U.S. patent application number 14/493950 was filed with the patent office on 2015-04-02 for diagnostic reagents.
The applicant listed for this patent is The Secretary of State For Environment, Food & Rural Affairs. Invention is credited to Gareth JONES, Hans VORDERMEIER.
Application Number | 20150093766 14/493950 |
Document ID | / |
Family ID | 42735123 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093766 |
Kind Code |
A1 |
JONES; Gareth ; et
al. |
April 2, 2015 |
DIAGNOSTIC REAGENTS
Abstract
There is provided a diagnostic reagent useful to determine
whether an animal has a tuberculosis infection or has been exposed
to a tuberculosis agent, for example a Mycobacterium. The reagent
is useful to distinguish between such an animal and an animal which
has been vaccinated against a tuberculosis infection.
Inventors: |
JONES; Gareth; (Surrey,
GB) ; VORDERMEIER; Hans; (Surrey, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Secretary of State For Environment, Food & Rural
Affairs |
Worcestershire |
|
GB |
|
|
Family ID: |
42735123 |
Appl. No.: |
14/493950 |
Filed: |
September 23, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13810945 |
Mar 22, 2013 |
|
|
|
PCT/GB2011/051343 |
Jul 18, 2011 |
|
|
|
14493950 |
|
|
|
|
Current U.S.
Class: |
435/7.24 ;
435/7.92; 530/326; 536/23.7 |
Current CPC
Class: |
G01N 33/5695 20130101;
A61K 49/0006 20130101; C07K 7/08 20130101; G01N 2333/7156 20130101;
C07K 14/35 20130101; G01N 2333/35 20130101 |
Class at
Publication: |
435/7.24 ;
435/7.92; 530/326; 536/23.7 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C07K 14/35 20060101 C07K014/35 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 19, 2010 |
GB |
1012072.3 |
Claims
1. A diagnostic reagent comprising a polypeptide comprising ammo
acid sequence SEQ ID NO:7 or a functional variant thereof,
characterised in that the reagent elicits a negative diagnostic
assay result when a tuberculosis infection assay is carried out on
a sample from an animal which has been vaccinated against infection
by a tuberculosis agent.
2-17. (canceled)
18. A method of detecting a tuberculosis infection in an animal, or
exposure of an animal to a tuberculosis agent, comprising the steps
of (i) contacting a population of cells from the animal with at
least one diagnostic reagent consisting of amino acid sequence SEQ
ID NO:7, or consisting of a polypeptide having 20-65 amino acids
and comprising SEQ ID NO:7, or comprising a polypeptide which is a
functional variant of SEQ ID NO:7 having 2, 4, or 5 amino acids
added to or deleted from the N- or C-terminus and/or having at
least 85% sequence identity to SEQ ID NO:7, characterised in that
the reagent elicits a negative diagnostic assay result when a
tuberculosis infection assay is carried out on a sample from an
animal which has been vaccinated against infection by a
tuberculosis agent using the live attenuated M. bovis strain
Bacille Calmette-Guerin (BCG); and (ii) determining whether the
cells of said population recognise the diagnostic reagent.
19. The method according to claim 18, wherein the population of
cells includes T-cells.
20. The method according to claim 18, comprising a cell-mediated
immunity 10 (CMI) assay.
21. The method according to claim 20, wherein the CMI assay detects
interferon gamma (IFN-.gamma.).
22. A method of detecting a tuberculosis infection in an animal, or
exposure of an animal to a tuberculosis agent, comprising
conducting a skin test on the animal using at least one diagnostic
reagent consisting of amino acid sequence SEQ ID NO:7, or
consisting of a polypeptide having 20-65 amino acids and comprising
SEQ ID NO:7, or comprising a polypeptide which is a functional
variant of SEQ ID NO:7 having 2, 4, or 5 amino acids added to or
deleted from the N- or C-terminus and/or having at least 85%
sequence identity to SEQ ID NO:7, characterised in that the reagent
elicits a negative diagnostic assay result when a tuberculosis
infection assay is carried out on a sample from an animal which has
been vaccinated against infection by a tuberculosis agent using the
live attenuated M. bovis strain Bacille Calmette-Guerin (BCG).
23-27. (canceled)
28. A nucleic acid encoding a polypeptide comprising at least one
polypeptide comprising the amino acid sequence of any of SEQ ID
NOs:1-10 and/or 50-69 or a functional variant thereof.
29-30. (canceled)
31. The method of claim 18, wherein said diagnostic reagent further
comprises at least one of the amino acid sequences SEQ ID NO:1, 2,
3, 4, 5, 6 or 9 or functional variants thereof, or comprising all
of SEQ ID NOs:1-9.
32. The method of claim 18, wherein said diagnostic reagent further
comprises a polypeptide of the amino acid sequence of SEQ ID NO:10
or a functional variant thereof.
33. The method of claim 18, wherein said diagnostic reagent further
comprises further comprising at least one of the amino acid
sequences SEQ ID NOs:11-69 or functional variants thereof.
34. The method of claim 22, wherein said diagnostic reagent further
comprises at least one of the amino acid sequences SEQ ID NO:1, 2,
3, 4, 5, 6 or 9 or functional variants thereof, or comprising all
of SEQ ID NOs:1-9.
35. The method of claim 22, wherein said diagnostic reagent further
comprises a polypeptide of the amino acid sequence of SEQ ID NO:10
or a functional variant thereof.
36. The method of claim 22, wherein said diagnostic reagent further
comprises further comprising at least one of the amino acid
sequences SEQ ID NOs:11-69 or functional variants thereof.
Description
FIELD OF INVENTION
[0001] The present invention relates to reagents for use in the
detection of tuberculosis infections, particularly tuberculosis in
mammals such as human beings and cattle, more particularly
infection by Mycobacteria such as M. tuberculosis and M. bovis. The
reagents are useful to differentiate between animals with a
tuberculosis infection and those which have been vaccinated against
infection, as a positive result is only obtained from infected
animals (or animals exposed to an infectious agent).
BACKGROUND
[0002] M. tuberculosis and M. bovis are important pathogens of man
and animals. M. tuberculosis is thought to infect up to a third of
the world's human population, remaining undetected during a latent
phase of infection and reactivating to cause 10 million cases of
tuberculosis and other diseases per year, resulting in 2 million
deaths (Corbett et al. (2003) Arch. Intern. Med. vol. 163 pp
1009-1021). M. bovis, which has more than 99.9% sequence identity
with M. tuberculosis, is the predominant causative agent of bovine
tuberculosis (BTB) and also causes disease in human. Cases of
bovine tuberculosis in cattle caused by M. tuberculosis have also
been reported, particularly in developing countries with high
incidence rates of human TB (see, for example, Berg et al. (2009)
PLoS ONE vol. 4 e5068). BTB represents a significant economic
burden to the agricultural industries of various countries
including the United Kingdom (Krebs (1997) "Bovine Tuberculosis in
Cattle & Badgers" HMSO, London, United Kingdom).
[0003] The primary diagnostic test used in the control and
surveillance of bovine TB is the tuberculin skin-test, a test that
has remained in the forefront of TB diagnosis in both man and
cattle for over 100 years. The development of the test arose
following the preparation of the first `tuberculin` by Robert Koch
in 1890. Whilst Koch's tuberculin failed to live up to its initial
claims of having curative properties, its diagnostic potential was
quickly realised. The most common formats of the test used in
cattle are the caudal fold test (CFT), the single intradermal
cervical tuberculin test (SIT) and the single intradermal
comparative cervical tuberculin test (SICCT) (Monaghan et al.
(1994) Vet. Microbiol. vol. 40 pp 111-24). Both test formats use a
purified protein derivative (PPD) tuberculin prepared from a
culture of M. bovis (PPD-B) as the primary diagnostic antigen.
Additionally, the SICCT test includes the use of a M. avium derived
PPD (PPD-A) to provide a measure of environmental sensitisation. It
is the more specific of the two tests (Plum (1931) Cornell Vet.
vol. 21 pp 68-76; Stenius (1938) Veterinary Record vol. 50 pp
633-7) and is therefore the adopted test format in the UK.
[0004] In addition to skin tests, blood-based diagnostic assays
that measure antigen-induced lymphokine production such as the
interferon gamma (IFN-.gamma.) are also under consideration. The
cytokine IFN-.gamma. appears to be critical in the development of
immunity to M. tuberculosis. For example, both mice with a
disrupted IFN-.gamma. gene and humans with mutated WN-.gamma.
receptor are highly susceptible to mycobacterial infections.
