Note: Descriptions are shown in the official language in which they were submitted.
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VACCINES AND THEIR USE
The present invention relates to vaccines and their use, and in particular to
vaccines for meningococcal disease.
The listing or discussion of a prior-published document in this specification
should not necessarily be talcen as an acknowledgement that the docuinent is
part
of the state of the art or is common general knowledge. The documents listed
in
the specification are hereby incorporated by reference.
Microbial infections remain a serious risk to human and animal health,
particularly
in light of the fact that many pathogenic microorganisms, particularly
bacteria, are
or inay become resistant to anti-microbial agents such as antibiotics.
Vaccination provides an alternative approach to combating microbial
infections,
but it is often difficult to identify suitable immunogens for use in vaccines
which
are safe and which are effective against a range of different isolates of a
pathogenic microorganism, particular a genetically diverse microorganism.
Although it is possible to develop vaccines which use as the irmnunogen
substantially intact microorganisms, such as live attenuated bacteria which
typically contain one or mutations in a virulence-determining gene, not all
microorganisms are amenable to this approach, and it is not always desirable
to
adopt this approach for a particular microorganism where safety cannot always
be
guaranteed. Also, some microorganisms express molecules which mimic host
proteins, and these are undesirable in a vaccine.
A particular group of i.nicroorganisms for which it is important to develop
further
vaccines is Neisseria 7neningitidis which causes meningococcal disease, a life
threatening infection which in the Europe, North America, developing countries
and elsewhere remains an important cause of childhood mortality despite the
iiitroduction of the conjugate serogroup C polysaccharide vaccine. This is
because
infections caused by serogroup B strains (NfnB), which express an a,2-8 linked
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polysialic acid capsule, are still prevalent. The term "serogroup" in relation
to N.
n2eningitidis refers to the polysaccharide capsule expressed on the bacterium.
The
common serogroup in the UK causing disease is B, while in Africa it is A.
Meningococcal septicaemia continues to carry a high case fatality rate; and
survivors are often left with major psychological and/or physical disability.
After a
non-specific prodromal illness, meningococcal septicaemia can present as a
fulminant disease that is refractory to appropriate anti-microbial therapy and
full
supportive measures. Therefore, the best approach to combating the public
health
menace of meningococcal disease is through prophylactic vaccination.
The non-specific early clinical signs and fulminant course of meningococcal
infection mean that therapy is often ineffective. Therefore vaccination is
considered the most effective. strategy to diminish the global disease burden
caused by this pathogen (Feavers (2000) ABC of meningococcal diversity. Nature
404, 451-2). Existing vaccines to prevent serogroup A, C, W135, and Y N.
nieningitidis infections are based on the polysaccharide capsule located on
the
surface of bacterium (Anderson et al (1994) Safety and immunogenicity of
meningococcal A and C polysaccharide conjugate vaccine in adults. Infect
Inznzun.
62, 3391-33955; Leach et al (1997) Induction of immunologic memory in
2o Gambian children by vaccination in infancy with a group *A plus group C
meningococcal polysaccharide-protein conjugate vaccine. Jbzfect Dis. 175, 200-
4;
Lieberman et al (1996). Safety and immmunogenicity of a serogroups A/C
Neisseria
7neningitidis oligosaccharide-protein conjugate vaccine in young children. A
randomized controlled trial. J. Anaerican Med. Assoc. 275, 1499-1503).
Progress
toward a vaccine against serogroup B infections has been more difficult as its
capsule, a hoinopolymer of a2-8 linked sialic acid, is a relatively poor
immunogen
in humans. This is because it shares epitopes expressed on a human cell
adhesion
molecule, N-CAM1 (Finne et al (1983) Antigenic similarities between brain
components and bacteria causing meningitis. hnplications for vaccine
development and pathogenesis. Lancet 2, 355-357). Indeed, generating immune
responses against the serogroup B capsule might actually prove harmful. Thus,
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there remains a need for new vaccines to prevent serogroup B N. n2eningitidis
infections.
The most validated immunologic correlate of protection against meningococcal
disease is the serum bactericidal assay (SBA). The SBA evaluates the ability
of
antibodies (usually IgG2a subclass) in seruin to mediate complement deposition
on the bacterial cell surface, assembly of the membrane attack complex, and
bacterial lysis. In the SBA, a known nuinber of bacteria are exposed serial
dilutions of the sera with a defined complement source. The number of
surviving
1o bacteria is determined, and the SBA is defined as the reciprocal of the
highest
dilution of serum that mediates 50% killing. The SBA is predictive of
protection
against serogroup C infections, and has been widely used as a surrogate for
immunity against NrnB infections. Importantly the SBA is a ready marker of
immunity for the pre-clinical assessment of vaccines, and provides a suitable
endpoint in clinical trials.
