasmid pRIT12845, described above. These DNA fragments were
inserted in Ty1 in a multistep construction and are joined by DNA
linkers.
EXAMPLE 2
Integration of Vectors Carrying HBsAg Expression Cassettes in the
Chromosomes of S. cerevisiae
S. cerevisiae strain 10S69d (ura3 Leu2 Trp1 gall, .alpha.) [Vrije
Universiteit Brussels] and the isogenic strain of opposite mating
type (10S69d a), obtained by mating type switching using a 2.mu.
plasmid carrying the HO gene [Cold Spring Harbor Laboratory] were
transformed with the linearized recombinant Ty vectors described in
Example 1. The linear Ty vectors (BglII or XhoI DNA fragments) were
purified after digestion of parent plasmids followed by
electrophoresis through 0.8% agarose gels. Samples containing 0.2
to 2 .mu.g of such DNA fragments and approximately 20 .mu.g of
sonicated salmon sperm DNA were used to transform approximately
10.sup.8 cells (spheroplasts), following the method of Hinnen et
al, Proc. Natl. Acad. Sci., 75:1929 (1978). Strains are cultivated
on YNB minimal medium with 2% glucose as carbon source. Tryptophan
and leucine were supplemented at 100 .mu.g/ml.
A series of URA3 transformants were obtained and purified. The URA3
gene is used in these transformations for selection of URA.sup.+
colonies (complementation of the ura3.sup.- mutation). One copy of
the URA3 gene is sufficient to allow the cell growth in the absence
of uracil in the medium. The majority of these transformants are
relatively stable. Reversion frequency to ura3 is lower than
10.sup.-4 after about 10 generations of growth on YEPD
non-selective medium consisting of 1% yeast extract, 2%
bactopeptone and 2% glucose.
Integration events were analyzed by Southern blot. Yeast DNA
isolation was performed as described by Davis et al, Meth.
Enzymol., 65:404 (1980). Southern blot analyses were performed as
described by Maniatis et al, "Molecular Cloning, A Laboratory
Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1982). Total DNA was prepared from series of candidates, digested
with EcoRI and after electrophoresis on agarose gels, transferred
to nitrocellulose filters. An HBV specific DNA probe, the nick
translated EcoRI-XbaI DNA fragment from the region coding for the
major (S) protein, was used for the detection of EcoRI genomic DNA
fragments carrying the expression cassette. Since there is only one
EcoRI site on the vector, each band corresponded to one vector
copy. One to several copies of vector can be integrated in the
transformants. One particular band present in a number of strains,
varied in intensity. Its size was compatible with the occurrence of
perfect tandem arrays of vectors. The plasmid pRIT13133 linearized
with EcoRI has the size expected for tandem EcoRI fragments of
integrated pRIT13133-L vector. It migrated identically to the
putative tandem fragment observed on Southern blots.
A series of transformants were crossed with the strain 10S69d (a),
and 2/2 segregations of the URA3 marker were observed, as well as
2/2 segregation of the EcoRI genomic fragments detected with the
HBsAg DNA probe. This showed that multiple integration events were
obtained at a single Ty locus. However, some of these genomic
fragments can be sometimes lost after meiosis. A series of
transformants were characterized, each carrying several vector
copies at one particular Ty locus.
Because many strains contain several copies of vector integrated at
single loci including frequently two or three genomic EcoRI
fragments homologous to the HBV probe in addition to the putative
tandem fragment, other arrangements of vectors may occur in
addition to tandem repeats.
Strains containing one to several copies of the expression
cassettes encoding the M or L proteins were obtained and
characterized in a similar way, using the vectors pRIT13009-L and
pRIT13134-L. Transformants carrying one or several copies of each
vector were identified. In each case, copy number was estimated by
Southern blot analyses of genomic DNA preparations as described
above for the vector pRIT13133-L. Digestions at restriction sites
that are unique in the vectors (EcoRI for pRIT13133-L, ClaI for
pRIT13009-L and pRIT13134-L) were performed, and the same HBV
specific DNA probe was used.
Analyses of genomic DNA demonstrates that HBsAg expression
cassettes are not rearranged after integration of the vector.
Internal PstI-SacII fragments from genomic DNA of transformants
carrying several vector copies have the expected size (using HBsAg,
TDH3 or ARG3 DNA probes). Transcriptional analyses were performed,
and showed synthesis of the three HBsAg mRNA species of the
expected sizes. No other vector transcripts, such as run-through
transcripts, were observed.
