U.S. patent application number 09/297171 was filed with the patent office on 2003-06-19 for immunogenic detoxified mutant e. coli lt-a toxin.
Invention is credited to GIULIANI, MARZIA MONICA, PIZZA, MARIAGRAZIA, RAPPUOLI, RINO.
Application Number | 20030113338 09/297171 |
Document ID | / |
Family ID | 10802210 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113338 |
Kind Code |
A1 |
PIZZA, MARIAGRAZIA ; et
al. |
June 19, 2003 |
IMMUNOGENIC DETOXIFIED MUTANT E. COLI LT-A TOXIN
Abstract
An immunogenic detoxified protein is provided which comprises
the amino acid sequence of subunit A of an E. coli heat labile
toxin (LT-A) or a fragment thereof in which at least amino acid
Ala-72 of the A subunit is mutated, preferably by substitution with
Arg. The toxoid is useful as vaccine against an enterotoxigenic
strain of E. coli and is produced by recombinant DNA means by
site-directed mutagenesis. It is also an effective adjuvant.
Inventors: |
PIZZA, MARIAGRAZIA; (SIENA,
IT) ; GIULIANI, MARZIA MONICA; (SIENA, IT) ;
RAPPUOLI, RINO; (CASTELNUOVO BERARDENGA, IT) |
Correspondence
Address: |
CHIRON CORPORATION
INTELLETUAL PROPERTY - R440
P.O. BOX 8097
EMERYVILLE,
CA
94662-8097
US
|
Family ID: |
10802210 |
Appl. No.: |
09/297171 |
Filed: |
April 27, 1999 |
PCT Filed: |
October 30, 1997 |
PCT NO: |
PCT/IB97/01440 |
Current U.S.
Class: |
424/184.1 ;
424/265.1; 514/2.8; 530/350 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 31/04 20180101; A61P 37/04 20180101; C07K 14/245 20130101;
A61K 38/00 20130101; A61P 1/12 20180101 |
Class at
Publication: |
424/184.1 ;
514/2; 424/265.1; 530/350 |
International
Class: |
A61K 039/00; A01N
037/18; C07K 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 1996 |
GB |
9622660.0 |
Claims
1. An immunogenic detoxified protein comprising the amino acid
sequence of subunit A of an E.coli heat labile toxin (LT-A), or a
fragment thereof, for use as a mucosal adjuvant, wherein amino acid
Ala-72 in said sequence or fragment is substituted with an arginine
residue.
2. An immunogenic composition comprising (i) a pharmaceutically
acceptable carrier and (ii) an immunogenic detoxified protein
comprising the amino acid sequence of subunit A of an E.coli heat
labile toxin (LT-A), or a fragment thereof, for use as a vaccine,
wherein amino acid Ala-72 in said sequence or fragment is
substituted with an arginine residue.
3. An immunogenic composition according to claim 2, further
comprising a second immunogenic antigen.
4. The use of an immunogenic detoxified protein comprising the
amino acid sequence of subunit A of an E.coli heat labile toxin
(LT-A), or a fragment thereof, in the manufacture of a vaccine,
wherein amino acid Ala-72 in said sequence or fragment is
substituted with an arginine residue.
5. The use of claims 4, wherein said vaccine comprises a second
immunogenic antigen.
6. A method of vaccinating a mammal, comprising administering (i)
an effective amount of an immunogenic detoxified protein comprising
the amino acid sequence of subunit A of an E.coli heat labile toxin
(LT-A), or a fragment thereof, wherein amino acid Ala-72 in said
sequence or fragment is substituted with an arginine residue,
optionally in association with (ii) a second immunogenic antigen.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to immunogenic detoxified heat
labile toxin proteins (LT) produced by enterotoxigenic strains of
Escherichia coli (E.coli) wherein at least amino acid Ala-72 of the
A subunit is mutated, and to their use in vaccines which are useful
in the prevention or treatment of enterotoxigenic E.coli infections
and as adjuvants for other immunogenic proteins. Toxoids of the
invention can be suitably produced using recombinant DNA techniques
by site-directed mutagenesis of DNA encoding the wild-type
toxins.
BACKGROUND TO THE INVENTION
[0002] Heat-labile enterotoxin (LT), produced by enterotoxigenic
strains of E. coli, and cholera toxin (CT), produced by V.cholerae
strains, are the causative agents of traveller's diarrhoea and
cholera, respectively [Spangler (1992) Microbiol Rev 56:622]. LT
and CT show 80% homology in the primary structure and an identical
tertiary structure. They are composed of two functionally distinct
domains: the enzymatically-active A subunit and the B pentamer,
which contains the receptor-binding site. The A subunit
ADP-ribosylates the target protein Gs, a GTP-binding protein which
regulates the intracellular levels of cAMP [Rappuoli & Pizza
(1991), in Sourcebook of bacterial protein toxins, Academic Press
NY]. Enhancement in cAMP levels can alter ion transport, inducing
secretion of water and chloride ions into the intestine.
[0003] CT and LT are both powerful immunogens and potent mucosal
adjuvants when co-administered with antigens at the mucosal level
[eg. Jackson et al. (1993) Infect Immun 61:4272; WO95/17211].
Immunogenicity and adjuvanticity of wild-type CT and LT have been
extensively studied in animals [eg. Rollwagen et al. (1993) Vaccine
11:1316], but their toxicity has precluded their use in humans. In
an attempt to overcome the problems generated by the use of active
holotoxins, two different approaches have been followed, one based
on the use of the B pentamer, the non-toxic domain of the holotoxin
[eg. Holmgren et al. (1992) Vaccine10:911], and the other based on
the generation of genetically detoxified derivatives of LT and CT
[eg. Fontana et al. (1995) Infect Immun 63:2356]. Site-directed
mutagenesis on both A and B subunits has provided a tool to explore
the basis of the immunological and adjuvant responses induced by
these molecules.
[0004] Examples of such experiments can be found in:
[0005] Harford et al. [Eur J Biochem (1989) 183:311] made LT-A
carrying Ser-61-Phe and Gly-79-Lys substitutions.
[0006] Tsuji et al. [J Biol Chem (1990) 265:22520] produced LT-A
carrying a Glu-112-Lys substitution.
[0007] Burnette et al. [Infect Immun (1991) 59:4266] produced CT-A
carrying a Arg-7-Lys substitution. This work can also be seen in
WO92/19265.
[0008] WO93/13202 described non-toxic CT and LT carrying mutations
at Val-53, Ser-63, Val-97, Tyr-104, and Pro-106
[0009] A mutant in the receptor binding site of the B subunit of
LT, the G33D mutant, has been reported to lack the immunological
properties of the native B subunit [Nashar et al. (1996) PNAS
93:226], suggesting that binding to the receptor is important for
the immunogenicity. It has also been shown that non-toxic
derivatives of LT carrying mutant A subunits retain the
immunological properties of wild-type LT [eg. Magagnoli et al.
(1996) 64:5434], suggesting that ADP-ribosylation activity is not
essential for immunogenicity.
[0010] In relation to adjuvanticity, much data has been generated
but many questions remain unanswered. Some studies have suggested
that LT-B and CT-B have adjuvant activity, but the conclusions
drawn have been compromised by contamination with active toxin
[Wilson et al. (1993) Vaccine 11:113]. Studies with recombinant
LT-B and CT-B have suggested that they behave as poor mucosal
adjuvants [eg. Douce et al. (1997) Infect. Immun. 65:2821]
[0011] Attempts to define the role of ADP-ribosylation activity in
the adjuvanticity of LT has generated conflicting results. Lycke et
al. [Eur J Immunol (1992) 22:2277] have described a non-toxic LT
derivative (LT-E112K) which, when administered with KLH by oral
route in mice, lacked the adjuvant properties of wild-type LT, thus
suggesting that adjuvant activity is linked to ADP-ribosylation
activity. LT derivatives such as LT-K7 and LT-K63 [eg. Douce et al.
(1997) supra; Douce et al. (1996) PNAS 92:1644; DiTommaso et al.
(1996) Infect Immun 64:974], however, which are devoid of toxicity
both in vitro and in vivo, have been shown to be able to elicit an
antibody response against a co-administered antigen in intranasally
immunised mice. LT-K63 has been shown to induce measle-specific CTL
response after intranasal immunisation with a synthetic peptide
[Partidos et al. (1996) Immunol 89:483], and strongly enhances
protection against H.pylori following intragastric immunisation
with H.pylori antigens. The antibody titres induced by these
non-toxic LT mutants were lower than those obtained with wild-type
toxin, however, and were only detected after two mucosal
immunisations [Douce et al. (1997) supra].
[0012] It is an object of the invention to provide forms of LT
which are detoxified, so that they might be suitable for use in
humans, but which retain the adjuvant and immunogenic properties of
LT as far as possible.
SUMMARY OF THE INVENTION
[0013] According to a first aspect of the present invention, there
is provided an immunogenic detoxified protein comprising the amino
acid sequence of subunit A of an E.coli heat labile toxin (LT-A),
or a fragment thereof, wherein at least amino acid Ala-72 in said
sequence or fragment is mutated.
[0014] As used herein, the term "detoxified" means that the toxoid
exhibits a reduction in toxicity relative to the wild-type toxin.