Most efforts at NniB vaccine development are directed toward defining
effective
protein subunits. There has been a major investment in 'Reverse vaccinology',
in
which genome sequences are interrogated for potentially surface expressed
proteins which are expressed as heterologous antigens and tested for their
ability
to generate meaningful responses in animals. However, this approach is limited
by
1) the computer algorithms for predicting surface expressed antigens, 2)
failure to
express many of potential immunogens, and 3) the total reliance on inurine
immune responses.
The key to a successful vaccine is to define antigen(s) that elicit protection
against
a broad range of disease isolates irrespective of serogroup or clonal group. A
genetic screening method (which we have termed Genetic Screening for
Immunogens or GSI) was used to isolate antigens that are conserved across the
genetic diversity of microbial strains and this is exemplified in relation to
meningococcal strains. This was done by identifying rnicrobial antigens, such
as
N. rneningitidis antigens, by GSI as described in more detail below; and
validated
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by assessing the function of the immune response elicited by the recombinant
antigens and by evaluating the protective efficacy of antigens (see Examples
and
see PCT/GB2004/005441 (published as WO 2005/060995 on 7 July 2005)
incorporated herein by reference). In essence, the GSI method relates to a
method
for identifying a polypeptide of a microorganism which polypeptide is
associated
with an immune response in an animal which has been subjected to the
microorganism, the method comprising the steps of (1) providing a plurality of
different niutants of the microorganism; (2) contacting the plurality of
mutant
microorganisms with antibodies from an aniinal which has raised an immune
1o response to the microorganism or a part thereof, under conditions whereby
if the
antibodies bind to the mutant microorganism the mutant microorganism is
killed;
(3) selecting surviving mutant microorganisms from step (2); (4) identifying
the
gene containing the mutation in any surviving l.nutant microorganism; and (5)
identifying the polypeptide encoded by the gene. It will be appreciated that
by the
way in which the polypeptides have been identified, they are highly relevant
as
antigenic polypeptides.
As described in more detail in the Exainples, particular genes identified by
the
GSI method are the NBM0341 (TspA), NMB0338, NMB1345, NMB0738,
NBM0792 (NadC family), NMB0279, NMB2050, NMB1335 (CreA), NMB2035,
NMB 13 51 (Fmu and Fmv), NMB 1574 (IIvC), NMB 129 8(rsuA), NMB 18 5 6
(LysR family), NMB0119, NMB1705 (rfak), NMB2065 (HeinK), NMB0339,
NMB0401 (putA), NMB1467 (PPX), NMB2056, NMB0808, NMB0774 (upp),
NMA0078, NMB0337 (branched-chain amino acid transferase), NMB0191 (ParA
fatnily), N.MB1710 (glutamate dehydrogenase (gdhA), NMB0062 (rfbA-1),
NIvIB1583 (hisB), NMB0377, NMB0264, NMB1333, NMB1036, NMB1176,
NMB 1359 and NMB 113 8 genes of Neisseria nieningitidis. The genome sequence
for N. nzeningitidis is available, for example from The Institute of Genoine
Research (TIGR); www.tigr.org.
Although these genes form part of the genoine that has been sPquenced, as far
as
the inventors are aware, they have not been isolated, the polypeptides they
encode
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have not been produced (and have not been isolated), and there is no
indication
that the polypeptides they encode may be useful as a component of a vaccine.
Thus, the invention includes the isolated genes as above and in the Examples
and
5 variants and fragments and fusions of such variants and fragments, and
includes
the polypeptides that the genes encode as described above, along with variants
and
fragment thereof, and fusions of such fragments and variants. Variants,
fraginents
and fusions are described in more detail below. Preferably, the variants,
fragments and fusions of the given genes above are ones which encode a
1o polypeptide which gives rise to neutralizing antibodies against N.
menii2gitidis.
Similarly, preferably, the variants, fragments and fusions of the polypeptide
whose
sequence is given above are ones which gives rise to neutralizing antibodies
against N. ineningitidis. The neutralising antibodies may be produced in any
animal with an immune systein, for exainple a rat, mouse or rabbit. The
invention
also includes isolated polynucleotides encoding the polypeptides whose
sequences
are given in the Example (preferably the isolated coding region) or encoding
the
variants, fragments or fusions. The invention also includes expression vectors
comprising such polynucleotides and host cells comprising such polynucleotides
and vectors (as is described in more detail below). The polypeptides described
in
the Examples are antigens identified by the method of the invention.
Molecular biological methods for use in the practice of the method of the
invention are well known in the art, for example from Sambrook & Russell
(2001)
.Molecular Cloning, a laboratory manual, third edition, Cold Spring Harbor
laboratory Press, Cold Spring Harbor, New Yorlc, incorporated herein by
reference.