However, specificity constraints are associated with the use of PPD
in such assays. These arise due to the crude mixture of M. bovis
proteins that PPD contains, many of which are cross-reactive with
the BCG vaccine strain and environmental mycobacterial species such
as M. avium and M. intracellulare.
[0005] The term "tuberculosis infection assay" used in the present
specification may refer to any of these diagnostic tests referred
to above.
[0006] Bovine TB is a significant and ongoing problem in the UK
(http://www.defra.gov.uk/food-farm/animals/diseases/tb, accessed 14
Jul. 2011). Cattle vaccination has been identified as one of the
most promising long term UK control strategies (Krebs (1997)
"Bovine Tuberculosis in Cattle & Badgers" HMSO) and the
development of an efficacious vaccine continues to be a research
priority. Currently, promising vaccines against bovine TB are based
on heterologous prime-boost combinations that include the live
attenuated. M. bovis vaccine strain Bacille Calmette-Guerin (BCG)
as one of their components (Hogarth et al. (2006) J. Pharm.
Pharmacol. vol. 58 pp 749-57). However, as in humans, vaccination
of cattle with BCG compromises the specificity of the tuberculin
skin-test since PPD contains cross reactive antigens shared by both
pathogenic and vaccine strains (Berggren (1981) Br. Vet. J. vol.
137 pp 88-94; Buddle et al. (1999) Clin. Diagn. Lab. Immunol. vol.
6 pp 1-5; Waddington & Ellwood (1972) Br. Vet. J. vol. 128 pp
541-52). Therefore, the development of diagnostic tests that can
differentiate vaccinated from infected animals, so-called DIVA
tests, are an essential pre-requisite to allow the inclusion of
BCG-based vaccination as part of bovine TB control strategies.
[0007] Previous studies have demonstrated that diagnostic reagents
which distinguish between vaccinated and infected cattle can be
developed using specific, defined antigens that are present in
virulent M. bovis but absent from the BCG. Genetic analysis of BCG
has revealed that several large genomic regions have been deleted
during attenuation and subsequent prolonged propagation in culture.
These regions have been characterised and antigens from one of
these regions, RD1, have been studied extensively in several
species including humans and cattle. For example, it has been
demonstrated that protein or peptide cocktails composed of two RDI
region antigens, ESAT-6 and CFP-10, can be used to distinguish
between M. bovis infected and BCG-vaccinated cattle. In humans,
synthetic ESAT-6 and CFP-10 peptides arc used in the
QuantiFERON.RTM.-TB Gold Test for TB diagnosis.
[0008] The practical application of such DIVA reagents have so far
been largely realised through their use in blood-based
interferon-.gamma. (1FN-.gamma.) release assays (IGRAs). For
example, WO2009/060184 disclosed several polypeptides including
epitopes from Rv3615c which were found to be useful to detect M.
bovis or M. tuberculosis infection, using such an assay. This
polypeptide has also been confirmed as useful to detect M.
tuberculosis infection in humans (Millington et al. (2011) Proc.
Natl. Acad. Sci. USA vol. 108 pp 5730-5735). Rv3615c is referred to
herein using the M. tuberculosis genome annotation
(http://genolist.pasteur.fr/TubercuList/, accessed 14 July. 2011).
In the M. bovis genome, it is annotated as Mb3645c
(http://genolist.pasteur.fr/BoviList/, accessed 14 Jul. 2011).
[0009] Given the high level of familiarity and wide-spread
application of the tuberculin skin-test by veterinarians and
clinicians, a DIVA skin-test format would provide a valuable
additional test platform. This might especially be the case where
the logistics of access to laboratory-based resources is
problematic. It is also notable that in recent years there has also
been renewed interest in a skin-test based DIVA test for human TB
with several reports demonstrating the skin-test potential of
ESAT-6 (Aggerbeck & Madsen (2006) Tuberculosis (Edinb.) vol. 86
pp 363-73; Arend et al. (2000) J. Infect. Dis. vol. 181 pp 1850-4;
Wu et al. (2008) Clin. Exp. Immunol. vol. 152 pp 81-7).
[0010] The present invention accordingly addresses the problem of
providing discriminatory diagnostic reagents for the detection of
mycobacterial infections, particularly in a DIVA skin-test
format.
SUMMARY OF INVENTION
[0011] According to a first aspect of the invention, there is
provided a diagnostic reagent comprising a polypeptide comprising
amino acid sequence SEQ TD NO:7 or a functional variant thereof.
The polypeptide may be a polypeptide which is not full length
Rv2346c or Rv1793. It may, for example, comprise between 15-65
amino acids, for example, between 15-60 amino acids, 15-50 amino
acids, 15-40 amino acids and may, for example, comprise at least
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
amino acids. This polypeptide elicits a positive result when used
in an assay to determine whether an animal has a tuberculosis
infection or has been exposed to a tuberculosis agent.
Advantageously, the diagnostic reagent can allow a user to
differentiate between an animal having a tuberculosis infection and
one which has been vaccinated against such an infection, as is
disclosed herein for the first time and as will be explained in
further detail below. Therefore, the reagent is characterised in
that the reagent elicits a negative diagnostic assay result when a
tuberculosis infection assay is carried out on a sample from an
animal which has been vaccinated against infection by a
tuberculosis agent. A negative result is also obtained when the
animal is unvaccinated and uninfected (or unexposed to a
tuberculosis agent). The animal may be a mammal, for example a cow,
a badger or a human being.
[0012] The diagnostic reagent may alternatively or further comprise
a polypeptide comprising amino acid sequence SEQ ID NO:8 or a
functional variant thereof. The polypeptide may be a polypeptide
which is not full length Rv3020c. It may, for example, comprise
between 15-65 amino acids, for example, between 15-60 amino acids,
15-50 amino acids, 15-40 amino acids and may, for example, comprise
at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29
or 30 amino acids. This polypeptide also elicits a positive result
when used in an assay to determine whether an animal has a
tuberculosis infection or has been exposed to a tuberculosis agent.
Again, the diagnostic reagent can allow a user to differentiate
between an animal having a tuberculosis infection and one which has
been vaccinated against such an infection. The reagent is,
therefore, characterised in that the reagent elicits a negative
diagnostic assay result when a tuberculosis infection assay is
carried out on a sample from an animal which has been vaccinated
against infection by a tuberculosis agent. A negative result is
also obtained when the animal is unvaccinated and uninfected (or
unexposed to a tuberculosis agent).
[0013] None of the polypeptides Rv2346c, Rv1793 or Rv3020c has
previously been identified as useful to differentiate between
tuberculosis-infected and vaccinated animals.
[0014] Optionally or additionally, the diagnostic reagent may
alternatively or further comprise at least one polypeptide each
comprising at least one of the amino acid sequences SEQ ID NO:1, 2,
3, 4, 5, 6, 9 or 10 or functional variants thereof.
[0015] In one embodiment, the diagnostic reagent may comprise all
of amino acid sequences SEQ ID NOs:1-9. This may be in the form of
individual polypeptides each having an amino acid sequence selected
from SEQ ID NOs:1-9, or may be one or more fusion proteins each
comprising two or more of SEQ ID NOs:1-9. These sequences were
included in peptide pool Sec#1 described herein and shown in Table
3 below.
[0016] The term "tuberculosis infection", as used throughout this
specification, indicates an infection in which the causative agent
is a Mycobacterium, for example, M. tuberculosis, M. bovis, and/or
M. africanum. In some cases, the infection can be the result of
exposure to a combination of these bacterial species. Likewise, the
term "tuberculosis agent" indicates an organism capable of causing
tuberculosic symptoms, typically a Mycobacterium, for example M.
tuberculosis, M. bovis, and/or M. africanum.
[0017] A vaccine which might be administered to an animal to
vaccinate against a tuberculosis infection includes the BCG
vaccine.
[0018] The diagnostic reagent may alternatively or further comprise
a polypeptide comprising at least one of the amino acid sequences
SEQ ID NOs:50-69 or a functional variant thereof. These sequences
were included in peptide pools #11 and #14 described herein and
shown in Table 2 below. For example, the diagnostic reagent may
comprise SEQ 1D NOs:7 and 50-59 and/or SEQ ID NOs:8 and 60-69.
[0019] In some embodiments, the diagnostic reagent may further
comprise at least one polypeptide each comprising at least one of
the amino acid sequences SEQ ID NOs:11-49 or functional variants
thereof. By way of non-limiting example, the diagnostic reagent may
comprise the polypeptides having amino acid sequences SEQ ID
NOs:11, 18, 22, 30 and 45, or may comprise the polypeptides having
amino acid sequences SEQ ID NOs:13, 15, 24, 46; in other words, the
diagnostic reagent may comprise any combination of polypeptides
which each have an amino acid sequence freely selected from those
indicated by SEQ ID NOs: 11-49.