The toxicity may be measured in mouse cells, CHO cells, by
evaluation of the morphological changes induced in Y1 cells, or
preferably by the rabbit ileal loop assay. As measured in Y1 cells,
for instance, "detoxified" means that the toxoid exhibits a
reduction in toxicity relative to the wild-type toxin of greater
than 10,000-fold.
[0015] Any residual toxicity should be sufficiently low for the
protein to be used in an effective immunogenic composition without
causing significant side effects. Certain mutants within the scope
of the invention may possess zero toxic activity.
[0016] As used herein, the term "residual toxicity" means that the
detoxified immunogenic protein may retain a measurable toxicity.
More particularly the level of toxicity may be optimised in a
benefit/side-effect trade-off to maximise immunogenicity and/or
adjuvanticity whilst maintaining a sufficiently low toxicity to be
tolerated by the subject after administration.
[0017] Thus, although these proteins are detoxified in the sense of
having a much lower toxicity than the wild-type protein, traces of
the enzymatic activity responsible for toxicity may remain. The
mutation causes a decrease in toxicity of which makes the toxoid
suitable for human use.
[0018] Most preferably the toxicity of the toxoid is reduced
relative to its natural occurring counterpart by greater than
10,000 fold as measured by the evaluation of the morphological
changes induced in Y1 cells or greater than 10 fold as measured by
the rabbit ileal loop assay, performed as described herein.
[0019] The term "toxoid" as used herein means a detoxified mutated
toxin protein.
[0020] In this specification, references to LT encompass the
various naturally occurring strain variants as well other variants
encompassing mutations which do not significantly alter the
properties of the subunits or of the assembled holotoxin. These
mutations may be, for instance, conservative amino acid changes
which do not affect the assembly of the holotoxin.
[0021] References to LT-A also encompass fragments of LT-A provided
that the fragment contains Ala-72.
[0022] Most importantly, the toxoid must remain immunologically
cross-reactive with the toxin from which it is derived.
[0023] The immunogenic protein may be LT subunit A toxoid, but is
preferably an assembled holotoxoid comprising a LT-A toxoid and
five LT-B subunits, which may themselves be wild-type or
mutated.
[0024] It will be appreciated that in derivatives of LT-A, such as
fragments, or in LT-A proteins of different E.coli strains, the
amino acid residue to be mutated is that which corresponds to
Ala-72 as defined for LT-A in Domenighini et al. [Molec. Microbiol.
(1995) 15:1165-1167]. Ala-72 is located on the second turn of the
alpha-helix in LT-A and faces the NAD binding site.
[0025] The mutation at Ala-72 may be a substitution, an insertion,
or a deletion. Preferably it is a substitution with a different
amino acid. The most preferred mutation is the substitution of
Ala-72 with arginine, for which the standard nomenclature is
A72R.
[0026] Whilst the A72R mutant retains residual toxicity, other
mutants within the scope of the invention may possess no toxicity,
for example mutants with one or more mutations at other sites, or
toxoids which are further detoxified by chemical means.
[0027] The or each amino acid substituted for a wild type amino
acid, whether at Ala-72 or elsewhere, may be a naturally occurring
amino acid or may be a modified or synthetic amino acid, provided
that the mutant retains the necessary immunogenic and detoxified
properties.
[0028] Substitutions which alter the amphotericity and/or
hydrophilicity of the protein whilst retaining as far as possible
the steric effect of the substituted amino acid are generally
preferred.
[0029] The toxoids of the invention may be synthesised chemically
using conventional peptide synthesis techniques, but are preferably
produced by recombinant DNA means.
[0030] Preferably the toxoid is obtained in substantially pure
form.
[0031] According to a second aspect of the invention, there is
provided an immunogenic composition comprising an immunogenic
detoxified protein of the first aspect of the invention and a
pharmaceutically acceptable carrier. This immunogenic composition
may be a vaccine against the enterotoxigenic strains of E.coli
itself and may thus optionally further comprise an adjuvant.
[0032] In addition to the properties typical of mutants devoid of
enzymatic activity, the mutant proteins also exhibit adjuvant
activity.
[0033] Additionally, the immunogenic composition may further
comprise a second antigen capable of raising an immunological
response to another disease. In such an alternative composition,
the immunogenic detoxified protein of the invention can act as a
mucosal adjuvant.
[0034] According to a third aspect of the invention, there is
provided the use of a toxoid of the first aspect as an
adjuvant.
[0035] According to a fourth aspect of the invention, there is
provided a method of vaccinating a mammal against an
enterotoxigenic strain of E.coli comprising administering an
immunologically effective amount of an immunogenic detoxified
protein according to the first aspect of the invention. Optionally,
the immunogenic detoxified protein of the invention may act as an
adjuvant for a second immunogenic protein administered separately,
sequentially or with the immunogenic detoxified protein of the
invention.
[0036] According to a fifth aspect of the invention, there is
provided a DNA sequence encoding an immunogenic detoxified protein
according to the first aspect of the invention.
[0037] Preferably the DNA sequence encodes both the detoxified
subunit A and subunit B in a polycistronic unit. Alternatively, the
DNA may encode only the detoxified subunit A.
[0038] According to a sixth aspect of the invention, there is
provided a vector comprising a DNA sequence according to the fifth
aspect of the invention.
[0039] According to a seventh aspect of the invention, there is
provided a host cell transformed with a vector according to the
sixth aspect of the invention.
[0040] The host cell may be any host capable of expressing a DNA
sequence according to the fifth aspect, but is preferably a
bacterium, most preferably E.coli, suitably transfected to produce
the desired immunogenic detoxified protein.
[0041] In a further embodiment of the seventh aspect of the
invention, the host cell may itself provide a protective species,
for example an E.coli strain mutated to a phenotype lacking wild
type LT and expressing an immunogenic detoxified protein of the
first aspect of the invention.
[0042] According to a eighth aspect of the invention, there is
provided a process for the production of an immunogenic detoxified
protein according to the first aspect of the invention comprising
culturing a host cell according to the seventh aspect of the
invention.
[0043] According to a ninth aspect of the invention, there is
provided a process for the production of DNA according to the fifth
aspect of the invention comprising the steps of subjecting a DNA
encoding a wild-type LT-A or a fragment thereof to site-directed
mutagenesis.
[0044] According to a tenth aspect of the invention, there is
provided a process for the formulation of an immunogenic
composition according to the second aspect comprising bringing an
immunogenic detoxified protein according to the first aspect of the
invention into association with a pharmaceutically acceptable
carrier, and optionally with an adjuvant.
[0045] According to a eleventh aspect of the invention, there is
provided a method for the prevention or treatment of disease in a
subject, comprising administering to said subject an
immunologically effective dose of an immunogenic composition
according to the second aspect.
[0046] Industrial Applicability
[0047] The immunogenic detoxified protein of the invention may
constitute the active component of a vaccine composition useful for
the prevention and treatment of infections by enterotoxigenic
strains of E.coli. The immunogenic detoxified protein may also be
used in a vaccine composition as a mucosal adjuvant. The
compositions are thus applicable for use in the pharmaceutical
industry.
DESCRIPTION OF THE DRAWINGS
[0048] FIGS. 1 to 5 show the biochemical and biological properties
of wild-type LT and of mutants LT-A72R (also shown as "LTR72") and
LTK63: FIG. 1 is a chromatographic profile on a superdex column,
with peak I corresponding to holotoxin and peak II to the EDTA in
the buffer; FIG. 2 is a Western blot of a trypsin digest
experiment; FIG. 3 shows the reults of in vitro ADP-ribosylation of
polyarginine; and FIGS. 4 and 5 show the results of in vitro and in
vivo toxicity experiments, respectively.
[0049] FIG. 6 shows serum anti-OVA antibody response after three
intranasal immunisations (indicated by arrows). FIG. 7 shows the
IgG subclasses after the third immunisation. FIG. 8 shows the IgA
levels after the third immunisation in serum samples (8A) and nasal
washes (8B) [mean titre and standard deviation are shown].
[0050] FIG. 9 shows serum anti-LT Ig (9A) and IgA (9B)
responses.
[0051] FIG. 10 shows OVA-driven proliferative responses. Background
values (no OVA added to clutures) varied between 1000 and 3000
cpm.
[0052] FIG. 11 shows the efficacy against systemic challenge of
intranasal immunisation with wild-type LT and with mutants A72R and
K63. Columns in 11B are mean titres and dots are individual titres.
Black dots show titres from mice immunised with LTB and not
protected against challenge.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0053] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See eg., Sambrook, et al., MOLECULAR CLONING; A
LABORATORY MANUAL, SECOND EDITION (1989); DNA CLONING, VOLUMES I
AND II (D. N Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J.
Gait ed, 1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S. J.
Higgins eds. 1984); TRANSCRIPTION AND TRANSLATION (B. D. Hames
& S. J. Higgins eds. 1984); ANIMAL CELL CULTURE (R. I. Freshney
ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B.
Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series,
METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS
FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987, Cold
Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.
155 (Wu and Grossman, and Wu, eds., respectively), Mayer and
Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR
BIOLOGY (Academic Press, London), Scopes, (1987), PROTEIN
PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition
(Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY,
VOLUMES I-IV (D. M. Weir and C. C. Blackwell eds 1986).
[0054] Standard abbreviations for nucleotides and amino acids are
used in this specification. All publications, patents, and patent
applications cited herein are incorporated by reference.