Variants of the gene may be made, for example by identifying related genes in
other microorganisms or in other strains of the microorganism, and cloning,
isolating or synthesizing the gene. Typically, variants of the gene are ones
which
have at least 70% sequence identity, more preferably at least 85% sequence
identity, most preferably at-least 95% sequence identity with the genes as
given
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above. Of course, replacements, deletions and insertions may be tolerated. The
degree of similarity between one nucleic acid sequence and another can be
determined using the GAP program of the University of Wisconsin Computer
Group.
Variants of the gene are also ones which hybridise under stringent conditions
to
the gene. By "stringent" we mean that the gene hybridises to the probe when
the
gene is immobilised on a membrane and the probe (which, in this case is >200
nucleotides in length) is in solution and the immobilised gene/hybridised
probe is
1o washed in 0.1 x SSC at 65 C for 10 min. SSC is 0.15 M NaCl/0.015 M Na
citrate.
Fragments of the gene (or the variant gene) may be made which are, for
example,
20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of the total of the
gene. Preferred fragments include all or part of the coding sequence. The
variant
and fragments may be fused to other, unrelated, polynucleotides.
The polynucleotide encodes a polypeptide which is iunm.unogenic and is
reactive
with the antibodies from an aniinal which has been subjected to the
microorganism from which the gene was identified.
The antigen may be the polypeptide as encoded by the gene identified above,
and
the sequence of the polypeptide may readily be deduced from the gene sequence.
In fiifther embodiments, the antigen may be a fragment of the identified
polypeptide or may be a variant of the identified polypeptide or may be a
fusion of
the polypeptide or fragment or variant.
Thus, a particular aspect of the invention provides a polypeptide coinprising
the
amino acid sequence selected from any one of SEQ ID Nos 2, 4, 6, 8, 10, 12,
14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58,
60, 62, 64, 66, 68; or a fragment or variant thereof or a fusion of such a
fiaginent
or variaa.lt. Thus, the invention provides the following isolated proteins, or
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fragments or variants thereof, or fusion of these: NMB0341, NMB1583,
NMB1345, NMB0738, NMB0792, NMB0279, NMB2050, NMB1335,
NMB2035, NMB1351, NMB1574, NMB1298, NMB1856, NMB0119,
NMB1705, NMB2065, NMB0339, NMB0401, NMB1467, NMB2056,
NM30808, NM1B0774, NMA0078, NMB0337, NMB0191, NMB1710,
NMB0062, NMB1333, NMB0377, NMB0264, NMB1036, NMB1176, NMB1359
and NMB1138 as described below.
Fragments of the identified polypeptide may be made which are, for example,
lo 20% or 30% or 40 % or 50% or 60% or 70% or 80% or 90% of the total of the
polypeptide. Typically, fragments are at least 10, 15, 20, 30, 40 , 50, 100 or
more
amino acids, but less than 500, 400, 300 or 200 amino acids. Variants of the
polypeptide may be made. By "variants" we include insertions, deletions and
substitutions, either conservative or non-conservative, where such changes do
not
substantially alter the normal function of the protein. By "conservative
substitutions" is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp,
Glu;
Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using
the
well known methods of protein engineering and site-directed mutagenesis.
A particular class of variants are those encoded by variant genes as discussed
above, for example from related microorganisms or other strains of the
microorganism. Typically the variant polypeptides have at least 70% sequence
identity, more preferably at least 85% sequence identity, most preferably at
least
95% sequence identity with the polypeptide identified using the method of the
invention.
The percent sequence identity between two polypeptides may be determined using
suitable computer programs, for exainple the GAP prograin of the University of
Wisconsin Genetic Computing Group and it will be appreciated that percent
identity is calculated in relation to polhTeptides whose sequence has been
aligned
optimally.
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The alignment may alternatively be carried out using the Clustal W program
(Thompson et al., (1994) Nucleic Acids Res 22, 4673-80). The paraineters used
may be as follows:
Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap
penalty; 3, number of top diagonals; 5. Scoring method: x percent.
Multiple alignment parameters: gap open penalty; 10, gap extension penalty;
0.05.
Scoring matrix: BLOSUM.
The fusions may be fusions with any suitable polypeptide. Typically, the
io polypeptide is one which is able to enhance the immune response to the
polypeptide it is fused to. The fi.i.sion partner may be a polypeptide that
facilitates
purification, for example by constituting a binding site for a moiety that can
be
immobilised in, for example, an affinity chromatography colulnn. Thus, the
fusion partner may comprise oligo-histidine or other amino acids which bind to
cobalt or niclcel ions. It may also be an epitope for a monoclonal antibody
such as
a Myc epitope.