[0020] Sequences SEQ ID NOs:11-21 are peptides which are fragments
of ESAT-6. Sequences SEQ ID NOs:22-31 are peptides which are
fragments of CFP-10. Sequences SEQ ID NOs:32-43 are peptides which
are fragments of Rv3615c. These sequences are discussed in
co-pending application no. PCT/GB2011/050843. SEQ ID NOs:44, 45 and
46 are full-length ESAT-6, CFP-10, and Rv3615c, respectively. SEQ
ID NO:47 is the protein MPB83, SEQ ID NO:48 is a fragment of MPB83
and SEQ ID NO:49 is the protein MPB70.
[0021] In one embodiment, the diagnostic reagent may comprise SEQ
1D NOs:7, 11-42 and 50-59. Alternatively or additionally, the
diagnostic reagent may comprise SEQ ID NOs:8, 11-42 and 60-69.
[0022] The diagnostic reagent may comprise individual peptide
sequences, or may comprise one or more fusion proteins each
comprising at least two amino acid sequences selected from SEQ ID
NOs:1-69, preferably comprising at least one of SEQ ID NOs:7 or
8.
[0023] The diagnostic reagent may be for use in a method of
detecting a tuberculosis infection in a mammal, or of detecting
exposure of an animal to a tuberculosis agent. Advantageously, the
method is capable of confirming such infection or exposure, as
differentiated from vaccination. The method may be a skin test such
as a caudal fold test (CFT), single intradermal test (SIT) or
single intradermal comparative cervical test (SICCT). A positive
result is obtained when the animal is infected with (or has been
exposed to) a tuberculosis agent and a negative result is obtained
if the animal is not so infected or exposed, even if the animal has
been vaccinated against infection by a tuberculosis agent.
[0024] In some embodiments, for example for use in a skin test, the
diagnostic reagent may be in the form of a sterile injectable
preparation which may be an aqueous or an oleaginous suspension, or
a suspension in a non-toxic parenterally-acceptable diluent or
solvent. The aqueous suspension may be prepared in, for example,
mannitol, water, Ringer's solution or isotonic sodium chloride
solution. Alternatively, it may be prepared in phosphate buffered
saline solution. The oleaginous suspension may be prepared in a
synthetic monoglyceride, a synthetic diglyceride, a fatty acid or a
natural pharmaceutically-acceptable oil. The fatty acid may be an
oleic acid or an oleic acid glyceride derivative. The natural
pharmaceutically-acceptable oil may be an olive oil, a castor oil,
or a polyoxyethylated olive oil or castor oil. The oleaginous
suspension may contain a long-chain alcohol diluent or dispersant,
for example, Ph. Hely.
[0025] Therefore, according to a second aspect of the invention,
there is provided a method of detecting a tuberculosis infection in
an animal, or exposure of an animal to a tuberculosis agent,
comprising the steps of [0026] (i) contacting a population of cells
from the animal with at least one diagnostic reagent as defined in
the first aspect of the invention; and [0027] (ii) determining
whether the cells of said population recognise the diagnostic
reagent.
[0028] Such a method is a "tuberculosis infection assay", as
referred to above and described herein. The population of cells may
include T-cells. Recognition of the diagnostic reagent by said
cells may be by way of, for example, binding of a T cell receptor
to the diagnostic reagent, for example, binding of the T cell
receptor to at least one polypeptide included in the diagnostic
reagent. The method may comprise a cell-mediated immunity (CMI)
assay, which may detect interferon gamma (IFN-.gamma.) as described
herein.
[0029] According to a related aspect of the invention, there is
provided a method of detecting a tuberculosis infection in an
animal, or exposure of an animal to a tuberculosis agent,
comprising conducting a skin test on the animal using at least one
diagnostic reagent according to the first aspect of the invention.
This is also considered to be a "tuberculosis infection assay" as
referred to above. "Using" and "usc" of polypeptides and diagnostic
reagents in the skin test included in the method typically involves
intradermal injection of the polypeptide(s) and/or diagnostic
reagent into the animal.
[0030] The skin test may be a CFT, SIT or SICCT test, as described
in the Office International des Epizooties (OIE) Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals
(ISBN-10:92-9044-718-4;
http://www.oie.int/eng/normes/mmanual/a_summary.htm,accessed 14
Jul. 2011). The manual provides information, definitions and
guidelines on positive test criteria.
[0031] The methods according to this aspect of the invention may be
for detecting M. bovis or M. tuberculosis infection in an animal,
for example (but not limited to) a mammal such as a cow, badger or
human being. Advantageously, because the diagnostic reagents are
able to differentiate between an infected animal and a vaccinated
animal, the user can be sure that a positive result from the method
is a true positive indicating infection, rather than a false
positive resulting from earlier vaccination of the animal.
Therefore, the methods in this aspect of the invention provide a
method of detecting a tuberculosis infection in an animal, or
exposure of an animal to a tuberculosis agent, comprising
conducting a skin test on the animal using at least one diagnostic
reagent according to the first aspect of the invention, wherein use
of the method on an animal which has been vaccinated against
infection by a tuberculosis agent results in a negative skin test
being obtained and use of the method on an animal infected with a
tuberculosis agent results in a positive skin test being
obtained.
[0032] According to a third aspect of the invention, there is
provided a diagnostic kit comprising a diagnostic reagent according
to the first aspect of the invention. The diagnostic kit may be for
use in one or more methods according to the second aspect of the
invention. Particularly where the kit is for use in a skin test
method, the diagnostic reagent may be in liquid form, as outlined
above, or may be in solid (for example, lyophilised) form. It may
be included in the kit in the form of at least one aliquot of
0.05-0.15 ml containing 1-15 .mu.g of each polypeptide contained in
the diagnostic reagent. For example, the kit may comprise aliquots
of about 0.05 ml, about 0.06 ml, about 0.07 ml, about 0.08 ml,
about 0.09 ml, about 0.1 ml, about 0.11 ml, about 0.12 ml, about
0.13 ml, about 0.14 ml or about 0.15 ml, containing 1-15 .mu.g,
3-12 .mu.g, 5-10 .mu.g of each protein, for example, about 5 .mu.g,
about 6 .mu.g, about 7 .mu.g, about 8 .mu.g, about 9 .mu.g or about
10 .mu.g of each polypeptide. Each aliquot may be contained in a
disposable injection device. The kit may further comprise at least
one sample of PPD. The diagnostic reagent is able to detect a
tuberculosis infection in an animal, or exposure of an animal to a
tuberculosis agent, for example it may be able to detect infection
by or exposure to M. bovis or M. tuberculosis. Particularly when
the diagnostic reagent comprises one or more of SEQ ID NOs:1-9
and/or 50-69, it enables a user to differentiate between a
tuberculosis infected animal and an animal which has been
vaccinated against a tuberculosis infection, by methods such as
described herein. As mentioned above, this enables the user to rely
on positive test results when using the diagnostic reagent as an
indication of tuberculosis infection rather than of
vaccination.
[0033] According to a fourth aspect of the invention, there is
provided a polypeptide (which may be isolated) comprising or
consisting of the amino acid sequence of any of SEQ ID NOs:1-10 or
50-69 or a functional variant thereof. The full length antigen
polypeptides listed in Table 3 may be excluded (e.g., Rv1038c,
Rv1197, Rv1792, etc.). Such a polypeptide may form a component of a
diagnostic reagent according to the first aspect of the invention.
The term "functional variant" indicates a polypeptide in which the
amino acid sequence differs from the base sequence from which it is
derived in that one or more amino acids within the sequence are
substituted for other amino acids. The variant is a functional
variant because the functional characteristics of the polypeptide
from which the variant is derived arc maintained. For example, a
similar immune response is elicited by exposure of an animal, or a
sample from an animal, to the variant polypeptide as compared to
the non-variant. In particular, any amino acid substitutions,
additions or deletions must not alter or significantly alter the
tertiary structure of one or more epitopes contained within the
polypeptide from which the variant is derived. The skilled person
is readily able to determine appropriate functional variants
without the application of inventive skill.
[0034] Amino acid substitutions may be regarded as "conservative"
where an amino acid is replaced with a different amino acid with
broadly similar properties. Non-conservative substitutions are
where amino acids are replaced with amino acids of a different
type.