EXAMPLE 3
Production of HBsAg (S,M) Mixed Structures Using Strains
Synthesizing Simultaneously the Different Surface Proteins
Having characterized series of transformants carrying different
copy numbers of a given expression cassette in the isogenic S.
cerevisiae haploids 10S69d (a) and 10S69d (.alpha.), selected
haploids were mated to obtain diploids carrying two different
expression cassettes, each in its preselected copy number.
A haploid transformant Y748 containing 4 to 5 copies of vector
pRIT13133-L (S expression cassette) and the haploid transformant
Y728 containing 1 copy of vector pRIT13009-L (M expression
cassette) were mated to give the diploid strain Y729 which thus
carries 4 to 5 copies of the S expression cassette together with 1
copy of the M expression cassette.
Cells, grown in YNB 2% glucose (with tryptophan 100 .mu.g/ml) to
mid-exponential phase, were collected, washed with cold water, and
resuspended in 50 mM sodium phosphate buffer, pH8.1, 4 mM ethylene
dinitrilo tetraacetic acid (EDTA), 1% Tween 20, 4 mM
polymethylsulfonamide (PMSF), and 10% propanol-2. The ratio of
buffer volume to wet weight of cells was 1-2 to 1. Cells were
broken by passing two times through a French Press at 20,000 psi,
and cellular debris removed by centrifugation for 30 minutes at
16,000 g. Crude cell extracts were centrifuged to equilibrium in
1.5 M CsCl, 25 mM sodium phosphate buffer, pH 7.4. Gradients were
fractionated.
Each fraction was analyzed for HBsAg antigenicity using the
AUSRIA.sup.R test [Abbott Lab] performed according to
manufacturer's instructions. The AUSRIA.sup.R test assays only
assembled surface proteins, not protein monomers. A peak of HBsAg
antigenicity is observed at a density of 1.18-1.19 g/cm.sup.3 in
the CsCl gradient of the Y748 extract, while the Y728 extract gives
a peak at a slightly higher density of 1.20-1.21 g/cm.sup.3. The
Y729 extract gives a peak of AUSRIA.sup.R activity of intermediate
density.
Each fraction was also analyzed for the presence of surface
proteins by immunoblot as follows: After sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli,
Nature, 227:680 (1970) and blotting to nitrocellulose according to
Towbin et al, Proc. Natl. Acad. Sci.. USA, 76:4350 (1979), the
sheet was incubated with a monoclonal antibody, RF6, [Royal Free
Hospital, London] directed against a denaturation and reduction
resistent epitope localized in the S moiety of the HBsAg envelope
proteins. Detection of the immune reaction was by the
biotin-streptavidin peroxidase method [Amersham, UK]. Immunoblot
analysis of the gradient fractions showed a perfect correlation
between the presence of HBV surface proteins and AUSRIA.sup.R
activity in each of the three gradients, indicating that the vast
majority of HBV surface proteins in the crude extract are present
in a lipoproteic structure.
The immunoblots show that the AUSRIA.sup.R active peak obtained
from the Y748 extract contains the expected 23 kd band
corresponding to the S protein. Additionally, the peak obtained
with Y728 contains a 33 kd and 30 kd band, the N-glycosylated and
unglycosylated form of the M protein. The AUSRIA.sup.R active peak
obtained with the extract from the diploid strain Y729 contains the
33, 30 and 23 kd species. The results with Y748 and Y728 are
identical to published results obtained when the S and M expression
cassettes were not integrated into the genome but present on 2.mu.
derived plasmids [Harford et al, "Expression of Hepatitis B Surface
Antigen in Yeast", In Develop. Biol. Standard. Second WHO/IABS
Symposium on Viral Hepatitis, Standardization in Immunoprophylaxis
of Infections by Hepatitis B Viruses, Athens, Greece (1982), Edt.
S. Karger, Basel 54: 125-130 (1983): Rutgers et al, cited above].
The S and M protein are expressed and assembled into S and M
protein particles, respectively.
The following procedure was performed to determine whether the Y729
derived AUSRIA.sup.R active material was a mixture of S and M
protein particles, each of homogeneous subunit composition, or a
single population of particles of mixed subunit composition. CsCl
peak fractions of each gradient containing the envelope proteins as
judged by immunoblot were pooled and dialyzed against phosphate
buffered saline (PBS). The concentration of the envelope proteins
in the dialyzed material was quantified by immunoblot of two-fold
serial dilutions of the samples. Based on this quantification,
samples containing equivalent amounts of S protein and also
equivalent amounts of M and/or L protein were prepared.