As discussed above, the variant polypeptides or polypeptide fragments, or
fusions
of these, are typically ones which give rise to neutralizing antibodies
against N.
n2eningitidis.
The invention also includes, therefore, a method of making an antigen as
described above, and antigens obtainable or obtained by the method.
The polynucleotides of the invention may be cloned into vectors, such as
expression vectors, as is well kn.own on the art. Such vectors maybe present
in
host cells, such as bacterial, yeast, mammalian and insect host cells. The
antigens
of the invention may readily be expressed from polynucleotides in a suitable
host
cell, and isolated therefrom for use in a vaccine.
Typical expression systems include the coimnercially available pET expression
vector series and E. coli host cells such as BL21. The polypeptides expressed
inay
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be purified by any method known in the art. Conveniently, the antigen is fused
to
a fusion partner that binds to an affinity column as discussed above, and the
fusion
is purified using the affinity coluinn (eg such as a niclcel or cobalt
affinity
coluirui).
It will be appreciated that the antigen or a polynucleotide encoding the
antigen
(such as a DNA molecule) is particularly suited for use as in a vaccine. In
that
case, the antigen is purified from the host cell it is produced in (or if
produced by
peptide synthesis purified from any contaminants of the synthesis). Typically
the
io antigen contains less that 5% of contaiuinating material, preferably less
than 2%,
1%, 0.5%, 0.1%, 0.01%, before it is formulated for use in a vaccine. The
antigen
desirably is substantially pyrogen free. Thus, the invention further includes
a
vaccine comprising the antigen, and method for making a vaccine comprising
combining the antigen with a suitable carrier, such as phosphate buffered
saline.
Whilst it is possible for an antigen of the invention to be administered
alone, it is
preferable to present it as a pharmaceutical formulation, together with one or
more
acceptable carriers. The carrier(s) must be "acceptable" in the sense of being
compatible with the antigen of the invention and not deleterious to the
recipients
thereof. Typically, the carriers will be water or saline which will be sterile
and
pyrogen free.
The vaccine may also conveniently include an adjuvant. Active irnmunisation of
the patient is preferred. In this approach, one or more antigens are prepared
in an
immunogenic formulation containing suitable adjuvants and carriers and
administered to the patient in lcnown ways. Suitable adjuvants include
Freund's
complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109
942,
EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran,
neutral oils (such as miglyol), vegetable oils (such as arachis oil),
liposomes,
Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189
141).
"Pluronic" is a Registered Trade Mark. The patient to be immunised is a
patient
requiring to be protected from infection with the microorga.l.iism.
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The invention also includes a pharmaceutical coinposition comprising a
polypeptide of the invention or variant or fraginent thereof, or fusion of
these, or a
polynucleotide of the invention or a variant or fraginent thereof or fusion of
these,
and a pharmaceutically acceptable carrier as discussed above.
5
The aforementioned antigens of the invention (or polynucleotides encoding such
antigens) or a formulation thereof may be adininistered by any conventional
method including oral and parenteral (eg subcutaneous or intramuscular)
injection.
The treatment may consist of a single dose or a plurality of doses over a
period of
l0 time.
It will be appreciated that the vaccine of the invention, depending on its
antigen
component (or polynucleotide), may be useful in the fields of human medicine
and
veterinary medicine.
Diseases caused by microorganisms are known in many animals, such as domestic
animals. The vaccines of the invention, when containing an appropriate antigen
or
polynucleotide encoding an antigen, are useful in man but also in, for
example,
cows, sheep, pigs, horses, dogs and cats, and in poultry such as chickens,
turkeys,
ducks and geese.
Thus, the invention also includes a method of vaccinating an individual
against a
microorganism, the method comprising administering to the individual an
antigen
(or polynucleotide encoding an antigen) or vaccine as described above. The
invention also includes the use of the antigen (or polynucleotide encoding an
antigen) as described above in the manufacture of a vaccine for vaccinating an
individual.
The antigen of the invention may be used as the sole antigen in a vaccine or
it may
be used in coinbination with other antigens whether directed at the saine or
different disease microorganisms. In relation to N. aeningitidis, the antigen
obtained which is reactive against NmB may be coinbined with components used
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in vaccines for the A and/or C serogroups. It may also conveniently be
combined
antigenic components which provide protection against Haenzoplailus and/or
Streptococcus pneunzoniae. The additional antigenic components may be
polypeptides or they may be other antigenic components such as a
polysaccharide.
Polysaccharides may also be used to enhance the immune response (see, for
example, Makela et al (2002) Expert Rev. Vaccines 1, 399-410).