[0055] In particular, the following amino acid abbreviations are
used:
1 Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp
Cysteine C Cys Glycine G Gly Glutamic Acid E Glu Glutamine Q Gln
Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys
Methionine N Met Phenylalanine F Phe Proline P Pro Serine S Ser
Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val
[0056] As mentioned above examples of the immunogenic detoxified
protein that can be used in the present invention include
polypeptides with minor amino acid variations from the natural
amino acid sequence of the protein other than at the sites of
mutation specifically mentioned.
[0057] A significant advantage of producing the immunogenic
detoxified protein by recombinant DNA techniques rather than by
isolating and purifying a protein from natural sources is that
equivalent quantities of the protein can be produced by using less
starting material than would be required for isolating the protein
from a natural source. Producing the protein by recombinant
techniques also permits the protein to be isolated in the absence
of some molecules normally present in cells. Indeed, protein
compositions entirely free of any trace of human protein
contaminants can readily be produced because the only human protein
produced by the recombinant non-human host is the recombinant
protein at issue. Potential viral agents from natural sources and
viral components pathogenic to humans are also avoided. Also,
genetically detoxified toxin are less likely to revert to a toxic
from than more traditional, chemically detoxified toxins.
[0058] Pharmaceutically acceptable carriers include any carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition. Suitable carriers are
typically large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, lipid aggregates
(such as oil droplets or liposomes) and inactive virus particles.
Such carriers are well known to those of ordinary skill in the art.
Additionally, these carriers may function as immunostimulating
agents (adjuvants).
[0059] Preferred aditional adjuvants to enhance effectiveness of
immunogenic compositions include, but are not limited to: aluminum
salts (alum) such as aluminium hydroxide, aluminium phosphate,
aluminium sulfate etc, oil emulsion formulations, with or without
other specific immunostimulating agents such as muramyl peptides or
bacterial cell wall components, such as for example (1) MF59
(Published International patent application WO90/14837, containing
5% Squalene, 0.5% Tween.RTM. 80, 0.5% Span.RTM. 85 (optionally
containing various amounts of MTP-PE (see below), although not
required) formulated into submicron particles using a
microfluidizer such as Model 110Y microfluidizer (Microfluidics,
Newton, Mass. 02164), (2) SAF, containing 10% squalene, 0.4% Tween
80, 5% pluronic-blocked polymer L121, and thr-MDP (see below)
either microfluidized into a submicron emulsion or vortexed to
generate a larger particle size emulsion, and (3) RIBI.TM. adjuvant
system (RAS) (Ribi Immunochem, Hamilton, Mont.) containing 2%
Squalene, 0.2% Tween.RTM. 80 and one or more bacterial cell wall
components from the group consisting of monophosphoryl lipid A
(MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS)
preferably MPL+CWS (Detox.TM.), muramyl peptides such as
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) etc., and
cytokines, such as interleukins (IL-1, IL-2 etc) macrophage colony
stimulating factor (M-CSF), tumour necrosis factor (TNF) etc.
Additionally, saponin adjuvants, such as Stimulon.TM. (Cambridge
Bioscience, Worcester, Mass.) may be used or particles generated
therefrom such as ISCOMS (immunostimulating complexes).
Furthermore, Complete Freunds Adjuvant (CFA) and Incomplete Freunds
Adjuvant (IFA) may be used. Alum and MF59 are preferred.
[0060] The immunogenic detoxified protein of the invention may be
used as an adjuvant for a second antigen in an immunologically
active composition for the treatment or vaccination of the human or
animal body.
[0061] The immunogenic compositions (eg the antigen,
pharmaceutically acceptable carrier and adjuvant) typically will
contain diluents, such as water, saline, glycerol, ethanol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles.
[0062] Typically, the immunogenic compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection may also be prepared. The preparation also may be
emulsified or encapsulated in liposomes for enhanced adjuvant
effect as discussed above under pharmaceutically acceptable
carriers.
[0063] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of the antigenic polypeptides, as
well as any other of the above-mentioned components, as needed. By
"immunologically effective amount", it is meant that the
administration of that amount to an individual, either in a single
dose or as part of a series, is effective for treatment or
prevention. This amount varies depending upon the health and
physical condition of the individual to be treated, the taxonomic
group of individual to be treated (eg., nonhuman primate, primate,
etc.), the capacity of the individual's immune system to synthesize
antibodies, the degree of protection desired, the formulation of
the vaccine, the treating doctor's assessment of the medical
situation, and other relevant factors. It is expected that the
amount will fall in a relatively broad range that can be determined
through routine trials.
[0064] The immunogenic compositions are conventionally administered
parenterally, eg. by injection either subcutaneously or
intramuscularly. Additional formulations suitable for other modes
of administration include oral and pulmonary formulations,
suppositories and transdermal applications. Dosage treatment may be
a single dose schedule or a multiple dose schedule. The vaccine may
be administered in conjunction with other immunoregulatory
agents.
[0065] The term "recombinant polynucleotide" as used herein intends
a polynucleotide of genomic, cDNA, semisynthetic, or synthetic
origin which, by virtue of its origin or manipulation: (1) is not
associated with all or a portion of a polynucleotide with which it
is associated in nature, (2) is linked to a polynucleotide other
than that to which it is linked in nature, or (3) does not occur in
nature.
[0066] The term "polynucleotide" as used herein refers to a
polymeric form of nucleotides of any length, either ribonucleotides
or deoxyribonucleotides. This term refers only to the primary
structure of the molecule. Thus, this term includes double- and
single-stranded DNA and RNA. It also includes known types of
modifications, for example, labels which are known in the art,
methylation, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (eg., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (eg., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example proteins (including for eg., nucleases, toxins,
antibodies, signal peptides, poly-L-lysine, etc.), those with
intercalators (eg., acridine, psoralen, etc.), those containing
chelators (eg., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, those with modified
linkages (eg., alpha anomeric nucleic acids, etc.), as well as
unmodified forms of the polynucleotide.
[0067] A "replicon" is any genetic element, eg., a plasmid, a
chromosome, a virus, a cosmid, etc. that behaves as an autonomous
unit of polynucleotide replication within a cell; i.e., capable of
replication under its own control. This may include selectable
markers.
[0068] A "vector" is a replicon in which another polynucleotide
segment is attached, so as to bring about the replication and/or
expression of the attached segment.
[0069] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding sequences to which
they are ligated. The nature of such control sequences differs
depending upon the host organism; in prokaryotes, such control
sequences generally include promoter, ribosomal binding site, and
transcription termination sequence; in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is necessary for expression,
and may also include additional components whose presence is
advantageous, for example, leader sequences and fusion partner
sequences.
[0070] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0071] An "open reading frame" (ORF) is a region of a
polynucleotide sequence which encodes a polypeptide; this region
may represent a portion of a coding sequence or a total coding
sequence.
[0072] A "coding sequence" is a polynucleotide sequence which is
translated into a polypeptide, usually via mRNA, when placed under
the control of appropriate regulatory sequences. The boundaries of
the coding sequence are determined by a translation start codon at
the 5'-terminus and a translation stop codon at the 3'-terminus. A
coding sequence can include, but is not limited to, cDNA, and
recombinant polynucleotide sequences.
[0073] "PCR" refers to the technique of polymerase chain reaction
as described in Saiki, et al., Nature 324:163 (1986); and Scharf et
al., Science (1986) 233:1076-1078; and U.S. Pat. Nos. 4,683,195;
and 4,683,202.
[0074] As used herein, x is "heterologous" with respect to y if x
is not naturally associated with y in the identical manner; i.e., x
is not associated with y in nature or x is not associated with y in
the same manner as is found in nature.
[0075] "Homology" refers to the degree of similarity between x and
y. The correspondence between the sequence from one form to another
can be determined by techniques known in the art. For example, they
can be determined by a direct comparison of the sequence
information of the polynucleotide. Alternatively, homology can be
determined by hybridization of the polynucleotides under conditions
which form stable duplexes between homologous regions (for example,
those which would be used prior to S.sub.1 digestion), followed by
digestion with single-stranded specific nuclease(s), followed by
size determination of the digested fragments.
[0076] As used herein, the term "polypeptidel" refers to a polymer
of amino acids and does not refer to a specific length of the
product; thus, peptides, oligopeptides, and proteins are included
within the definition of polypeptide. This term also does not refer
to or exclude post expression modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations and the
like. Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring.
[0077] A polypeptide or amino acid sequence "derived from" a
designated nucleic acid sequence refers to a polypeptide having an
amino acid sequence identical to that of a polypeptide encoded in
the sequence, or a portion thereof wherein the portion consists of
at least 3-5 amino acids, and more preferably at least 8-10 amino
acids, and even more preferably at least 11-15 amino acids, or
which is immunologically identifiable with a polypeptide encoded in
the sequence. This terminology also includes a polypeptide
expressed from a designated nucleic acid sequence.
[0078] The protein may be used for producing antibodies, either
monoclonal or polyclonal, specific to the protein. The methods for
producing these antibodies are known in the art.
[0079] "Recombinant host cells", "host cells," "cells," "cell
cultures," and other such terms denote, for example,
microorganisms, insect cells, and mammalian cells, that can be, or
have been, used as recipients for recombinant vector or other
transfer DNA, and include the progeny of the original cell which
has been transformed. It is understood that the progeny of a single
parental cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original
parent, due to natural, accidental, or deliberate mutation.
Examples for mammalian host cells include Chinese hamster ovary
(CHO) and monkey kidney (COS) cells.