Immuneprecipitation was performed with a monoclonal antibody (Mab)
specific for either the preS2 region, Mab S2.4 or Mab S2.9
[SmithKline Biologicals, Rixensart, Belgium], the N-glycosylated
form of the yeast derived L and M protein preS2 region, Mab Q19/10
[University of Gottingen, FRG] or the preS1 region, Mab S1.1
[SmithKline Biologicals, Rixensart, Belgium] of the envelope
proteins. These domain specific Mabs were prepared by standard
techniques, e.g., by fusing spleen cells from an animal immunized
with 22 nm particles derived from plasma, with a continuous cell
line such as a myeloma line, and selecting clones which produce
Mabs specific for a given region.
Immune complexes were captured by formalin-fixed Staphylococcus
aureus (Staph A) cells [Immuno-precipitin, BRL, Gaithersburg, Md.,
USA]. Rabbit anti-mouse serum was used as sandwich between the Mab
and the Staph A cells. Staph A cells were pretreated as recommended
by the supplier and finally washed and resuspended (10% w/v) in
PBS. To the samples (500 .mu.l each) of envelope protein particles
and a control sample of PBS only, washed Staph A cells (20 .mu.l)
were added and the mixture was incubated for 30 minutes at room
temperature.
After centrifugation, preS specific Mab was added to the
supernatant and incubation was for 2 hours at room temperature.
Then 20 .mu.l of Staph A-rabbit anti-mouse complex was added and
incubation continued overnight at 4.degree. C. Immune complexes
were collected by centrifugation, washed five times with PBS
containing 0.05% Tween 20 and the final pellet was resuspended in
sample buffer for SDS-PAGE. The immune precipitates were then
analyzed by immunoblot as described above.
As a control, S protein particles were mixed with M protein
particles in a ratio comparable to the one observed for the
proteins present in the Y729 derived particles. The S protein
derived from Y729 cells is coprecipitated with the M protein, while
the S protein derived from Y748 cells or the S protein present in
the mixture of S and M protein particles is not precipitated. The
immuneprecipitation reaction removes the majority of S protein from
the solution of the Y729 derived particles. In contrast, the S
protein remains in the supernatant of the immuneprecipitation
reaction on the mixture of the S and M protein particles.
Immuneprecipitation with Mab Q19/10 which is specific for a
N-glycosylation dependent preS2 epitope gave identical results.
This demonstrates that at least a large amount of S protein
synthesized in Y729 cells is coassembled with M protein into
particles of mixed (S,M) subunit composition.
EXAMPLE 4
Formation of (S,L) and (S,M,L) Mixed Structures
Co-expression of S and L protein was analyzed as described above.
Yeast extracts from the S. cerevisiae haploid transformant Y957
containing 5 to 7 copies of vector pRIT13133-L (S expression
cassette), the haploid transformant Y1034 containing 3 or 4 copies
of vector pRIT13134-L (L expression cassette), and the diploid
Y1044 formed by mating these haploids, were subjected to CsCl
equilibrium centrifugation.
AUSRIA.sup.R activity was found in a peak at a density of 1.18,
1.20 and 1.19 g/cm.sup.3 for the Y957, Y1034 and Y1044 extracts,
respectively. Immunoblots confirmed the HBsAg antigenicity profile
and showed the AUSRIA.sup.R peaks to contain the 23 kd S protein
(Y957 extract), the 38 kd and 45 kd unglycosylated and glycosylated
forms of the L protein (Y1034 extract), and both the S and L
protein species in the Y1044 derived peak. While the L protein
species from the Y1034 extract bands at a higher density than the
Y957 derived S protein, the L protein species derived from Y1044
have shifted their position to a lower density coinciding with the
S protein position.
Immuneprecipitation of the CsCl derived lipoprotein structures with
the preS-specific Mab described in Example 3 showed that the S
protein derived from Y1044 cells is co-precipitated with the L
proteins while the S protein derived from Y957 cells or the S
protein present in the mixture of S and L lipoprotein structures is
not precipitated. Analysis of the supernatants of the immune
precipitation reaction showed that the S protein is efficiently
removed from the solution of Y1044 derived lipoprotein structures.
Identical results were obtained with Mab Q19/10, demonstrating that
co-expression of S and L proteins leads to coassembly into mixed
(S,L) lipoprotein structures.
Formation of (S,M,L) mixed structures was demonstrated in a similar
way. An established (S,M) producing haploid strain was mated with
an L producing strain. Immuneprecipitations with anti-preS1 and
anti-preS2 antibodies were performed as described above for the
demonstration of (S,M) and (S,L) structures. Results show the
presence of (S,M,L) mixed structures in the extract of the diploid
strain.