It is particularly preferred in the above vaccines and methods of vaccination
if the
antigen is the polypeptide encoded by any of the genes as described above (and
in
1o the Examples), or a variant or fragment or fusion as described above (or a
polynucleotide encoding said antigen), and that the disease to be vaccinated
against is Neisseria nzeningitidis infection (meningococcal disease).
The invention will now be described in greater detail by reference to the
following
non-limiting Examples.
Example 1: Genetic screening for immunogens (GSI) in N. naeningitidis
The application of GSI in this example involves screening libraries of
insertional
mutants of N. nzeningitidis for strains which are less susceptible to killing
by
bactericidal antibodies. GSI is described in more detail in PCT/GB2005/005441
(published as WO 2005/060995 on-7 July 2005).
We have demonstrated the effectiveness of GSI by screening a library of
mutants
of the sequenced NniB isolate, MC58, with sera raised in mice against a
capsule
minus of the same strain. A total of 40,000 mutants was analysed with sera
raised
in mice by intraperitoneal immunisation with the homologous strain; the SBA of
this sera is around 2,000 against the wild-type strain. Surviving mutants were
detected when the library was exposed to serum at a 1:560 dilution (which
kills all
wild-type bacteria). To establish whether the transposon insertion in the
surviving
mutants was responsible for the ability to withstand killing, the lnutations
were
backcrossed into the parental strain, and the backcrossed inutants were
confirmed
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as being more resistant to killing than the wild-type in the SBA. The sequence
of
the gene affected by the transposon was examined by isolating the transposon
insertion site by inarker rescue. We found that two of the genes affected were
TspA and NMB0338. TspA is a surface antigen which elicits strong CD4+ T cell
responses and is recognized by sera from patients (Kizil et al (1999) I72fect
bnmun.
67, 3533-41). NMB0338 is a gene of previously unknown function which
encodes a polypeptide that is predicted to contain two transmeinbrane domains,
and is located at the cell surface. The amino acid sequence encoded by NMB0338
is:
MERNGVFGKIVGNRILRMSSEHAAASYPKPCKSFKLAQSWFRVRSCLGGVFIYGA
NMKLIYTVIKIIILLLFLLLAVINTDAVTFSYLPGQKFDLPLIVVLFGAFVVGII
FGMFALFGRLLSLRGENGRLRAEVKKNARLTGKELTAPPAQNAPESTKQP
There are several practical advantages of using Nm.B for GSI aside from the
public
health imperative: a) the bacterium is genetically tractable; b) killing of
the
bacterium by effector immune mechanism is straightforward to assay; c) the
genome sequences are available for three isolates of different serogroups and
clonal lineages (IV-A, ET-5, and ET-37 for serogroups A, B, and C,
respectively);
and d) well-characterised clinical resources are available for this work.
GSI has two potential limitations. First, targets of bactericidal antibodies
may be
essential. This is unlikely as all known targets of bactericidal antibodies in
NmB
are non-essential, and no currently licensed bacterial vaccine.targets an
essential
gene product. Second, sera will contain antibodies to multiple antigens, and,
loss
of a single antigen may not affect the sundval of mutants. We have already
shown
that even during selection with sera raised against the homologus strain,
relevant
antigens were still identified using appropriate dilutions of sera.
m~
e major advantages oir rv5Tl are iLMa~ ~ ~ ~ ~) the l ~u'~g11 ~ tlu V'll~h--N
~,
ut steps do not involve
in
technically demanding or costly procedures, (such as protein
expression/purification and immunisation), and 2) human samples can be used in
the assay rather than relying solely on animal data. GSI will rapidly pinpoint
the
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subset of surface proteins that elicit bactericidal activity, allowing more
detailed
analysis of a smaller number of candidates.
l. Identification of targets of bactericidal antibodies using GSI
Murine sera raised against heterologous strains, and human sera, are used to
identify cross-reactive antigens. The sera are obtained from:
i) mice immunised by the systemic route with heterologous strains: the
strains will be selected and/or constructed to avoid isolates with the
same immunotype and sub-serotype.
ii) acute and convalescent sera from patients infected with known isolates
ofN. meningitidis (provided by Dr R. Wall, Northwick Park)
iii) pre- and post-immunisation samples (provided by the Meningococcal
Reference Laboratory) from volunteers receiving defined outer
membrane vesicle (OMVs) vaccines derived from the NmB isolate,
H44/76.
Each'of these sources of sera has specific advantages and disadvantages.
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Serum source Advantages Disadvantages
Murine 1) Defined antigenic exposure. 1) Animal source of
2) Use of genetically modified strains to material
generate immune response.