[0080] Specifically, as used herein, "cell line," refers to a
population of cells capable of continuous or prolonged growth and
division in vitro. Often, cell lines are clonal populations derived
from a single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants. The term "cell lines" also
includes immortalized cells. Preferably, cell lines include
nonhybrid cell lines or hybridomas to only two cell types.
[0081] As used herein, the term "microorganism" includes
prokaryotic and eukaryotic microbial species such as bacteria and
fungi, the latter including yeast and filamentous fungi.
[0082] "Transformation", as used herein, refers to the insertion of
an exogenous polynucleotide into a host cell, irrespective of the
method used for the insertion, for example, direct uptake,
transduction, f-mating or electroporation. The exogenous
polynucleotide may be maintained as a non-integrated vector, for
example, a plasmid, or alternatively, may be integrated into the
host genome.
[0083] By "genomic" is meant a collection or library of DNA
molecules which are derived from restriction fragments that have
been cloned in vectors. This may include all or part of the genetic
material of an organism.
[0084] By "cDNA" is meant a complementary DNA sequence that
hybridizes to a complementary strand of DNA.
[0085] By "purified" and "isolated" is meant, when referring to a
polypeptide or nucleotide sequence, that the indicated molecule is
present in the substantial absence of other biological
macromolecules of the same type. The term "purified" as used herein
preferably means at least 75% by weight, more preferably at least
85% by weight, more preferably still at least 95% by weight, and
most preferably at least 98% by weight, of biological
macromolecules of the same type present (but water, buffers, and
other small molecules, especially molecules having a molecular
weight of less than 1000, can be present).
[0086] Once the appropriate coding sequence is isolated, it can be
expressed in a variety of different expression systems; for example
those used with mammalian cells, baculoviruses, bacteria, and
yeast.
[0087] i. Mammalian Systems
[0088] Mammalian expression systems are known in the art. A
mammalian promoter is any DNA sequence capable of binding mammalian
RNA polymerase and initiating the downstream (3') transcription of
a coding sequence (eg. structural gene) into mRNA. A promoter will
have a transcription initiating region, which is usually placed
proximal to the 5' end of the coding sequence, and a TATA box,
usually located 25-30 base pairs (bp) upstream of the transcription
initiation site. The TATA box is thought to direct RNA polymerase
II to begin RNA synthesis at the correct site. A mammalian promoter
will also contain an upstream promoter element, usually located
within 100 to 200 bp upstream of the TATA box. An upstream promoter
element determines the rate at which transcription is initiated and
can act in either orientation [Sambrook et al. (1989) "Expression
of Cloned Genes in Mammalian Cells." In Molecular Cloning: A
Laboratory Manual, 2nd ed.].
[0089] Mammalian viral genes are often highly expressed and have a
broad host range; therefore sequences encoding mammalian viral
genes provide particularly useful promoter sequences. Examples
include the SV40 early promoter, mouse mammary tumor virus LTR
promoter, adenovirus major late promoter (Ad MLP), and herpes
simplex virus promoter. In addition, sequences derived from
non-viral genes, such as the murine metallotheionein gene, also
provide useful promoter sequences. Expression may be either
constitutive or regulated (inducible), depending on the promoter
can be induced with glucocorticoid in hormone-responsive cells.
[0090] The presence of an enhancer element (enhancer), combined
with the promoter elements described above, will usually increase
expression levels. An enhancer is a regulatory DNA sequence that
can stimulate transcription up to 1000-fold when linked to
homologous or heterologous promoters, with synthesis beginning at
the normal RNA start site. Enhancers are also active when they are
placed upstream or downstream from the transcription initiation
site, in either normal or flipped orientation, or at a distance of
more than 1000 nucleotides from the promoter [Maniatis et al.
(1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of
the Cell, 2nd ed.]. Enhancer elements derived from viruses may be
particularly useful, because they usually have a broader host
range. Examples include the SV40 early gene enhancer [Dijkema et al
(1985) EMBO J. 4:761] and the enhancer/promoters derived from the
long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.
(1982b) Proc. Natl. Acad. Sci. 79:6777] and from human
cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally,
some enhancers are regulatable and become active only in the
presence of an inducer, such as a hormone or metal ion
[Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et
al. (1987) Science 236:1237].
[0091] A DNA molecule may be expressed intracellularly in mammalian
cells. A promoter sequence may be directly linked with the DNA
molecule, in which case the first amino acid at the N-terminus of
the recombinant protein will always be a methionine, which is
encoded by the ATG start codon. If desired, the N-terminus may be
cleaved from the protein by in vitro incubation with cyanogen
bromide.
[0092] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provides for secretion of the foreign protein in
mammalian cells. Preferably, there are processing sites encoded
between the leader fragment and the foreign gene that can be
cleaved either in vivo or in vitro. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the secretion of the protein from the cell. The
adenovirus triparite leader is an example of a leader sequence that
provides for secretion of a foreign protein in mammalian cells.
[0093] Usually, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-transcriptional
cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349;
Proudfoot and Whitelaw (1988) "Termination and 3' end processing of
eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and
D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These
sequences direct the transcription of an mRNA which can be
translated into the polypeptide encoded by the DNA. Examples of
transcription terminater/polyadenylation signals include those
derived from SV40 [Sambrook et al (1989) "Expression of cloned
genes in cultured mammalian cells." In Molecular Cloning: A
Laboratory Manual].
[0094] Some genes may be expressed more efficiently when introns
(also called intervening sequences) are present. Several cDNAs,
however, have been efficiently expressed from vectors that lack
splicing signals (also called splice donor and acceptor sites) [see
eg., Gothing and Sambrook (1981) Nature 293:620]. Introns are
intervening noncoding sequences within a coding sequence that
contain splice donor and acceptor sites. They are removed by a
process called "splicing,"following polyadenylation of the primary
transcript [Nevins (1983) Annu. Rev. Biochem. 52:441; Green (1986)
Annu. Rev. Genet. 20:671; Padgett et al. (1986) Annu. Rev. Biochem.
55:1119; Krainer and Maniatis (1988) "RNA splicing." In
Transcription and splicing (ed. B. D. Hames and D. M. Glover)].
[0095] Usually, the above described components, comprising a
promoter, polyadenylation signal, and transcription termination
sequence are put together into expression constructs. Enhancers,
introns with functional splice donor and acceptor sites, and leader
sequences may also be included in an expression construct, if
desired. Expression constructs are often maintained in a replicon,
such as an extrachromosomal element (eg., plasmids) capable of
stable maintenance in a host, such as mammalian cells or bacteria.
Mammalian replication systems include those derived from animal
viruses, which require trans-acting factors to replicate. For
example, plasmids containing the replication systems of
papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or
polyomavirus, replicate to extremely high copy number in the
presence of the appropriate viral T antigen. Additional examples of
mammalian replicons include those derived from bovine
papillomavirus and Epstein-Barr virus. Additionally, the replicon
may have two replication systems, thus allowing it to be
maintained, for example, in mammalian cells for expression and in a
procaryotic host for cloning and amplification. Examples of such
mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.
(1989) Mol. Cell. Biol. 9:946 and pHEBO [Shimizu et al. (1986) Mol.
Cell. Biol. 6:1074].
[0096] The transformation procedure used depends upon the host to
be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0097] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalized cell lines available
from the American Type Culture Collection (ATCC), including but not
limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (eg., Hep G2), and a number of other
cell lines.
[0098] ii. Baculovirus Systems
[0099] The polynucleotide encoding the protein can also be inserted
into a suitable insect expression vector, and is operably linked to
the control elements within that vector. Vector construction
employs techniques which are known in the art.
[0100] Generally, the components of the expression system include a
transfer vector, usually a bacterial plasmid, which contains both a
fragment of the baculovirus genome, and a convenient restriction
site for insertion of the heterologous gene or genes to be
expressed; a wild type baculovirus with a sequence homologous to
the baculovirus-specific fragment in the transfer vector (this
allows for the homologous recombination of the heterologous gene in
to the baculovirus genome); and appropriate insect host cells and
growth media.
[0101] After inserting the DNA sequence encoding the protein into
the transfer vector, the vector and the wild type viral genome are
transfected into an insect host cell where the vector and viral
genome are allowed to recombine. The packaged recombinant virus is
expressed and recombinant plaques are identified and purified.
Materials and methods for baculovirus/insect cell expression
systems are commercially available in kit form from, inter alia,
Invitrogen, San Diego Calif. ("MaxBac" kit). These techniques are
generally known to those skilled in the art and fully described in
Summers and Smith, Texas Agricultural Experiment Station Bulletin
No. 1555 (1987) (hereinafter "Summers and Smith").
[0102] Prior to inserting the DNA sequence encoding the protein
into the baculovirus genome, the above described components,
comprising a promoter, leader (if desired), coding sequence of
interest, and transcription termination sequence, are usually
assembled into an intermediate transplacement construct (transfer
vector). This construct may contain a single gene and operably
linked regulatory elements; multiple genes, each with its owned set
of operably linked regulatory elements; or multiple genes,
regulated by the same set of regulatory elements. Intermediate
transplacement constructs are often maintained in a replicon, such
as an extrachromosomal element (eg., plasmids) capable of stable
maintenance in a host, such as a bacterium. The replicon will have
a replication system, thus allowing it to be maintained in a
suitable host for cloning and amplification.