[0035] By "conservative substitution" is meant the substitution of
an amino acid by another amino acid of the same class, in which the
classes arc defined as follows:
TABLE-US-00001 Class Amino acid examples Nonpolar: Ala, Val, Leu,
Ile, Pro, Met, Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr,
Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His.
[0036] As is well known to those skilled in the art, altering the
primary structure of a polypeptide by a conservative substitution
may not significantly alter the activity of that polypeptide
because the side-chain of the amino acid which is inserted into the
sequence may be able to form similar bonds and contacts as the side
chain of the amino acid which has been substituted out. This is so
even when the substitution is in a region which is critical in
determining the peptide's conformation.
[0037] As mentioned above, non-conservative substitutions are
possible provided that these do not disrupt the tertiary structure
of an epitope within the polypeptide, for example, which do not
interrupt the immunogenicity (for example, the antigenicity) of the
peptide. In particular, the addition or deletion of a small number
(for example, up to about 10, or up to about 5, for example about
1, 2, 3, 4 or 5) of amino acids to the N- or C-terminus of a
polypeptide may not adversely affect the immunogenicity of the
peptide and such variants to an amino acid sequence are
particularly envisaged as being included within the term
"functional variant" of a polypeptide.
[0038] Broadly speaking, fewer non-conservative substitutions will
be possible without altering the biological activity of the
polypeptide. Suitably, variants may be at least about 50%
identical, about 60% identical, for example at least about 75%
identical, such as at least about 85%, 90%, 95%, 96%, 97%, 98% or
about 99% identical to the base sequence, determined as a
percentage of the full length of the longest of the polypeptides
being compared.
[0039] Sequence identity between amino acid sequences can be
determined by comparing an alignment of the sequences. When an
equivalent position in the compared sequences is occupied by the
same amino acid, then the molecules are identical at that position.
Scoring an alignment as a percentage of identity is a function of
the number of identical amino acids at positions shared by the
compared sequences. When comparing sequences, optimal alignments
may require gaps to be introduced into one or more of the sequences
to take into consideration possible insertions and deletions in the
sequences. Sequence comparison methods may employ gap penalties so
that, for the same number of identical molecules in sequences being
compared, a sequence alignment with as few gaps as possible,
reflecting higher relatedness between the two compared sequences,
will achieve a higher score than one with many gaps. Calculation of
maximum percent identity involves the production of an optimal
alignment, taking into consideration gap penalties. The percentage
sequence identity may be determined using the BLASTP software,
publicly available at http://blast.ncbi.nlm.nih.gov/Blast.cgi
(accessible on 14 Jul. 201 1), using default parameter settings.
Comparison should be determined for the full length sequence of the
polypeptide, to avoid high sequence identity over a short fragment
of the polypeptide.
[0040] The invention also encompasses fusion proteins comprising
more than one of SEQ ID NOs:1-10 and/or 50-69. The full length
antigen proteins listed in Table 3 may be excluded (e.g., Rv1038c,
Rv1 197, Rv1792, etc.).
[0041] According to a fifth aspect of the invention, there is
provided a nucleic acid encoding a polypeptide comprising at least
one of the amino acid sequences SEQ ID NOs:1-10 or 50-69 or a
functional variant thereof. The DNA sequence encoding the
full-length antigen proteins listed in Table 3 may be excluded
(e.g., the Rv1038c gene, the Rv1197 gene, etc.). The nucleic acid
may form part of a vector and there is also provided a cell
transformed with such a vector.
[0042] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of the words, for
example "comprising" and "comprises", mean "including but not
limited to" and do not exclude other moieties, additives,
components, integers or steps. Throughout the description and
claims of this specification, the singular encompasses the plural
unless the context otherwise requires. In particular, where the
indefinite article is used, the specification is to be understood
as contemplating plurality as well as singularity, unless the
context requires otherwise.
[0043] Features of each aspect of the invention may be as described
in connection with any of the other aspects.
[0044] Other features of the present invention will become apparent
from the following examples. Generally speaking, the invention
extends to any novel one, or any novel combination, of the features
disclosed in this specification (including the accompanying claims
and drawings). Thus, features, integers, characteristics, compounds
or chemical moieties described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein, unless incompatible therewith.
[0045] Moreover, unless stated otherwise, any feature disclosed
herein may be replaced by an alternative feature serving the same
or a similar purpose.
BRIEF DESCRIPTION OF FIGURES
[0046] Particular non-limiting examples of the present invention
will now be described with reference to the following Figures, in
which:
[0047] FIG. 1 shows the responder frequency of 23 TB-reactor (TB)
and 8 BCG-vaccinated (BCG) animals to the most frequently
recognized secretome peptide pools;
[0048] FIG. 2 shows the responder frequencies of 22 TB-reactor
animals (open bars) and 23 BCG-vaccinated animals (filled bars) to
individual secretome peptides (* indicates peptides selected for
inclusion into the Sec#1 peptide pool);
[0049] FIG. 3 (A) shows the responder frequency of 22 TB-reactor
and 21 BCG-vaccinated animals to the Sec#1 peptide pool (p<0.05,
Fisher's Exact Test) and FIG. 3(B) shows IFN-.gamma. responses
(.DELTA.OD .sub.450) from 8 TB-reactor and 3 BCG-vaccinated animals
to both the Sec#1 and Sec#2 peptide pools, with the dashed
horizontal line representing the cut off for a positive
response;
[0050] FIG. 4 shows skin-test responses to peptide cocktails in
cattle naturally exposed to M. bovis infection; and
[0051] FIG. 5 shows skin-test responses of a reference peptide
cocktail containing peptides of ESAT6, CFP10 and Rv3615c either
alone or in combination with a peptide cocktail of either Rv2346c
(peptide pool #11) or Rv3020c (peptide pool #14) (significance
between groups determined by repeated measure ANOVA, *p<0.05),
in cattle naturally exposed to M. bovis infection.
EXAMPLES
[0052] Materials and Methods
[0053] Cattle
[0054] All animals were housed at the Veterinary Laboratories
Agency at the time of blood sampling and procedures were conducted
within the limits of a United Kingdom Home Office Licence under the
Animal (Scientific Procedures) Act 1986, which were approved by the
local ethical review committee. The following groups of animals
were used in this study:
[0055] (i) Tuberculosis Reactors (TB-Reactors)
[0056] Heparinised blood samples were obtained from naturally
infected, SICCT-positive reactors from herds known to have bovine
tuberculosis (BTB) as determined by the Animal Health Agency.
Heparinised blood samples were also obtained from 4 animals who
were experimentally infected ca. 6 months with an M. bovis field
strain from Great Britain (AF 2122/97) by intratracheal
instillation of 1.times.10.sup.3 CFU as previously described (Dean
et al. (2005) Infect. Immun. vol. 73 pp 6467-6471). A detailed post
mortem examination of 36 TB-reactor animals revealed visible
TB-lesions in all but four animals, confirming the presence of
active disease.
[0057] (ii) BCG-Vaccinated
[0058] Heparinised blood samples were obtained from animals
vaccinated with BCG as previously described (Vordermeier et al.
(1999) Clin. Diagn. Lab. Immunol. vol. 6 pp 675-682). Briefly,
calves (ca. 6 months of age) from BTB-free herds were vaccinated
with BCG Pasteur by subcutaneous injection of 1 X 10.sup.6 CFU into
the side of the neck.
[0059] Production and Preparation of Peptides and Antigens
[0060] 119 proteins secreted, or potentially secreted, from
Mycobacterium bovis were selected for antigen screening as
previously described (Jones et al. (2010) Infect. Immun. vol. 78 pp
1326-1332). Peptides were synthesized (JPT Peptide Technologies
GmbH, Berlin, Germany) in pools of 20-mers overlapping by 12 amino
acids for each of the genes of interest. In total, 379 peptide
pools containing a total of 4129 peptides were evaluated. These
peptide pools were dissolved in RPMI 1640 (Gibco, UK) containing
20% dimethyl sulfoxide (DMSO) to obtain a concentration of 1
mg/ml/peptide, and the peptide pools were used to stimulate whole
blood at a final concentration of 5 .mu.g/ml/peptide. Peptides that
comprised the pools for some antigens were synthesized individually
(Mimotopes Pty Ltd, Clayton, Australia), dissolved in RPMI 1640
containing 20% DMSO to obtain a concentration of 5 mg/ml and used
individually to stimulate whole blood at a final concentration of
10 .mu.g/ml, or formulated into additional peptide pools at a
concentration of 10 .mu.g/ml/peptide. Peptides from ESAT-6 and
CFP-10 were formulated to obtain a peptide cocktail as previously
described (Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol.
vol. 8 pp 571-578) and were used at a final concentration of 5
.mu.g/ml/peptide. This peptide cocktail was used as a `gold
standard` with which to compare the immunogenicities of the other
antigens.