3) Naive samples available
4) Examine individuals responses
Patient sera 1) Human material 1) Background imm.unity
2) Known strain exposure 2) Limited material
3) Acute and convalescent sera available
Sera following 1) Human material 1) Background immunity
irnmunisation 2) Defined antigenic exposure 2) Limited material
with H4476 3) Pre and post immunisation sera
OMVs available
4) Examine individuals responses
a) Sera from animals immunised with heterologous strains (ie the sequenced
serogroup A or C strains) are used in GSI to select the library of MC58
mutants.
We have shown that immunisation with live, attenuated NnzB elicits cross-
reactive
bactericidal antibody responses against serogroup A and C strains. The antigen
absent in inutants with enhanced survival in the face of human sera are
identified
by marker rescue of the disrupted gene.
b) Mutations are identified that confer resistance against killing by
lo heterologous sera, and it is determined whether the gene product is also a
target
for killing of the sequenced, serogroup A and C strains, Z2491 and FAM1 8
respectively. The genome databases are inspected for homologues of the genes.
If
a homologue is present, the transposon irisertion is amplified from the MC58
mutant and introduced into the serogroup A and C strains by transformation.
The
relative sunlival of the inutant and wild-type strain of each serogroup are
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compared. Thus, GSI can quicldy give information whether the targets of
bactericidal activity are conserved and accessible in diverse strains of N.
nzeningitidis, irrespective of serogroup, immunotype and subserotype.
5 c) Mutants with enhanced survival against sera raised in mice are tested
using
human sera from either convalescent patients or vaccinees receiving
heterologous
OMV vaccines (derived from H44/76). This addresses the iinportant question of
whether the targets are capable of eliciting bactericidal antibodies in human.
With
other vaccine approaches, this information is only gained at the late,
expensive
1o stage of clinical trials that requires GMP manufacture of vaccine
candidates.
The advantages are that GSI is a high-throughput analysis performed using
simple,
available techniques. Antigens which elicit bactericidal antibodies in humans
and
which mediate killing of multiple strains can be identified rapidly as GSI is
15 flexible with respect to the bacterial strain and sera used. Mutants
selected using
human sera are analysed in the same way as those selected by murine sera.
2. Assessnient of the antibody response of recombinant GSI antigens
Proteins which are targets of bactericidal antibodies that are recognised by
sera
from convalescent patients and vaccines are expressed in E. coli using
commercially available vectors. The corresponding open reading fraines are
ainplified by PCR from MC58, and ligated into vectors such as pCR Topo CT or
pBAD/His, to allow protein expression under the control of a T7 or arabinose-
inducible promoter, respectively. Purification of the recoinbinant proteins
from
total cellular protein is performed via the His Tag fused to the C terminus of
the
protein on a Nickel or Cobalt coluxnn.
Adult New Zealand White rabbits are iuninunized on two occasions separated by
four v,reeks by subcutaneous injection with 25 g of purified protein with
Freund's
incomplete adjuvant. Sera from animals will be checlced prior to immunisation
for
pre-existing anti Njn antibodies by whole cell ELISA. Animals which have an
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initial seruin titre of <1:2 are used for inununisation experiments. Post-
immunisation serum are obtained two weel:s after the second immunisation. To
confirm that specific antibodies have been raised, pre- and post-immunisation
serum is tested by i) Western analysis against tlie purified protein and ii)
ELISA
using cells from the wild-type and the corresponding mutant (generated by
GSI).
SBAs will be performed against MC58 (the homologous strain), and the
sequenced serogroup A and C strains with the rabbit immune serum. The assay
will be performed in triplicate on at least two occasions. SBAs of >8 will be
lo considered significant. The results provide evidence of whether the protein
candidates can elicit bactericidal antibodies as recombinant proteins.
3. Establishing the protective efficacy of GSI antigens
All the candidates are tested for their ability to protect animals against
live
bacterial challenge as this allows any aspect of immunity (cellular or
humoral) to
be assessed in a single assay. We have established a model of active
immunisation
and protection against live bacterial infection. In this model, adult mice are
immunised on days 0 and 21, and on day 28 receive live bacterial challenge of
106
or 107 CFU of MC58 intraperitoneally in iron dextran (as the supplemental iron
source). The model is similar to that described for evaluation of the
protective
efficacy of irnmunisation with Tbps Danve et al (1993) Tjaccine 11, 1214-1220.
Non-immunised animals develop bacteraemia within 4 hours of infection, and
show signs of systemic illness by 24 hours. We have already been able to
deinonstrate the protective efficacy of both attenuated Nfn strains and a
protein
antigen against live meningococcal challenge; PorA is an outer membrane
protein
that elicits bactericidal antibodies, but which is not a lead vaccine
candidate
because of extensive antigenic variation (Bart et al (1999) hzfect Imm.un. 67,
3 832-
3 846.