EXAMPLE 5
Control Over the Relative Amounts of the Envelope Proteins by
Variation of the Relative Copy Numbers of the Expression
Cassettes
As described in the above examples, Ty1 linear expression vectors
were found to be integrated at a series of Ty1 chromosomal loci.
Haploid transformants, each carrying several vector copies at a
particular locus have been obtained by screening series of
transformants. Therefore, an increase in copy number can be
obtained by classical genetics, allowing variation of the relative
copy number of the different expression cassettes. Therefore, the
mixed particle composition may be varied and controlled by
increasing the number of expression cassettes in each vector.
EXAMPLE 6
Antigenicity and Immunogenicity Testing of Particles
Particles of this invention containing two different proteins (M
and S peptides) on the same particle were tested for their
antigenicity and their immunogenicity in Balb/c mice. In these
different assays, HBS (S) and HBS (M) particles were used
respectively as negative or positive controls. The antigenicity was
analyzed with anti-S, RF1 and RF6 [H. Thomas, Royal Free Hospital,
London] and anti-preS2, S2.7, S2.10, and S2.5 [Smith Kline
Biologicals] Mabs in ELISA tests.
The particles in serial dilutions were captured by anti-S
polyclonal antibodies [Enzygnost-Behring] and detected by the
different Mab. HBS (S), HBS (M) and HBS (S,M) particles react very
well with the two anti-S monoclonal antibodies, RF1 and RF6. For
the preS2 epitopes, only the HBS (M) and HBS (S,M) present a
reactivity with the S2.5, S2.7 and S2.10 Mab. The HBS (S) did not
give a reaction above the background value.
The immunogenicity of HBS(S,M) particles of this invention [batch
87/04 (3) in Table II below] was studied in Balb/c mice after
injection of 1 .mu.g of antigen (protein)/mouse at days 0 and 30.
The bleeding of mice was done at day 45 and the different assays
performed on individual sera. Controls included HBS(S) particles
[batch HB0158 (1) in Table II below] and HBS(M) particles [batch
35-26 (2) in Table II below] adsorbed on Al(OH).sub.3. Anti-HBS
antibodies were measured with the AusAb kit [Abbott, USA]and
expressed in mIU/ml using the Hollinger formula [Hollinger et al,
in Szumness et al eds, "Viral Hepatitis", Philadelphia: Franklin
Institute Press 451-466 (1982)].
Anti-preS2 antibodies were measured using an ELISA-inhibition
assay. Briefly, serial dilutions of the serum were incubated with
HBS (M) particles adsorbed to polystyrene wells of a microplate.
After washing, the Mab S2-5 (anti-PreS2 Mab) conjugated to
peroxidase (PO) was added to each well. The anti-preS2 antibody
present in the serum analyzed competes with S2.5-PO for the same
epitope if their antibody specificity is the same. The OD of each
unknown sample was transformed in equivalent Mab (mcg/ml) using a
standard curve obtained with the purified S2.5.
The individual anti-HBS antibody titers after two injections of
antigen (the particles or controls identified above) in Balb/c mice
are shown in Table II below. The individual anti-preS2 antibody
titers after injection of antigen in Balb/c mice are shown in the
Table III below. In these Tables, if the titer was below the
cut-off value, it was given a value of 5 for the calculation of
GMT.
TABLE II Anti-HBS Antibody Titers (mIU/ml) Negative HB0158 35-26
87/04 Mouse Controls (1) (2) (3) 1 6.4 36950 48889 184254 2 3.7
6844 9250 114780 3 0.0 30403 966 38199 4 2.6 3172 3715 60993 5 5.9
3438 82950 69028 6 10.1 120449 3853 7596 7 8.8 38361 4055 42553 8
2.3 17819 3254 127568 9 0.5 9297 35819 174571 10 4.2 7655 6530
43291 GMT 3.3 14762 8326 63464
TABLE II Anti-HBS Antibody Titers (mIU/ml) Negative HB0158 35-26
87/04 Mouse Controls (1) (2) (3) 1 6.4 36950 48889 184254 2 3.7
6844 9250 114780 3 0.0 30403 966 38199 4 2.6 3172 3715 60993 5 5.9
3438 82950 69028 6 10.1 120449 3853 7596 7 8.8 38361 4055 42553 8
2.3 17819 3254 127568 9 0.5 9297 35819 174571 10 4.2 7655 6530
43291 GMT 3.3 14762 8326 63464
The above results show that (S,M) mixed particles are immunogenic
and induce the production of antibodies against S and preS
epitopes.