[0103] Currently, the most commonly used transfer vector for
introducing foreign genes into AcNPV is pAc373. Many other vectors,
known to those of skill in the art, have also been designed. These
include, for example, pVL985 (which alters the polyhedrin start
codon from ATG to ATT, and which introduces a BamHI cloning site 32
basepairs downstream from the ATT; see Luckow and Summers, Virology
(1989) 17:31.
[0104] The plasmid usually also contains the polyhedrin
polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol.,
42:177) and a procaryotic ampicillin-resistance (amp) gene and
origin of replication for selection and propagation in E. coli.
[0105] Baculovirus transfer vectors usually contain a baculovirus
promoter. A baculovirus promoter is any DNA sequence capable of
binding a baculovirus RNA polymerase and initiating the downstream
(5' to 3') transcription of a coding sequence (eg. structural gene)
into mRNA. A promoter will have a transcription initiation region
which is usually placed proximal to the 5' end of the coding
sequence. This transcription initiation region usually includes an
RNA polymerase binding site and a transcription initiation site. A
baculovirus transfer vector may also have a second domain called an
enhancer, which, if present, is usually distal to the structural
gene. Expression may be either regulated or constitutive.
[0106] Structural genes, abundantly transcribed at late times in a
viral infection cycle, provide particularly useful promoter
sequences. Examples include sequences derived from the gene
encoding the viral polyhedron protein, Friesen et al., (1986) "The
Regulation of Baculovirus Gene Expression," in: The Molecular
Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127
839 and 155 476; and the gene encoding the p10 protein, Vlak et
al., (1988), J. Gen. Virol. 69:765.
[0107] DNA encoding suitable signal sequences can be derived from
genes for secreted insect or baculovirus proteins, such as the
baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409).
Alternatively, since the signals for mammalian cell
posttranslational modifications (such as signal peptide cleavage,
proteolytic cleavage, and phosphorylation) appear to be recognized
by insect cells, and the signals required for secretion and nuclear
accumulation also appear to be conserved between the invertebrate
cells and vertebrate cells, leaders of non-insect origin, such as
those derived from genes encoding human .alpha.-interferon, Maeda
et al., (1985), Nature 315:592; human gastrin-releasing peptide,
Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human
IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404;
mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human
glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be
used to provide for secretion in insects.
[0108] A recombinant polypeptide or polyprotein may be expressed
intracellularly or, if it is expressed with the proper regulatory
sequences, it can be secreted. Good intracellular expression of
nonfused foreign proteins usually requires heterologous genes that
ideally have a short leader sequence containing suitable
translation initiation signals preceding an ATG start signal. If
desired, methionine at the N-terminus may be cleaved from the
mature protein by in vitro incubation with cyanogen bromide.
[0109] Alternatively, recombinant polyproteins or proteins which
are not naturally secreted can be secreted from the insect cell by
creating chimeric DNA molecules that encode a fusion protein
comprised of a leader sequence fragment that provides for secretion
of the foreign protein in insects. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the translocation of the protein into the
endoplasmic reticulum.
[0110] After insertion of the DNA sequence and/or the gene encoding
the expression product precursor of the protein, an insect cell
host is co-transformed with the heterologous DNA of the transfer
vector and the genomic DNA of wild type baculovirus--usually by
co-transfection. The promoter and transcription termination
sequence of the construct will usually comprise a 2-5 kb section of
the baculovirus genome. Methods for introducing heterologous DNA
into the desired site in the baculovirus virus are known in the
art. (See Summers and Smith supra; Ju et al. (1987); Smith et al.,
Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For
example, the insertion can be into a gene such as the polyhedrin
gene, by homologous double crossover recombination; insertion can
also be into a restriction enzyme site engineered into the desired
baculovirus gene. Miller et al., (1989), Bioessays 4:91.The DNA
sequence, when cloned in place of the polyhedrin gene in the
expression vector, is flanked both 5' and 3' by polyhedrin-specific
sequences and is positioned downstream of the polyhedrin
promoter.
[0111] The newly formed baculovirus expression vector is
subsequently packaged into an infectious recombinant baculovirus.
Homologous recombination occurs at low frequency (between about 1%
and about 5%); thus, the majority of the virus produced after
cotransfection is still wild-type virus. Therefore, a method is
necessary to identify recombinant viruses. An advantage of the
expression system is a visual screen allowing recombinant viruses
to be distinguished. The polyhedrin protein, which is produced by
the native virus, is produced at very high levels in the nuclei of
infected cells at late times after viral infection. Accumulated
polyhedrin protein forms occlusion bodies that also contain
embedded particles. These occlusion bodies, up to 15 .mu.m in size,
are highly refractile, giving them a bright shiny appearance that
is readily visualized under the light microscope. Cells infected
with recombinant viruses lack occlusion bodies. To distinguish
recombinant virus from wild-type virus, the transfection
supernatant is plaqued onto a monolayer of insect cells by
techniques known to those skilled in the art. Namely, the plaques
are screened under the light microscope for the presence
(indicative of wild-type virus) or absence (indicative of
recombinant virus) of occlusion bodies. "Current Protocols in
Microbiology" Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);
Summers and Smith, supra; Miller et al. (1989).
[0112] Recombinant baculovirus expression vectors have been
developed for infection into several insect cells. For example,
recombinant baculoviruses have been developed for, inter alia:
Aedes aegypti, Autographa californica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni (PCT Pub.
No. WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright
(1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol.
3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell.
Dev. Biol. 25:225).
[0113] Cells and cell culture media are commercially available for
both direct and fusion expression of heterologous polypeptides in a
baculovirus/expression system; cell culture technology is generally
known to those skilled in the art. See, eg., Summers and Smith
[supra].
[0114] The modified insect cells may then be grown in an
appropriate nutrient medium, which allows for stable maintenance of
the plasmid(s) present in the modified insect host. Where the
expression product gene is under inducible control, the host may be
grown to high density, and expression induced. Alternatively, where
expression is constitutive, the product will be continuously
expressed into the medium and the nutrient medium must be
continuously circulated, while removing the product of interest and
augmenting depleted nutrients. The product may be purified by such
techniques as chromatography, eg., HPLC, affinity chromatography,
ion exchange chromatography, etc.; electrophoresis; density
gradient centrifugation; solvent extraction, or the like. As
appropriate, the product may be further purified, as required, so
as to remove substantially any insect proteins which are also
secreted in the medium or result from lysis of insect cells, so as
to provide a product which is at least substantially free of host
debris, eg., proteins, lipids and polysaccharides.
[0115] In order to obtain protein expression, recombinant host
cells derived from the transformants are incubated under conditions
which allow expression of the recombinant protein encoding
sequence. These conditions will vary, dependent upon the host cell
selected. However, the conditions are readily ascertainable to
those of ordinary skill in the art, based upon what is known in the
art.
[0116] iii. Bacterial Systems
[0117] Bacterial expression techniques are known in the art. A
bacterial promoter is any DNA sequence capable of binding bacterial
RNA polymerase and initiating the downstream (3") transcription of
a coding sequence (eg. structural gene) into mRNA. A promoter will
have a transcription initiation region which is usually placed
proximal to the 5' end of the coding sequence. This transcription
initiation region usually includes an RNA polymerase binding site
and a transcription initiation site. A bacterial promoter may also
have a second domain called an operator, that may overlap an
adjacent RNA polymerase binding site at which RNA synthesis begins.
The operator permits negative regulated (inducible) transcription,
as a gene repressor protein may bind the operator and thereby
inhibit transcription of a specific gene. Constitutive expression
may occur in the absence of negative regulatory elements, such as
the operator. In addition, positive regulation may be achieved by a
gene activator protein binding sequence, which, if present is
usually proximal (5') to the RNA polymerase binding sequence. An
example of a gene activator protein is the catabolite activator
protein (CAP), which helps initiate transcription of the lac operon
in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev.
Genet. 18:173]. Regulated expression may therefore be either
positive or negative, thereby either enhancing or reducing
transcription.
[0118] Sequences encoding metabolic pathway enzymes provide
particularly useful promoter sequences. Examples include promoter
sequences derived from sugar metabolizing enzymes, such as
galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and
maltose. Additional examples include promoter sequences derived
from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.
(1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids
Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publ. Nos. 036 776 and 121
775]. The g-laotamase (bla) promoter system [Weissmann (1981) "The
cloning of interferon and other mistakes." In Interferon 3 (ed. I.
Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature
292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also
provide useful promoter sequences.
[0119] In addition, synthetic promoters which do not occur in
nature also function as bacterial promoters. For example,
transcription activation sequences of one bacterial or
bacteriophage promoter may be joined with the operon sequences of
another bacterial or bacteriophage promoters, creating a synthetic
hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac
promoter is a hybrid trp-lac promoter comprised of both trp
promoter and lac operon sequences that is regulated by the lac
repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983)
Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter
can include naturally occurring promoters of non-bacterial origin
that have the ability to bind bacterial RNA polymerase and initiate
transcription. A naturally occurring promoter of non-bacterial
origin can also be coupled with a compatible RNA polymerase to
produce high levels of expression of some genes in prokaryotes. The
bacteriophase T7 RNA polymerase/promoter system is an example of a
coupled promoter system [Studier et al. (1986) J. Mol. Biol.