[0061] Bovine tuberculin (PPD-B) was supplied by the Tuberculin
Production Unit at the Veterinary Laboratories Agency, Weybridge,
Surrey, UK and was used at a final concentration of 10 .mu.g/ml.
Staphylococcal enterotoxin B (SEB; Sigma-Aldritch, UK) was included
as a positive control at a final concentration of 1 .mu.g/ml, while
whole blood was incubated with RPMI 1640 alone as a negative
control.
[0062] IFN-.gamma. Enzyme-Linked Immunosorbent Assay (ELISA)
[0063] Whole blood aliquots (250 .mu.l) were added in duplicate to
antigen in 96-well plates and incubated at 37.degree. C. in the
presence of 5% CO.sub.2 for 24 hours, following which plasma
supernatants were harvested and stored at -80.degree. C. until
required. Quantification of IFN-.gamma. in the plasma supernatants
was determined using the Bovigam ELISA kit (Prionics AG,
Switzerland). A result was considered positive if the optical
density at 450 nm (OD.sub.450) with antigen minus the OD.sub.450
without antigen (.DELTA.OD .sub.450) was .gtoreq.0.01 in both of
the duplicate wells.
[0064] Skin test evaluation of Rv2346c (peptide pool #11) or
Rv3020c (peptide pool #14) Skin-test evaluation of defined protein
and peptide antigens was performed in cattle naturally exposed to
M. bovis infection (n=17). These cattle were recruited as a result
of providing positive responses to the single intradermal cervical
comparative tuberculin (SICCT) skin-test during routine field
surveillance operations.
[0065] Antigens were administered at 8 sites per animal (4 on each
side of the neck). Antigens were administered in a volume of 100
.mu.l. All defined protein or peptide antigen cocktails were
administered at a concentration of 5 ug/ml per cocktail component.
The skin thickness was measured at the injection site immediately
prior to intradermal administration of antigen. Skin thickness at
the injection site was re-measured 72 hours after antigen
administration and the increase in skin-thickness over this time is
determined. This method is also suitable for determining the
reaction in cattle uninfected with M. bovis, whether vaccinated or
unvaccinated against such infect ion.
[0066] Purified recombinant protein antigens were supplied by
Lionex GmbH. The composition of the cocktail of ESAT-6, CFP-10 and
Rv3615c was SEQ ID NOs:11-43. A combination of 11 peptides
providing a complete sequence overlap for each of the antigens
Rv3020c and Rv2346c was used, as listed in Table 2 below (SEQ ID
NOs:7 and 50-59 provide complete sequence overlap for Rv2346c and
SEQ ID NOs:8 and 60-69 provide complete sequence overlap for
Rv3020c). The Rv3020c and Rv2346c cocktails (i.e. peptide pools #14
and #11, respectively, as shown in Table 2) were tested
individually and in combination with a cocktail containing the
overlapping peptides of ESAT-6, CFP-10 and Rv3615c (i.e., SEQ ID
NOs:11-43), which is itself the subject of co-pending patent
application PCT/GB201 1/050843.
[0067] Results
[0068] To test the hypothesis that M. bovis secreted proteins are
likely to contain immunogenic antigens that can be used to increase
the specificity of diagnostic tests, 379 pools of overlapping
peptides (4129 peptides in total representing 119 antigens) were
screened for their ability to stimulate an 1FN-.gamma. response in
vitro using whole blood from both TB-reactor (n=23) and
BCG-vaccinated animals (n=8). As expected, all TB-reactor and
BCG-vaccinated animals responded to PPD-B and to the positive
control antigen SEB, whilst 22 TB-reactor animals (96%) and 2
BCG-vaccinated animals (25%) responded to the ESAT-6/CFP-10 peptide
cocktail (data not shown). Of the 379 peptide pools, approximately
half (n=184) failed to induce IFN-.gamma. in any of the TB-reactor
or BCG-vaccinated animals. For the remaining 195 peptide pools, 163
and 77 were recognized by TB-reactor and BCG-vaccinated animals
respectively, with 45 being recognised by both groups of animals
(Table 1). Encouragingly, with regards to differential diagnostic
reagents, 118 different peptide pools were recognized by TB-reactor
animals but failed to induce an IFN-.gamma. response in any of the
BCG-vaccinated animals studied.
[0069] A hierarchy of responses to the different peptide pools was
noted, with responder frequencies ranging from 4% to 65% in the
TB-reactor animals, and 13% to 38% in the BCG-vaccinated animals
(Table 1).
TABLE-US-00002 TABLE 1 Recognition of the secreted antigen peptide
pools. Number of peptide pools Number of peptide pools recognized
(%) not recognized (%) BCG- BCG- TB-reactors vaccinated TB-reactors
vaccinated All peptide pools 163 (43%) 77 (20%) 216 (57%) 302 (80%)
(379 in total) TB-reactor pools 163 (100%) 45 (28%) N.A. 118 (72%)
(163 in total)
[0070] FIG. 1 details the responder frequencies for the top 8 most
frequently recognised peptide pools, i.e. those that induced an
IFN-.gamma. response in more than half of the TB-reactor animals
studied. Strikingly, all but one peptide pool (#30-2) represented
antigens belonging to the ESAT-6 protein family. Interestingly,
peptide pools #11 and #14 were not recognized by any of the
BCG-vaccinated animals, suggesting that they contain peptides with
potential application as DIVA reagents. The peptides included in
these pools are indicated in Table 2.
TABLE-US-00003 TABLE 2 Sequences of peptide pools #11 and #14. SEQ
SEQ ID ID Sequence NO Sequence NO Peptide Pool #11 (Rv2346c pool)
Peptide Pool #14 (Rv3020c pool) MTINYQFGDVDAHGAMIRAQ 50
MSLLDAHIPQLIASHTAFAA 60 DVDAHGAMIRAQAGLLEAEH 51
PQLIASHTAFAAKAGLMRHT 61 IRAQAGLLEAEHQAIVRDVL 52
AFAAKAGLMRHTIGQAEQQA 62 EAEHQAIVRDVLAAGDFWGG 53
MRHTIGQAEQQAMSAQAFHQ 63 RDVLAAGDFWGGAGSVACQE 7 EQQAMSAQAFHQGESAAAFQ
64 FWGGAGSVACQEFITQLGRN 54 AFHQGESAAAFQGAHARFVA 65
ACQEFITQLGRNFQVTYEQA 55 AAFQGAHARFVAAAAKVNTL 66
LGRNFQVIYEQANAHGQKVQ 56 RFVAAAAKVNTLLDIAQANL 67
YEQANAHGQKVQAAGNNMAQ 57 VNTLLDIAQANLGEAAGTYV 68
QKVQAAGNNMAQTDSAVGSS 58 QANLGEAAGTYVAADAAAAS 8 VQAAGNNMAQTDSAVGSSWA
59 EAAGTYVAADAAAASSYTGF 69
[0071] Although 6 out of the top 8 most frequently recognized
peptide pools induced IFN-.gamma. responses in some BCG-vaccinated
animals, the inventors next reasoned that a fine detail
investigation of the immunogenicity of the components of these
pools may reveal additional individual peptides with potential use
as DIVA reagents. To this end, overlapping peptides contained
within these peptide pools were screened individually for their
ability to induce IFN-.gamma. production in both TB-reactor (n=22)
and BCG-vaccinated (n=23) animals. In these experiments, 19
TB-reactor animals (86%) but no BCG-vaccinated animals (0%)
responded to the peptide cocktail (data not shown). Fifty-three
individual peptides were identified as immunogenic in TB-reactor
animals, with responder frequencies ranging from 5% to 50% (FIG.
2). Of these peptides, six (peptides #17, #32, #48, #58, #67 and
#69) also induced IFN-.gamma. responses in BCG-vaccinated animals
with responder frequencies ranging from 4% to 17% (FIG. 2).