EXAMPLE 7
Bordetella pertussis Toxin--Production of the Complete Toxin in
vivo in S. cerevisiae
The B. pertussis toxin is assembled as a hexamer, containing five
different subunits: S1, S2, S3, S4 and 5, S4 being present in two
copies per toxin [Tamura et al, Biochem., 21:5516 (1982)]. Genes
encoding the five subunits are constructed in expression cassettes
for expression in S. cerevisiae by conventional methods. The same
or different promoters are used, and the resulting expression
cassettes are inserted into 5 different Ty vectors according to the
invention. These vectors are introduced independently in S.
cerevisiae strains using similar methods to those described for
HBsAg expression cassettes.
Appropriate expression levels for the 5 subunits are obtained by
using different appropriate promoters and/or by adapting each
vector copy number in order to obtain the correct subunit
dosage.
EXAMPLE 8
HIV gp160--Simultaneous Production in S. cerevisiae of Antigens
From Different Isolates and Formation of "Rosettes" Containing a
Mixture of These Proteins
The gp120 or gp160 HIV envelope proteins are likely essential
constituents in an effective AIDS vaccine. The variations observed
in different viral isolates are important and seem to be
concentrated in the envelope protein-encoding region. An efficient
vaccine may be developed according to the present invention taking
into account this extensive heterogeneity and including envelope
antigens encoded by different viral isolates. The gp160 antigen of
HIV could assemble in "rosettes" similar to those reported for type
C retrovirus envelope proteins, [see, Schneider et al, J. Virol.,
29:624 (1979); Schneider et al, Z. Naturforsch, 36:353 (1981);
Schneider et al, J. Gen. Virol., 64:559 (1983)]. The gp160 protein
produced in S. cerevisiae according to this invention may be
obtained as such structures.
Thus using the methods described above a series of gp160 isolates
may be produced simultaneously in the same strain of S. cerevisiae,
in order to obtain "rosettes" structures containing a mixture of
these different gp160 proteins. A series of Ty vectors, each
containing an expression cassette encoding one isolate of gp160 may
be constructed and introduced into S. cerevisiae strains, using
conventional methods of construction of expression cassettes and
following the methods of the present invention.
EXAMPLE 9
Recipient Yeast Strains for Transformation with Ty Linear Vectors
Carrying the CUP1 Selective Marker
Two S. cerevisiae cupe1.tangle-solidup. haploid strains (CUP1 gene
disrupted in a strain containing a single copy of that gene) are
preferred as recipient yeast strains in the method of the present
invention. These strains have the respective genomes: EJ
cup1.tangle-solidup. 3d (ura 3, leu 2, trp 1, gal
1.tangle-solidup., cup 1.tangle-solidup., a) and EJ cup
1.tangle-solidup.7b (ura 3, trp 1, gal 1.tangle-solidup., cup
1.tangle-solidup., .alpha.). These strains were constructed from a
strain JWG 32-11 C (trp1, leu 2-3, 2-112, ura 3-52, can.sup.R,
Cup1: URA 3, .alpha.). A stable ura 3 spontaneous mutant was
selected and was subsequently crossed with a prototrophic strain
3962 C (a) [J. M. Wiame]. Segregants EJ cup 1.tangle-solidup. 3d
and EJ cup 1.tangle-solidup.7b were selected for their high
efficiency of transformation with Ty linear vectors.
These strains were used for integration of vectors pRIT13034-L and
pRIT13134-L, previously described herein. URA3 transformants were
isolated, and screened for resistance to copper toxicity. The more
resistant transformants appeared to contain 2 to 5 copies of
integrated vectors. By classical genetics, strains were obtained
carrying higher vector copy numbers, as described above for the
strain 10S69d transformed with pRIT13133-L. TRP+revertants of LEU2
segregants carrying several copies of vectors integrated at several
loci were shown to produce high levels of S, L or (S, L) particles,
and grew well in fermentors.
The above description and examples are illustrative and not
limiting of the invention. For example, the X and Y polypeptides
may be other than those described in the examples above. The S, M
and L proteins may be employed in modified form to construct a more
desireable mixed particle for vaccine use. In addition other
polypeptides may be employed in the methods of the invention to
generate mixed particles of appropriate composition for vaccine
use. Additional known vector components may be employed to replace
the specific marker genes, promoters and linker sequences and the
like employed in the examples. This invention encompasses all
improvements and modifications falling within the scope of the
following claims.
* * * * *