189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In
addition, a hybrid promoter can also be comprised of a
bacteriophage promoter and an E. coli operator region (EPO Publ.
No. 267 851).
[0120] In addition to a functioning promoter sequence, an efficient
ribosome binding site is also useful for the expression of foreign
genes in prokaryotes. In E. coli, the ribosome binding site is
called the Shine-Dalgarno (SD) sequence and includes an initiation
codon (ATG) and a sequence 3-9 nucleotides in length located 3-11
nucleotides upstream of the initiation codon [Shine et al. (1975)
Nature 254:34]. The SD sequence is thought to promote binding of
mRNA to the ribosome by the pairing of bases between the SD
sequence and the 3' and of E. coli 16S rRNA [Steitz et al. (1979)
"Genetic signals and nucleotide sequences in messenger RNA." In
Biological Regulation and Development: Gene Expression (ed. R. F.
Goldberger)]. To express eukaryotic genes and prokaryotic genes
with weak ribosome-binding site [Sambrook et al. (1989) "Expression
of cloned genes in Escherichia coli." In Molecular Cloning: A
Laboratory Manual].
[0121] A DNA molecule may be expressed intracellularly. A promoter
sequence may be directly linked with the DNA molecule, in which
case the first amino acid at the N-terminus will always be a
methionine, which is encoded by the ATG start codon. If desired,
methionine at the N-terminus may be cleaved from the protein by in
vitro incubation with cyanogen bromide or by either in vivo on in
vitro incubation with a bacterial methionine N-terminal peptidase
(EPO Publ. No. 219 237).
[0122] Fusion proteins provide an alternative to direct expression.
Usually, a DNA sequence encoding the N-terminal portion of an
endogenous bacterial protein, or other stable protein, is fused to
the 5' end of heterologous coding sequences. Upon expression, this
construct will provide a fusion of the two amino acid sequences.
For example, the bacteriophage lambda cell gene can be linked at
the 5' terminus of a foreign gene and expressed in bacteria. The
resulting fusion protein preferably retains a site for a processing
enzyme (factor Xa) to cleave the bacteriophage protein from the
foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins
can also be made with sequences from the lacZ [Jia et al. (1987)
Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff
et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EPO Publ. No.
324 647] genes. The DNA sequence at the junction of the two amino
acid sequences may or may not encode a cleavable site. Another
example is a ubiquitin fusion protein. Such a fusion protein is
made with the ubiquitin region that preferably retains a site for a
processing enzyme (eg. ubiquitin specific processing-protease) to
cleave the ubiquitin from the foreign protein. Through this method,
native foreign protein can be isolated [Miller et al. (1989)
Bio/Technology 7:698].
[0123] Alternatively, foreign proteins can also be secreted from
the cell by creating chimeric DNA molecules that encode a fusion
protein comprised of a signal peptide sequence fragment that
provides for secretion of the foreign protein in bacteria [U.S.
Pat. No. 4,336,336]. The signal sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell. The protein is either
secreted into the growth media (gram-positive bacteria) or into the
periplasmic space, located between the inner and outer membrane of
the cell (gram-negative bacteria). Preferably there are processing
sites, which can be cleaved either in vivo or in vitro encoded
between the signal peptide fragment and the foreign gene.
[0124] DNA encoding suitable signal sequences can be derived from
genes for secreted bacterial proteins, such as the E. coli outer
membrane protein gene (ompA) [Masui et al. (1983), in: Experimental
Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J.
3:2437] and the E. coli alkaline phosphatase signal sequence (phoA)
[Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an
additional example, the signal sequence of the alpha-amylase gene
from various Bacillus strains can be used to secrete heterologous
proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad.
Sci. USA 79:5582; EPO Publ. No. 244 042].
[0125] Usually, transcription termination sequences recognized by
bacteria are regulatory regions located 3' to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA.
Transcription termination sequences frequently include DNA
sequences of about 50 nucleotides capable of forming stem loop
structures that aid in terminating transcription. Examples include
transcription termination sequences derived from genes with strong
promoters, such as the trp gene in E. coli as well as other
biosynthetic genes.
[0126] Usually, the above described components, comprising a
promoter, signal sequence (if desired), coding sequence of
interest, and transcription termination sequence, are put together
into expression constructs. Expression constructs are often
maintained in a replicon, such as an extrachromosomal element (eg.,
plasmids) capable of stable maintenance in a host, such as
bacteria. The replicon will have a replication system, thus
allowing it to be maintained in a procaryotic host either for
expression or for cloning and amplification. In addition, a
replicon may be either a high or low copy number plasmid. A high
copy number plasmid will generally have a copy number ranging from
about 5 to about 200, and usually about 10 to about 150. A host
containing a high copy number plasmid will preferably contain at
least about 10, and more preferably at least about 20 plasmids.
Either a high or low copy number vector may be selected, depending
upon the effect of the vector and the foreign protein on the
host.
[0127] Alternatively, the expression constructs can be integrated
into the bacterial genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to the
bacterial chromosome that allows the vector to integrate.
Integrations appear to result from recombinations between
homologous DNA in the vector and the bacterial chromosome. For
example, integrating vectors constructed with DNA from various
Bacillus strains integrate into the Bacillus chromosome (EPO Publ.
No. 127 328). Integrating vectors may also be comprised of
bacteriophage or transposon sequences.
[0128] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of bacterial strains that have been transformed.
Selectable markers can be expressed in the bacterial host and may
include genes which render bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin),
and tetracycline [Davies et al. (1978) Annu. Rev.Microbiol.
32:469]. Selectable markers may also include biosynthetic genes,
such as those in the histidine, tryptophan, and leucine
biosynthetic pathways.
[0129] Alternatively, some of the above described components can be
put together in transformation vectors. Transformation vectors are
usually comprised of a selectable market that is either maintained
in a replicon or developed into an integrating vector, as described
above.
[0130] Expression and transformation vectors, either
extra-chromosomal replicons or integrating vectors, have been
developed for transformation into many bacteria. For example,
expression vectors have been developed for, inter alia, the
following bacteria: Bacillus subtilis [Palva et al. (1982) Proc.
Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953;
PCT Publ. No. WO 84/04541], Escherichia coli [Shimatake et al.
(1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et
al. (1986) J. Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829
and 136 907], Streptococcus cremoris [Powell et al. (1988) Appl.
Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al.
(1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans
[U.S. Pat. No. 4,745,056].
[0131] Methods of introducing exogenous DNA into bacterial hosts
are well-known in the art, and usually include either the
transformation of bacteria treated with CaCl.sub.2 or other agents,
such as divalent cations and DMSO. DNA can also be introduced into
bacterial cells by electroporation. Transformation procedures
usually vary with the bacterial species to be transformed. See eg.,
[Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al.
(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259
and 063 953; PCT Publ. No. WO 84/04541, Bacillus], [Miller et al.
(1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J.
Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc.
Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res.
16:6127; Kushner (1978) "An improved method for transformation of
Escherichia coli with ColE1-derived plasmids. In Genetic
Engineering: Proceedings of the International Symposium on Genetic
Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970)
J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318;
Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173
Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38,
Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203,
Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698;
Harlander (1987) "Transformation of Streptococcus lactis by
electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.
Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powell et
al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987)
Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].
[0132] iv. Yeast Expression
[0133] Yeast expression systems are also known to one of ordinary
skill in the art. A yeast promoter is any DNA sequence capable of
binding yeast RNA polymerase and initiating the downstream (3')
transcription of a coding sequence (eg. structural gene) into mRNA.
A promoter will have a transcription initiation region which is
usually placed proximal to the 5' end of the coding sequence. This
transcription initiation region usually includes an RNA polymerase
binding site (the "TATA Box") and a transcription initiation site.
A yeast promoter may also have a second domain called an upstream
activator sequence (UAS), which, if present, is usually distal to
the structural gene. The UAS permits regulated (inducible)
expression. Constitutive expression occurs in the absence of a UAS.
Regulated expression may be either positive or negative, thereby
either enhancing or reducing transcription.
[0134] Yeast is a fermenting organism with an active metabolic
pathway, therefore sequences encoding enzymes in the metabolic
pathway provide particularly useful promoter sequences. Examples
include alcohol dehydrogenase (ADH) (EPO Publ. No. 284 044),
enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),
hexokinase, phosphofructokinase, 3-phosphoglyceratemutase- , and
pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeast PHO5 gene,
encoding acid phosphatase, also provides useful promoter sequences
[Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
[0135] In addition, synthetic promoters which do not occur in
nature also function as yeast promoters. For example, UAS sequences
of one yeast promoter may be joined with the transcription
activation region of another yeast promoter, creating a synthetic
hybrid promoter. Examples of such hybrid promoters include the ADH
regulatory sequence linked to the GAP transcription activation
region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of
hybrid promoters include promoters which consist of the regulatory
sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined
with the transcriptional activation region of a glycolytic enzyme
gene such as GAP or PyK (EPO Publ. No. 164 556). Furthermore, a
yeast promoter can include naturally occurring promoters of
non-yeast origin that have the ability to bind yeast RNA polymerase
and initiate transcription. Examples of such promoters include,
inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA
77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al.
(1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al.
(1979) "The Expression of Bacterial Antibiotic Resistance Genes i
the Yeast Saccharomyces cerevisiae," in: Plasmids of Medical,
Environmental and Commercial Importance (eds. K>N> Timmis and
A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier
et al. (1980) Curr. Genet. 2:109;].