[0072] In order to evaluate whether a combination of individual
peptides from different secretome antigens is sufficient to
differentially induce an IFN-.gamma. response in TB-reactor
animals, a peptide pool (Sec#1) consisting of 10 peptides was
constructed. Firstly, peptides #42 and #55 (SEQ ID NOs:7 & 8,
respectively) were selected as they were the two most frequently
recognised peptides (responder frequencies of 50% and 36%
respectively) and also because they belonged to peptide pools not
recognised by BCG-vaccinated animals (pools #11 and #14
respectively, FIG. 1). A further four peptides (peptides #20, #29,
#33 and #64) were next selected as they were recognised in
TB-reactor animals that failed to respond to peptides #42 or #55
(data not shown). Lastly, a further four peptides (peptides #16,
#19, #25 and #57) were included due to their location in regions of
homology between multiple ESAT-6 proteins (see Table 3).
TABLE-US-00004 TABLE 3 Identification of peptides in pools Sec#1
and Sec#2 SEQ Pool Peptide ID Sequence Located in antigens: Sec#1
Pep#16 1 MWASAQNISGAGWSGMAEAT Rv1038c, Rv1197, Rv1792, Rv2347c,
Rv3620c Pep#19 2 MTQMNQAFRNIVNMLHGVRD Rv1038c, Rv3620c Pep#20 3
RNIVNMLHGVRDGLVRDANN Rv1038c, Rv1197, Rv1792, Rv2347c, Rv3620c
Pep#25 4 MAQMNQAFRNIVNMLHGVRD Rv1197, Rv2347c Pep#29 5
EAEHQAIIRDVLTASDFWGG Rv1198 Pep#33 6 LGRNFQVIYEQANAHGQKVQ Rv1198,
Rv2346c, Rv3619c, Rv1037c, Rv1793 Pep#42 7 RDVLAAGDFWGGAGSVACQE
Rv2346c, Rv1793 Pep#55 8 QANLGEAAGTYVAADAAAAS Rv3020c Pep#57 9
DVDAHGAMIRAQAGSLEAEH Rv3619c, Rv1037c Pep#64 10
SAELPDWLAANRGLAPGGHA Rv3803c Sec#2 As above but omitting Pep#64
[0073] As shown in FIG. 3A, the responder frequency to the Sec#1
peptide pool was significantly greater in TB-reactor animals
(p<0.05, Fisher's Exact Test), with 14 out of 22 (64%)
TB-reactor animals recognising the peptide pool compared with 6 out
of 21 (29%) BCG-vaccinated animals. In order to optimise the
peptide pool for use as a DIVA reagent, the individual peptide
components of the Sec#1 peptide pool were re-screened for their
ability to induce an IFN--.gamma. response in BCG-vaccinated
animals. These experiments identified only a single peptide
(peptide #64) as being immunogenic in some BCG-vaccinated animals
(data not shown). Thus, a second peptide pool (Sec#2) was
constructed that lacked this peptide and the ability of both Sec#1
and Scc#2 to induce IFN-.gamma. was compared in both TB-reactor
animals (n=8) and BCG-vaccinated animals (n=3) which had previously
demonstrated to recognise the former peptide pool. Omitting peptide
#64 from the pool had little effect on the responder frequency for
TB-reactor animals, with 7 of the 8 animals still producing
IFN-.gamma. .quadrature.above the cut off (FIG. 3B). Overall, 7 out
of 13 (54%) TB-reactor animals produced IFN-.gamma. in response to
Sec#2 (data not shown). In contrast, removal of peptide #64
completely abrogated the response in all BCG-vaccinated animals
tested (FIG. 3B).
[0074] The skin-test data is shown in FIG. 4. One animal failed to
induce a skin-reaction in the comparative tuberculin skin-test and,
similarly, this same animal provided no skin-test response to any
of the defined antigen combinations. Addition of the peptide
cocktail of Rv3020c (SEQ ID NOs:8 and 60-69) to the reference
cocktail containing peptides of ESAT-6, CFP-10 and Rv3615c (SEQ ID
NOs: 11-43) demonstrated significantly stronger responses than the
reference cocktail alone, see FIG. 5.
[0075] Discussion
[0076] The results presented herein have significant importance
with regards to the development of DIVA reagents. Screening of 119
proteins secreted, or potentially secreted, by M. Bovis revealed
three unique peptide pools that were frequently recognized by M.
bovis-infected cattle but failed to induce an IFN-.gamma. response
in any BCG-vaccinated animals studied. Two of these peptide pools
consisted of overlapping peptides that represented the full amino
acid sequence for two individual antigens, Rv2346c and Rv3020c,
whilst the third (Sec#2) consisted of a cocktail of 9 peptides
derived from multiple antigens.
[0077] The underlying mechanism for the differential recognition of
Rv2346c and Rv3020c remains unclear. Firstly, both genes are
located in the genomes of M. bovis (strain AF2122/97) and, more
importantly, in BCG Pasteur (strain 1173P2) which was used to
immunise the cattle. Secondly, genome analysis revealed identical
amino acid sequences of the two proteins between the two strains.
Thus, the lack of immune responses to Rv2346c and Rv3020c seen in
BCG-vaccinated animals is unlikely to be explained by deletions or
amino acid sequence alterations within these two proteins in the
BCG Pasteur strain used for vaccination.
[0078] It is unlikely that IFN-.gamma. responses to a single
protein antigen will be sufficient for the detection of M. bovis
infection of cattle. Indeed, the QuantiFERON.RTM.-TB Gold Test for
human M. tuberculosis infection utilizes synthetic peptides from
two different M. tuberculosis antigens (ESAT-6 and CFP-10). With
this in mind, the inventors developed a cocktail (Sec#2) containing
individual peptides isolated from various peptide pools
representing the most frequently recognised antigens.
Interestingly, these antigens were found to belong to the ESAT-6
protein family, highlighting the immunodominance of these antigens.
The Sec#2 cocktail contained several immunodominant peptides with
restricted expression amongst the ESAT-6 proteins; for example,
peptide #55 is located only within Rv3020c while peptide #42 is
located in Rv2346c and Rv1793 (Table 3). However, given the high
degree of amino acid similarity between the members of the ESAT-6
protein family, several of these peptide sequences represented
multiple antigens. For example, peptides #16 and #20 are located in
Rv1038c, Rv1197, Rv1792, Rv2347c and Rv3620c, while peptide #33 is
located in Rv1198, Rv2346c, Rv3619c, Rv1037c and Rv1793. Thus,
without wishing to be bound by theory, targeting these shared
sequences not only reduces the number of different components
within the DIVA reagent but may also exploit a potential greater
antigenic load for these regions.
[0079] The ESAT-6/CFP-10 peptide cocktail used in the studies
presented herein has been developed as a DIVA reagent in cattle,
with reported sensitivities of approximately 78% in M.
bovis-infected animals (Sidders et al. (2008) Infect. Immun. vo. 76
pp 3932-3939; Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol.
vol. 8 pp 571-578). Thus, one area of research of high importance
is the identification of reagents that may complement the
ESAT-6/CFP-10 peptide cocktail in the diagnosis of bovine TB.
[0080] Recently, the inventors have demonstrated that 4 out of 7
(57%) M. bovis-infected animals that failed to recognise the
ESAT-6/CFP-10 peptide cocktail did mount an IFN-.gamma. response to
the antigen Rv3615c, theoretically increasing diagnostic
sensitivity to 91% without compromising specificity in
BCG-vaccinated animals (Sidders et al. (2008) Infect. Immun. vo. 76
pp 3932-3939). In the current study, 5 out of 13 (38%) TB-reactor
animals recognized Rv3615c (data not shown), results similar to
those previously reported (Sidders et al. (2008)). All of these 5
animals recognized the Sec#2 peptide cocktail, which also induced
responses in a further two animals (overall responder frequency of
54%), suggesting that the Scc#2 peptide cocktail may be as good, if
not better, at complementing ESAT-6/CFP-10 in the diagnosis of
bovine TB without compromising specificity in BCG-vaccinatcd
animals.
[0081] Finally, skin test data showed that the peptides improved
the sensitivity of skin test detection of M. bovis infected cattle
when using a cocktail of ESAT-6, CFP-10 and Rv3615c peptides.
[0082] In summary, the results of this study demonstrate that
cocktails of synthetic peptides derived from secreted or
potentially secreted antigens have the capacity to distinguish
between M. bovis-infected and BCG-vaccinated animals in blood-based
screening assays.