3o
Six week old, BALB/c mice (group size, 35 animals) receive 25 g of
recoinbinant protein with Freund's incomplete adjuvant subcutaneously on days
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day 0 and 21, then are challenged with 106 (15 animals) or 107 (15 animals)
CFU
of MC58 intraperitoneally on day 28. Two challenge doses are used to examine
the vaccine efficacy at a high and low challenge dose; sera are obtained on
day 28
from the remaining five animals in each group, and from five animals before
the
first immunisation and stored at -70 C for ffiirther immunological assays.
Animals
in control groups receive either i) adjuvant alone, ii) recombinant refolded
PorA,
and iii) a live, attenuated Nnz strain. To reduce the overall number of
animals in
control groups, sets of five candidates will be tested at one time (number of
groups
= 5 candidates + 3 controls). Survival of animals in the groups is compared by
1o Mann Whitney U Test. Vdith group sizes of 15 mice/dose, the experiments are
powered to show a 25% difference in survival between groups.
For vaccines which show significant protection against challenge, a repeat
experiment is performed to confirm the finding. Furthermore, to establish that
vaccination with a candidate also elicits protection against bacteraemia,
levels of
bacteraeinia are determined during the second experiment; blood is sampled at
22
hr post-infection in immunised and un-immunised animals (bacteraemia is
maximal at this time). The results are analysed using a two-tailed Student-T
test to
determine if there is a significant reduction in bacteraemia in vaccinated
animals.
Furtlaer materials and niethods used
Mutagenesis ofNeissei=ia nzeningitidis
For work with Neisseria nieningitidis, mutants were constructed by in vitro
mutagenesis. Genomic DNA from N. fneningitidis was subjected to mutagenesis
with a Tn5 derivative containing a marker encoding resistance to kanamycin,
and
an origin of replication which is functional in E. eoli. These elements are
bound
by coinposite Tn5 ends. Transposition reactions were carried out with a
hyperactive variant of Tn5 and the DNA repaired with T4 DNA polymerase and
ligase in the presence of ATP and nucleotides. The repaired DNA was used to
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transform N. nzeningitidis to kanamycin resistance. Southern analysis
confismed
that each mutant contained a single insertion of the transposon only.
Serunz bactericidal assays (SBAs)
Bacteria were grown overnight on solid media (brain heart infusion media with
Levanthals supplement) and then re-strealced to solid media for four hours on
the
morning of experiments. After this time, bacteria were harvested into
phosphate
buffered saline and enumerated. SBAs were performed in a 1 ml volume,
lo containing a complement source (baby rabbit or human) and approximately 105
colony forming units. The bacteria were collected at the end of the incubation
and
plated to solid media to recover surviving bacteria.
Isolating the transposon insertion sites
Genornic DNA will be recovered from mutants of interest by standard methods
and digested with PvuII, EcoRV, and DraI for three hours, then purified by
phenol
extraction. The DNA will then be self-ligated in a 100 microlitre volume
overni.ght at 16 C in the presence of T4 DNA ligase, precipitated, then used
to
2o transform E. coli to kanamycin resistance by electroporation.
ExaMle 2: Further screening and results thereof
GSI has been used to screen a library of approximately 40,000 insertional
mutants
of MC58. The library was constructed by in vitro Tn5 mutagenesis, using a
transposon harbouring the origin of replication from pACYC184.
MC58 was chosen as it is a serogroup B isolate of N. nzeningitidis, and the
complete genome sequence of this strain is known.
The library is always screened in parallel with the wild-type strain as a
control,
and the number of colonies recovered from the library aiid the wild-type is
shown.