[0136] A DNA molecule may be expressed intracellularly in yeast. A
promoter sequence may be directly linked with the DNA molecule, in
which case the first amino acid at the N-terminus of the
recombinant protein will always be a methionine, which is encoded
by the ATG start codon. If desired, methionine at the N-terminus
may be cleaved from the protein by in vitro incubation with
cyanogen bromide.
[0137] Fusion proteins provide an alternative for yeast expression
systems, as well as in mammalian, baculovirus, and bacterial
expression systems. Usually, a DNA sequence encoding the N-terminal
portion of an endogenous yeast protein, or other stable protein, is
fused to the 5' end of heterologous coding sequences. Upon
expression, this construct will provide a fusion of the two amino
acid sequences. For example, the yeast or human superoxide
dismutase (SOD) gene, can be linked at the 5' terminus of a foreign
gene and expressed in yeast. The DNA sequence at the junction of
the two amino acid sequences may or may not encode a cleavable
site. See eg., EPO Publ. No. 196 056. Another example is a
ubiquitin fusion protein. Such a fusion protein is made with the
ubiquitin region that preferably retains a site for a processing
enzyme (eg. ubiquitin-specific processing protease) to cleave the
ubiquitin from the foreign protein. Through this method, therefore,
native foreign protein can be isolated (see, eg., PCT Publ. No. WO
88/024066).
[0138] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provide for secretion in yeast of the foreign
protein. Preferably, there are processing sites encoded between the
leader fragment and the foreign gene that can be cleaved either in
vivo or in vitro. The leader sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell.
[0139] DNA encoding suitable signal sequences can be derived from
genes for secreted yeast proteins, such as the yeast invertase gene
(EPO Publ. No. 012 873; JPO Publ. No. 62,096,086) and the A-factor
gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast
origin, such as an interferon leader, exist that also provide for
secretion in yeast (EPO Publ. No. 060 057).
[0140] A preferred class of secretion leaders are those that employ
a fragment of the yeast alpha-factor gene, which contains both a
"pre" signal sequence, and a "pro" region. The types of
alpha-factor fragments that can be employed include the full-length
pre-pro alpha factor leader (about 83 amino acid residues) as well
as truncated alpha-factor leaders (usually about 25 to about 50
amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EPO
Publ. No. 324 274). Additional leaders employing an alpha-factor
leader fragment that provides for secretion include hybrid
alpha-factor leaders made with a presequence of a first yeast, but
a pro-region from a second yeast alphafactor. (See eg., PCT Publ.
No. WO 89/02463.)
[0141] Usually, transcription termination sequences recognized by
yeast are regulatory regions located 3' to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA. Examples
of transcription terminator sequence and other yeast-recognized
termination sequences, such as those coding for glycolytic
enzymes.
[0142] Usually, the above described components, comprising a
promoter, leader (if desired), coding sequence of interest, and
transcription termination sequence, are put together into
expression constructs. Expression constructs are often maintained
in a replicon, such as an extrachromosomal element (eg., plasmids)
capable of stable maintenance in a host, such as yeast or bacteria.
The replicon may have two replication systems, thus allowing it to
be maintained, for example, in yeast for expression and in a
procaryotic host for cloning and amplification. Examples of such
yeast-bacteria shuttle vectors include YEp24 [Botstein et al.
(1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad.
Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol.
Biol. 158:157]. In addition, a replicon may be either a high or low
copy number plasmid. A high copy number plasmid will generally have
a copy number ranging from about 5 to about 200, and usually about
10 to about 150. A host containing a high copy number plasmid will
preferably have at least about 10, and more preferably at least
about 20. Enter a high or low copy number vector may be selected,
depending upon the effect of the vector and the foreign protein on
the host. See eg., Brake et al., supra.
[0143] Alternatively, the expression constructs can be integrated
into the yeast genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to a yeast
chromosome that allows the vector to integrate, and preferably
contain two homologous sequences flanking the expression construct.
Integrations appear to result from recombinations between
homologous DNA in the vector and the yeast chromosome [Orr-Weaver
et al. (1983) Methods in Enzymol. 101:228-245]. An integrating
vector may be directed to a specific locus in yeast by selecting
the appropriate homologous sequence for inclusion in the vector.
See Orr-Weaver et al., supra. One or more expression construct may
integrate, possibly affecting levels of recombinant protein
produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750].
The chromosomal sequences included in the vector can occur either
as a single segment in the vector, which results in the integration
of the entire vector, or two segments homologous to adjacent
segments in the chromosome and flanking the expression construct in
the vector, which can result in the stable integration of only the
expression construct.
[0144] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of yeast strains that have been transformed. Selectable
markers may include biosynthetic genes that can be expressed in the
yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418
resistance gene, which confer resistance in yeast cells to
tunicamycin and G418, respectively. In addition, a suitable
selectable marker may also provide yeast with the ability to grow
in the presence of toxic compounds, such as metal. For example, the
presence of CUP1 allows yeast to grow in the presence of copper
ions [Butt et al. (1987) Microbiol. Rev. 51:351].
[0145] Alternatively, some of the above described components can be
put together into transformation vectors. Transformation vectors
are usually comprised of a selectable marker that is either
maintained in a replicon or developed into an integrating vector,
as described above.
[0146] Expression and transformation vectors, either
extrachromosomal replicons or integrating vectors, have been
developed for transformation into many yeasts. For example,
expression vectors have been developed for, inter alia, the
following yeasts:Candida albicans [Kurtz, et al. (1986) Mol. Cell.
Biol. 6:142], Candida maltose [Kunze, et al. (1985) J. Basic
Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J.
Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.
202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol.
158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J.
Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology
8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic
Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol.
Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555],
Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad.
Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163],
Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706],
and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet.
10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].
[0147] Methods of introducing exogenous DNA into yeast hosts are
well-known in the art, and usually include either the
transformation of spheroplasts or of intact yeast cells treated
with alkali cations. Transformation procedures usually vary with
the yeast species to be transformed. See eg., [Kurtz et al. (1986)
Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol.
25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol.
132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;
Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De
Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et
al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al.
(1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic
Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia];
[Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et
al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse
(1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985)
Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49;
Yarrowia].
[0148] 1. Preparation of LT-A72R mutant
[0149] a. Source of LT DNA
[0150] The 1.5 kb SmaI-EcoRI fragment from plasmid pEWD299,
containing the LT-A gene and the LTpromoter region [Pronk et al.
(1985) J. Biol. Chem. 260:13580; Spicer et al. (1981) Proc. Natl.
Acad. Sci. USA 78:50] was subcloned into the Bluescript KS vector
(Stratagene). The resultant vector, termed BS-LT-A, was used for
site-directed mutagenesis [Zoller & Smith (1982) Nucl. Acid.
Res. 10:6487)].
[0151] b. Methods of mutation
[0152] Site-directed mutagenesis was performed according to the
method of Zoller & Smith [supra] on single-stranded DNA of
BS-LT-A vector. The oligonucleotide used:
[0153] .sup.5'GCTCACTTACGTGGACAGTCT.sup.3' (oligoLT-A72R)
[0154] mutates the codon for Ala-72 (GCA) to an Arg codon
(CGT).
[0155] The mutated SmaI-EcoRI fragment containing the mutated codon
was ligated with the 0.57 kb EcoRI-HindIII fragment [Pronk et al.;
Spicer et al.] containing the gene coding for LT-B and cloned into
Bluescript KS vector (Stratagene) to generate vector BS-LTA72R.
[0156] c. Expression and purification of the LT-A72R mutant.
[0157] E.coli were transformed with BS-LTA72R vector and grown in
LB broth in a 5 litre fermenter. The mutant protein LT-A72R was
purified from the periplasm [Pronk et al.; Magagnoli et al. (1996)
Infect Immun 64:5434] using controlled pore glass (CPG 350, Serva)
and A5M Agarose columns and then by gel filtration using Sephacryl
S-200.TM..
[0158] An aliquot of the purified material was analysed on a
Superdex 200HR column. FIG. 1 shows the elution profile of LT-A72R
in comparison with wild-type LT and mutant LTK63 (Ser-63-Lys
mutant--see WO93/13202 and also Pizza et al. (1994) J.Exp. Med.
6:2147).
[0159] LT-A72R elutes as a single peak corresponding to the
fully-assembled holotoxin, with a profile identical to that of
wild-type LT and LTK63. This demonstrates that the A72R mutation
does not alter the structure of the A subunit, that it is
efficiently exported to the periplasm, and correctly assembled as
AB.sub.5. 100 .mu.l aliquots of A72R stored at 4.degree. C. were
analysed in the same way every two months for a year and no change
in elution profile was detected.
[0160] 2. Physical characterisation
[0161] a. Trypsin digestion
[0162] 45 .mu.g toxin (LT, LT-A72R, LTK63) was treated with 9 .mu.g
trypsin in a final volume of 150 .mu.l 10 mM Tris, pH 7.5, at
37.degree. C. 30 .mu.l samples were collected after 5 and 30
minutes incubation and the reaction was stopped with 3.6 .mu.g
trypsin inhibitor. 10 .mu.l 4.times.concentrated electrophoresis
sample buffer was added to each sample and the mixture was heated
to 95.degree. C. for 10 minutes. Proteins were loaded onto 15% SDS
minigels and either stained with Coomassie brilliant blue R-250 or
transferred onto a nitrocellulose membrane, which was subsequently
incubated with a rabbit anti-LT polyclonal serum at a dilution of
1:300.