Sequence CWU 1
1
69120PRTMycobacterium bovis 1Met Trp Ala Ser Ala Gln Asn Ile Ser
Gly Ala Gly Trp Ser Gly Met 1 5 10 15 Ala Glu Ala Thr 20
220PRTMycobacterium bovis 2Met Thr Gln Met Asn Gln Ala Phe Arg Asn
Ile Val Asn Met Leu His 1 5 10 15 Gly Val Arg Asp 20
320PRTMycobacterium bovis 3Arg Asn Ile Val Asn Met Leu His Gly Val
Arg Asp Gly Leu Val Arg 1 5 10 15 Asp Ala Asn Asn 20
420PRTMycobacterium bovis 4Met Ala Gln Met Asn Gln Ala Phe Arg Asn
Ile Val Asn Met Leu His 1 5 10 15 Gly Val Arg Asp 20
520PRTMycobacterium bovis 5Glu Ala Glu His Gln Ala Ile Ile Arg Asp
Val Leu Thr Ala Ser Asp 1 5 10 15 Phe Trp Gly Gly 20
620PRTMycobacterium bovis 6Leu Gly Arg Asn Phe Gln Val Ile Tyr Glu
Gln Ala Asn Ala His Gly 1 5 10 15 Gln Lys Val Gln 20
720PRTMycobacterium bovis 7Arg Asp Val Leu Ala Ala Gly Asp Phe Trp
Gly Gly Ala Gly Ser Val 1 5 10 15 Ala Cys Gln Glu 20
820PRTMycobacterium bovis 8Gln Ala Asn Leu Gly Glu Ala Ala Gly Thr
Tyr Val Ala Ala Asp Ala 1 5 10 15 Ala Ala Ala Ser 20
920PRTMycobacterium bovis 9Asp Val Asp Ala His Gly Ala Met Ile Arg
Ala Gln Ala Gly Ser Leu 1 5 10 15 Glu Ala Glu His 20
1020PRTMycobacterium bovis 10Ser Ala Glu Leu Pro Asp Trp Leu Ala
Ala Asn Arg Gly Leu Ala Pro 1 5 10 15 Gly Gly His Ala 20
1116PRTMycobacterium bovis 11Met Thr Glu Gln Gln Trp Asn Phe Ala
Gly Ile Glu Ala Ala Ala Ser 1 5 10 15 1216PRTMycobacterium bovis
12Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser 1
5 10 15 1316PRTMycobacterium bovis 13Ala Ile Gln Gly Asn Val Thr
Ser Ile His Ser Leu Leu Asp Glu Gly 1 5 10 15 1416PRTMycobacterium
bovis 14Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr Lys Leu
Ala 1 5 10 15 1516PRTMycobacterium bovis 15Lys Gln Ser Leu Thr Lys
Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 1 5 10 15
1616PRTMycobacterium bovis 16Ala Ala Trp Gly Gly Ser Gly Ser Glu
Ala Tyr Gln Gly Val Gln Gln 1 5 10 15 1716PRTMycobacterium bovis
17Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 1
5 10 15 1816PRTMycobacterium bovis 18Lys Trp Asp Ala Thr Ala Thr
Glu Leu Asn Asn Ala Leu Gln Asn Leu 1 5 10 15 1916PRTMycobacterium
bovis 19Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala
Gly 1 5 10 15 2016PRTMycobacterium bovis 20Ala Arg Thr Ile Ser Glu
Ala Gly Gln Ala Met Ala Ser Thr Glu Gly 1 5 10 15
2115PRTMycobacterium bovis 21Gln Ala Met Ala Ser Thr Glu Gly Asn
Val Thr Gly Met Phe Ala 1 5 10 15 2218PRTMycobacterium bovis 22Met
Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly 1 5 10
15 Asn Phe 2316PRTMycobacterium bovis 23Gln Glu Ala Gly Asn Phe Glu
Arg Ile Ser Gly Asp Leu Lys Thr Gln 1 5 10 15 2418PRTMycobacterium
bovis 24Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu
Ser 1 5 10 15 Thr Ala 2517PRTMycobacterium bovis 25Ile Asp Gln Val
Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg 1 5 10 15 Gly
2618PRTMycobacterium bovis 26Gly Ser Leu Gln Gly Gln Trp Arg Gly
Ala Ala Gly Thr Ala Ala Gln 1 5 10 15 Ala Ala 2718PRTMycobacterium
bovis 27Ala Gly Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala
Ala 1 5 10 15 Asn Lys 2818PRTMycobacterium bovis 28Val Val Arg Phe
Gln Glu Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp 1 5 10 15 Glu Ile
2920PRTMycobacterium bovis 29Gln Lys Gln Glu Leu Asp Glu Ile Ser
Thr Asn Ile Arg Gln Ala Gly 1 5 10 15 Val Gln Tyr Ser 20
3018PRTMycobacterium bovis 30Asn Ile Arg Gln Ala Gly Val Gln Tyr
Ser Arg Ala Asp Glu Glu Gln 1 5 10 15 Gln Gln 3116PRTMycobacterium
bovis 31Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln Met Gly
Phe 1 5 10 15 3220PRTMycobacterium bovis 32Met Thr Glu Asn Leu Thr
Val Gln Pro Glu Arg Leu Gly Val Leu Ala 1 5 10 15 Ser His His Asp
20 3320PRTMycobacterium bovis 33Pro Glu Arg Leu Gly Val Leu Ala Ser
His His Asp Asn Ala Ala Val 1 5 10 15 Asp Ala Ser Ser 20
3420PRTMycobacterium bovis 34Ser His His Asp Asn Ala Ala Val Asp
Ala Ser Ser Gly Val Glu Ala 1 5 10 15 Ala Ala Gly Leu 20
3520PRTMycobacterium bovis 35Asp Ala Ser Ser Gly Val Glu Ala Ala
Ala Gly Leu Gly Glu Ser Val 1 5 10 15 Ala Ile Thr His 20
3620PRTMycobacterium bovis 36Ala Ala Gly Leu Gly Glu Ser Val Ala
Ile Thr His Gly Pro Tyr Cys 1 5 10 15 Ser Gln Phe Asn 20
3720PRTMycobacterium bovis 37Ala Ile Thr His Gly Pro Tyr Cys Ser
Gln Phe Asn Asp Thr Leu Asn 1 5 10 15 Val Tyr Leu Thr 20
3820PRTMycobacterium bovis 38Ser Gln Phe Asn Asp Thr Leu Asn Val
Tyr Leu Thr Ala His Asn Ala 1 5 10 15 Leu Gly Ser Ser 20
3920PRTMycobacterium bovis 39Val Tyr Leu Thr Ala His Asn Ala Leu
Gly Ser Ser Leu His Thr Ala 1 5 10 15 Gly Val Asp Leu 20
4020PRTMycobacterium bovis 40Leu Gly Ser Ser Leu His Thr Ala Gly
Val Asp Leu Ala Lys Ser Leu 1 5 10 15 Arg Ile Ala Ala 20
4120PRTMycobacterium bovis 41Gly Val Asp Leu Ala Lys Ser Leu Arg
Ile Ala Ala Lys Ile Tyr Ser 1 5 10 15 Glu Ala Asp Glu 20
4220PRTMycobacterium bovis 42Arg Ile Ala Ala Lys Ile Tyr Ser Glu
Ala Asp Glu Ala Trp Arg Lys 1 5 10 15 Ala Ile Asp Gly 20
4320PRTMycobacterium bovis 43Ala Lys Ile Tyr Ser Glu Ala Asp Glu
Ala Trp Arg Lys Ala Ile Asp 1 5 10 15 Gly Leu Phe Tyr 20
4495PRTMycobacterium bovis 44Met Thr Glu Gln Gln Trp Asn Phe Ala
Gly Ile Glu Ala Ala Ala Ser 1 5 10 15 Ala Ile Gln Gly Asn Val Thr
Ser Ile His Ser Leu Leu Asp Glu Gly 20 25 30 Lys Gln Ser Leu Thr
Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45 Glu Ala Tyr
Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 50 55 60 Leu
Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly 65 70
75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala
85 90 95 45100PRTMycobacterium bovis 45Met Ala Glu Met Lys Thr Asp
Ala Ala Thr Leu Ala Gln Glu Ala Gly 1 5 10 15 Asn Phe Glu Arg Ile
Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val 20 25 30 Glu Ser Thr
Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly 35 40 45 Thr
Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys 50 55
60 Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly
65 70 75 80 Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu
Ser Ser 85 90 95 Gln Met Gly Phe 100 46103PRTMycobacterium bovis
46Met Thr Glu Asn Leu Thr Val Gln Pro Glu Arg Leu Gly Val Leu Ala 1
5 10 15 Ser His His Asp Asn Ala Ala Val Asp Ala Ser Ser Gly Val Glu
Ala 20 25 30 Ala Ala Gly Leu Gly Glu Ser Val Ala Ile Thr His Gly
Pro Tyr Cys 35 40 45 Ser Gln Phe Asn Asp Thr Leu Asn Val Tyr Leu
Thr Ala His Asn Ala 50 55 60 Leu Gly Ser Ser Leu His Thr Ala Gly
Val Asp Leu Ala Lys Ser Leu 65 70 75 80 Arg Ile Ala Ala Lys Ile Tyr
Ser Glu Ala Asp Glu Ala Trp Arg Lys 85 90 95 Ala Ile Asp Gly Leu
Phe Thr 100 47220PRTMycobacterium bovis 47Met Ile Asn Val Gln Ala
Lys Pro Ala Ala Ala Ala Ser Leu Ala Ala 1 5 10 15 Ile Ala Ile Ala
Phe Leu Ala Gly Cys Ser Ser Thr Lys Pro Val Ser 20 25 30 Gln Asp
Thr Ser Pro Lys Pro Ala Thr Ser Pro Ala Ala Pro Val Thr 35 40 45
Thr Ala Ala Met Ala Asp Pro Ala Ala Asp Leu Ile Gly Arg Gly Cys 50
55 60 Ala Gln Tyr Ala Ala Gln Asn Pro Thr Gly Pro Gly Ser Val Ala
Gly 65 70 75 80 Met Ala Gln Asp Pro Val Ala Thr Ala Ala Ser Asn Asn
Pro Met Leu 85 90 95 Ser Thr Leu Thr Ser Ala Leu Ser Gly Lys Leu
Asn Pro Asp Val Asn 100 105 110 Leu Val Asp Thr Leu Asn Gly Gly Glu
Tyr Thr Val Phe Ala Pro Thr 115 120 125 Asn Ala Ala Phe Asp Lys Leu
Pro Ala Ala Thr Ile Asp Gln Leu Lys 130 135 140 Thr Asp Ala Lys Leu
Leu Ser Ser Ile Leu Thr Tyr His Val Ile Ala 145 150 155 160 Gly Gln
Ala Ser Pro Ser Arg Ile Asp Gly Thr His Gln Thr Leu Gln 165 170 175
Gly Ala Asp Leu Thr Val Ile Gly Ala Arg Asp Asp Leu Met Val Asn 180
185 190 Asn Ala Gly Leu Val Cys Gly Gly Val His Thr Ala Asn Ala Thr
Val 195 200 205 Tyr Met Ile Asp Thr Val Leu Met Pro Pro Ala Gln 210
215 220 4820PRTMycobacterium bovis 48Gly Leu Val Cys Gly Gly Val
His Thr Ala Asn Ala Thr Val Tyr Met 1 5 10 15 Ile Asp Thr Val 20
49193PRTMycobacterium bovis 49Met Lys Val Lys Asn Thr Ile Ala Ala
Thr Ser Phe Ala Ala Ala Gly 1 5 10 15 Leu Ala Ala Leu Ala Val Ala
Val Ser Pro Pro Ala Ala Ala Gly Asp 20 25 30 Leu Val Gly Pro Gly
Cys Ala Glu Tyr Ala Ala Ala Asn Pro Thr Gly 35 40 45 Pro Ala Ser
Val Gln Gly Met Ser Gln Asp Pro Val Ala Val Ala Ala 50 55 60 Ser
Asn Asn Pro Glu Leu Thr Thr Leu Thr Ala Ala Leu Ser Gly Gln 65 70
75 80 Leu Asn Pro Gln Val Asn Leu Val Asp Thr Leu Asn Ser Gly Gln
Tyr 85 90 95 Thr Val Phe Ala Pro Thr Asn Ala Ala Phe Ser Lys Leu
Pro Ala Ser 100 105 110 Thr Ile Asp Glu Leu Lys Thr Asn Ser Ser Leu
Leu Thr Ser Ile Leu 115 120 125 Thr Tyr His Val Val Ala Gly Gln Thr
Ser Pro Ala Asn Val Val Gly 130 135 140 Thr Arg Gln Thr Leu Gln Gly
Ala Ser Val Thr Val Thr Gly Gln Gly 145 150 155 160 Asn Ser Leu Lys
Val Gly Asn Ala Asp Val Val Cys Gly Gly Val Ser 165 170 175 Thr Ala
Asn Ala Thr Val Tyr Met Ile Asp Ser Val Leu Met Pro Pro 180 185 190
Ala 5020PRTMycobacterium bovis 50Met Thr Ile Asn Tyr Gln Phe Gly
Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile Arg Ala Gln 20
5120PRTMycobacterium bovis 51Asp Val Asp Ala His Gly Ala Met Ile
Arg Ala Gln Ala Gly Leu Leu 1 5 10 15 Glu Ala Glu His 20
5220PRTMycobacterium bovis 52Ile Arg Ala Gln Ala Gly Leu Leu Glu
Ala Glu His Gln Ala Ile Val 1 5 10 15 Arg Asp Val Leu 20
5320PRTMycobacterium bovis 53Glu Ala Glu His Gln Ala Ile Val Arg
Asp Val Leu Ala Ala Gly Asp 1 5 10 15 Phe Trp Gly Gly 20
5420PRTMycobacterium bovis 54Phe Trp Gly Gly Ala Gly Ser Val Ala
Cys Gln Glu Phe Ile Thr Gln 1 5 10 15 Leu Gly Arg Asn 20
5520PRTMycobacterium bovis 55Ala Cys Gln Glu Phe Ile Thr Gln Leu
Gly Arg Asn Phe Gln Val Ile 1 5 10 15 Tyr Glu Gln Ala 20
5620PRTMycobacterium bovis 56Leu Gly Arg Asn Phe Gln Val Ile Tyr
Glu Gln Ala Asn Ala His Gly 1 5 10 15 Gln Lys Val Gln 20
5720PRTMycobacterium bovis 57Tyr Glu Gln Ala Asn Ala His Gly Gln
Lys Val Gln Ala Ala Gly Asn 1 5 10 15 Asn Met Ala Gln 20
5820PRTMycobacterium bovis 58Gln Lys Val Gln Ala Ala Gly Asn Asn
Met Ala Gln Thr Asp Ser Ala 1 5 10 15 Val Gly Ser Ser 20
5920PRTMycobacterium bovis 59Val Gln Ala Ala Gly Asn Asn Met Ala
Gln Thr Asp Ser Ala Val Gly 1 5 10 15 Ser Ser Trp Ala 20
6020PRTMycobacterium bovis 60Met Ser Leu Leu Asp Ala His Ile Pro
Gln Leu Ile Ala Ser His Thr 1 5 10 15 Ala Phe Ala Ala 20
6120PRTMycobacterium bovis 61Pro Gln Leu Ile Ala Ser His Thr Ala
Phe Ala Ala Lys Ala Gly Leu 1 5 10 15 Met Arg His Thr 20
6220PRTMycobacterium bovis 62Ala Phe Ala Ala Lys Ala Gly Leu Met
Arg His Thr Ile Gly Gln Ala 1 5 10 15 Glu Gln Gln Ala 20
6320PRTMycobacterium bovis 63Met Arg His Thr Ile Gly Gln Ala Glu
Gln Gln Ala Met Ser Ala Gln 1 5 10 15 Ala Phe His Gln 20
6420PRTMycobacterium bovis 64Glu Gln Gln Ala Met Ser Ala Gln Ala
Phe His Gln Gly Glu Ser Ala 1 5 10 15 Ala Ala Phe Gln 20
6520PRTMycobacterium bovis 65Ala Phe His Gln Gly Glu Ser Ala Ala
Ala Phe Gln Gly Ala His Ala 1 5 10 15 Arg Phe Val Ala 20
6620PRTMycobacterium bovis 66Ala Ala Phe Gln Gly Ala His Ala Arg
Phe Val Ala Ala Ala Ala Lys 1 5 10 15 Val Asn Thr Leu 20
6720PRTMycobacterium bovis 67Arg Phe Val Ala Ala Ala Ala Lys Val
Asn Thr Leu Leu Asp Ile Ala 1 5 10 15 Gln Ala Asn Leu 20
6820PRTMycobacterium bovis 68Val Asn Thr Leu Leu Asp Ile Ala Gln
Ala Asn Leu Gly Glu Ala Ala 1 5 10 15 Gly Thr Tyr Val 20
6920PRTMycobacterium bovis 69Glu Ala Ala Gly Thr Tyr Val Ala Ala
Asp Ala Ala Ala Ala Ser Ser 1 5 10 15 Tyr Thr Gly Phe 20
* * * * *
References