[0163] FIG. 2 shows the results of this Western blot analysis.
After 5 minutes incubation, trypsin causes almost immediate nicking
of the A subunit into A1 and A2 domains, and nicking is complete by
30 minutes. No sensitivity differences were detected between LT,
LT-A72R, or LTK63.
[0164] b. ADP-ribosylation of polyarginine
[0165] LT (wild-type and mutant) was analysed as described by Lai
et al. [BBRC 102:1021, 1981] and the results are shown in FIG.
3.
[0166] The ADP-ribosylation activity of LT-A72R is at least 2
orders of magnitude lower than that of wild-type LT: the minimal
concentration to obtain a detectable activity was 0.5 .mu.g for LT,
and 85 .mu.g for LT-A72R. In contrast, LTK63 is totally devoid of
activity.
[0167] c. Toxicity
[0168] In vitro toxicity was assessed by following the
morphological changes caused by LT on Y1 adrenal cells [Donta et
al. (1973) Science 183:334]. The assay was performed in microtitre
plates using 50000 cells/well with twofold dilutions of LT, LT-A72R
and LTK63, starting from 80 pg/well for wild-type and 40 .mu.g/well
for mutants. Morphological changes were read after 48 hours
incubation.
[0169] In vivo toxicity was assessed using an ileal loop assay [De
(1959) Nature 183:1533]. Two New Zealand adult rabbits (ca. 2.5 kg)
were used for each assay and 1 ml samples of toxin (wild-type,
LT-A72R, LTK63) were injected into each loop at various
concentrations, with a control loop receiving 1 ml PBS. After 18-20
hours, the volume of liquid accumulated in each loop was measured
with a syringe and the length of each loop was measured again. The
results, from 4-6 repeats, expressed as volume of liquid per unit
length of the loop (ml/cm) are shown in FIG. 5.
[0170] In agreement with the ADP-ribosylation results, LT-A72R had
10.sup.-5 toxicity of wild-type LT on Y1 cells (FIG. 4) and was
approximately 20.times. less toxic than wild-type in the ileal loop
assay (FIG. 5). As previously reported, LTK63 was completely
non-toxic by both assays.
[0171] 3. Immunological characterisation
[0172] a. Mucosal adjuvanticity
[0173] The adjuvanticity of LT-A72R was tested using the protocol
of Douce et al. [PNAS 92:1644-1648 (1995)]. Groups of ten BALB/c
mice (female, 4-6 weeks old) were immunised intranasally with 1
.mu.g toxin (LT, LT-A72R, LTB, or LK-K63) and 10 .mu.g OVA. The
animals were lightly anaesthetised and immunised on day 0, 21 and
35 with a 15 .mu.l volume per nostrel. Immune responses were
followed in serum samples taken on days 0, 20, 34 and 52. The
animals were sacrificed and nasal lavages were performed by
repeated flushing and aspiration of 1 ml PBS containing 0.1%
BSA.
[0174] LT-specific antibodies were measured using a GM1 capture
ELISA. Each well on 96-well plates was first coated with 150 ng GM1
ganglioside by overnight incubation at 4.degree. C. Wells were then
washed three times with PBS (+0.05% Tween-20) and 50 ng toxin was
added to each well. Plates were incubated at 2 hours at 37.degree.
C. OVA-specific antibodies were assessed by coating each well with
60 .mu.g/ml OVA and incubated overnight at 4.degree. C.
[0175] The plates were washed and wells saturated with PBS (+1%
BSA) for 1 hour at 37.degree. C. Sera from individual mice were
tested starting from a dilution of 1:50 in PBS; nasal lavages were
tested starting from undiluted. Plates were incubated at 37.degree.
C. for 2 hours. Specific Ig were measured using horseradish
peroxidase-conjugated rabbit anti-mouse Ig. Antibodies were then
revealed by adding o-phenylenediamine as a substrate. After 10
minutes the reaction was blocked by adding 12.5%
H.sub.2SO.sub.4.
[0176] IgG subclasses were determined with IgG1, IgG2a, IgG2b, or
IgG3 biotinylated antibodies. Peroxidase-streptavadin was then
added to each well (1:1000) and the plates were incubated at
37.degree. C. for 1 hour. Bound antibody was visualised as
above.
[0177] Absorbances were read at 490 nm and ELISA titres were
determined arbitrarily as the reciprocal of the last dilution which
gave an OD.sub.490.gtoreq.0.3 above the level measured in
pre-immune sera.
[0178] Titres of specific IgA in the sera and in the mucosal
lavages were measured using biotin-conjugated goat anti-mouse IgA
.alpha. chain specific antibody, followed by
streptavadin-peroxidase. Bound antibodies were revealed using the
OPD substrate as described above. ELISA titres were determined
arbitrarily as the reciprocal of the last dilution which gave an
OD.sub.490.gtoreq.0.2 above that of the non-immunised controls.
[0179] Values were always normalised using a positive control
sample in each plate.
[0180] As shown in FIG. 6, wild-type LT and LT-A72R induced the
highest anti-OVA antibody response. LTK63 induced an intermediate
level, and LTB gave a low response. The antigen-specific antibody
response was detectable after a single immunisation for wild-type
LT and the A72R mutant, whereas (as previously described) LTK63
required at least two immunisations to induce serum anti-OVA
antibodies.
[0181] OVA-specific IgG isotypes were measured in pools of sera of
the last bleeding (FIG. 7). High titres of anti-OVA IgG1, IgG2a and
IgG2b were induced in the groups receiving wild-type LT or the A72R
mutant as an adjuvant, although the dominant isotype with the
mutant was IgG2a. OVA-specific IgG3 antibodies were never
detectable. LTB was not a good adjuvant.
[0182] In all experiments, OVA-specific IgA was never detected
after just 1 or two intranasal immunisations. Following a third
immunisation, however, a serum IgA response was detected with
wild-type LT and with the A72R and K63 mutants, but not with LTB
(FIG. 8A). A similar pattern was seen at the mucosal level (FIG.
8B), and after three immunisations OVA-specific IgA was found in
the nasal washes of the wild-type group and the A72R and K63 mutant
groups, but not in the control or LTB groups.
[0183] Serum anti-LT antibody responses are shown in FIG. 9. The
mice mounted responses after the first immunisation, which was
significantly boosted by the second. The A72R mutant was more
immunogenic that the non-toxic K63 mutant, and both were much more
immunogenic than LTB.
[0184] b. OVA-driven proliferative response
[0185] 14-20 days after 2 or 3 intranasal immunisations with OVA+LT
(wild-type or mutant), 2 or 3 mice per group were sacrificed and
spleens removed. Spleen cell suspensions were obtained and
resuspended in complete DMEM (10% FCS, 2 mM L-glutamine, 15 mM
Hepes, 100U penicillin/streptomycin, 50 mM 2-mercapto-ethanol).
2.times.10.sup.5 spleen cells/well were seeded in U-bottomed
96-well plates and cultured in the presence of different
concentrations of OVA for 5 days. [.sup.3H]-thymidine was added (1
.mu.Ci/well) 16 hours before the end of culture. Cells were then
harvested with a cell harvester, and [.sup.3H]-thymidine
incorporation was evaluated by liquid scintillation counting (FIG.
10).
[0186] It is clear that intranasal co-administration of antigen
with wild-type or with A72R or K63 mutants induced a priming of
OVA-specific T cells in vivo, which was much stronger than that
detectable after immunisation with OVA alone or with OVA+LTB.
[0187] c. In vivo challenge
[0188] The LD.sub.50 for LT was determined by inoculating groups of
10 BALE/c mice (female, 9 weeks old) intraperitoneally with LT
(12.5 .mu.g, 25 .mu.g, 50 .mu.g, or 100 .mu.g) or PBS. After 7 days
of observation, LD.sub.50 was determined as 20.4 .mu.g.
[0189] Four week old BALB/c mice were immunised intranasally at day
0 and 21 with 1 .mu.g toxin (LT, LT-A72R, LT-K63, or LTB) and
challenged with LT (2.times.LD.sub.50) at day 35. They were
observed for death for 7 days. Sera were collected at days 0, 20
and 35 and anti-LT titres analysed by ELISA (as above).
[0190] All mice immunised with wild-type or mutant LT survived to
the challenge, whereas only 30% of those receiving LTB survived.
All mice in the control group died (FIG. 11A).
[0191] Sera of mice immunised with wild-type LT, or with the A72R
or K63 mutants, contained very high and comparable levels of
anti-LT antibodies (FIG. 11B). In mice receiving LTB, however, the
titres were 10-20.times. lower. The three LTB mice who survived to
the LT challenge (open circles) had significantly higher titres
than those in the dead animals.
[0192] 4. Conclusions
[0193] ADP-ribosylation activity is not necessary for the
adjuvanticity of LT, but the presence of low levels of enzymatic
activity may be useful to induce a faster and higher immune
response to co-administered antigens.
[0194] The A72R mutant of LT is an effective mucosal adjuvant. In
addition, the mutant retains the immunogenicity of wild-type LT and
is able to induce protective immunity against LT. It may therefore
also be useful for anti-diarrhoea vaccination.
[0195] It will, of course, be understood that the invention is
described above by way of example only and modifications may be
made within the scope and spirit of the invention.
* * * * *