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Selection witla nzuf~ine seM
Initially the library was analysed using sera from animals immunised with the
attenuated strain YH102. Adult mice (Balb/C) received 108 colony forming units
intra-peritoneally on three occasions, and sera was collected 10 days after
the final
immunisation,
The screen identified several mutants with enhanced resistance to serum
lcilling:
lo This was confirmed by isolating individual mutants, reconstructing the
mutation in
the original genetic background, and re-testing the individual mutants for
their
susceptibility to complement mediated lysis against the wild-tye. The
transposon
insertions are in the following gene:
NMB0341 (TspA) DNA sequence
ATGCCCGCCGGCCGACTGCCCCGCCGATGCCCGATGATGACGAAATTTACAGACTGTACG
CGGTCAAACCGTATTCAGCCGCC_AACCCACAGGGG?1TACATCTTG_AAAACAACAGACAA
ATCAAACTGATTGCCGCCTCCGTCGCAGTTGCCGCATCCTTTCAGGCACATGCTGGACTG
GGCGGACTGAATATCCAGTCCAACCTTGACGAACCCTTTTCCGGCAGCATTACCGTAACC
GGCGAAGAAGCCAAAGCCCTGCTAGGCGGCGGCAGCGTTACCGTTTCCGAAAAAGGCCTG
ACCGCCAAAGTCCACAAGTTGGGCGACAAAGCCGTCATTGCCGTTTCTTCCGAACAGGCA
GTCCGCGATCCCGTCCTGGTGTTCCGCATCGGCGCAGGCGCACAGGTACGCGAATACACC
GCCATCCTCGATCCTGTCGGCTACTCGCCCAAAACCAAATCTGCACTTTCAGACGGCAAG
ACACACCGCAAAACCGCTCCGACAGCAGAGTCCCAAGAAAATCAAAACGCCAAAGCCCTC
CGCAAAACCGAT.AAAAAAGACAGCGCGAACGCAGCCGTC.AAACCGGCATACAACGGCAAA
ACCCATACCGTCCGCAAAGGCGAAACGGTCAAACAGATTGCCGCCGCCATCCGCCCG.AAA
CACCTGACGCTCGAACAGGTTGCCGATGCGCTGCTGAAGGCAAACCCAAATGTTTCCGCA
CACGGCAGACTGCGTGCGGGCAGCGTGCTTCACATTCCGAATCTGAACAGGATCAAAGCG
GAACAACCCAAACCGCAAACGGCGAAACCCAAAGCCGAAACCGCATCCATGCCGTCCGAA
CCGTCCAAACAGGCAACGGTAGAGAA.ACCGGTTGAAAAACCTGAAGCAAAAGTTGCCGCG
CCCGAAGCAAAAGCGGAAAAACCGGCCGTTCGACCCGAACCTGTACCCGCTGCAAATACT
GCCGCATCGGAAACCGCTGCCGAATCCGCCCCCC.AAGAAGCCGCCGCTTCTGCCATCGAC
ACGCCGACCGACGAAACCGGTAACGCCGTTTCCGAACCTGTCGAACAGGTTTCTGCCGAA
GfiAGAAACCGAAAGCGGACTGTTTGACGGTCTGTTCGGCGGTTCGTACACCTTGCTGCTT
GCCGGCGGAGGCGCGGCATTAATCGCCCTGCTGCTGCTTTTGCGCCTTGCCCAATCCAAA
CGCGCGCGCCGTACCGAAGAATCCGTCCCTGAGGAAGAGCCTGACCTTGACGACGCGGCA
GACGACGGCATAGAAATCACCTTTGCCGAAGTCGAAACTCCGGCAACGCCCGAACCCGCT
CCGAAAAACGATGTAAACGACACACTTGCCTTAGATGGGGAATCTGAAGAAGAGTTATCG
GCAAAACAAACGTTCGATGTCGAAACCGATACGCCTTCCAACCGCATCGACTTGGATTTC
GACAGCCTGGCAGCCGCGCAAAACGGCATTTTATCCGGCGCACTTACGCAGGATGAAGAA
ACCCAAAAACGCGCGGATGCCGATTGGAACGCCATCGAATCCACAGACAGCGTGTACGAG
CCCGAGACCTTCAACCCGTACAACCCTGTCGAAATCGTCATCGACACGCCCGAACCGGAA
TCTGTCGCCCAAACTGCCGAAAACAAACCGGAAACCGTCGATACCGATTTCTCCGACAAC
CTGCCCTCAAACAACCATATCGGCACAGAAGAAACAGCTTCCGCAAAACCTGCCTCACCC
TCCGGACTGGCAGGCTTCCTGAAGGCTTCCTCGCCCGAAACCP_TCTTGGAAA.AAACAGTT
GCCGAAGTCCAAACACCGGAAGAGTTGCACGATTTCCTGAAAGTGTACGAAACCGATGCC
GT CGCGGAAACT GCGCC TGAAACGCCC GATT T CAACGCCGCCGCAGAC GATT T G T CCGCA
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TTGCTTCAACCTGCCGAAGCACCGTCCGTTGAGGAAARTATAACGGAAACCGTTGCCGAA
ACACCCGACTTCAACGCCACCGCAGACGATTTGTCCGCATTACTTCAACCTTCTAAAGTA
CCTGCCGTTGAGGAAAATGCAGCGGAAACCGTTGCCGATGATTTGTCCGCACTGTTGCAA
CCTGCTGAAGCACCGGCCGTTGAGGAAAATGTAA.CGGAAACCGTTGCCGAAACACCCGAT
5 TTC_AACGCCACCGCAGACGATTTGTCCGCATTACTTCAACCTTCTGAAGCACCTGCCGTT
GAGGAAAATGCAGCGGAAACCGTTGCCGATGATTTGTCCGCACTGTTGCAZ-1CCTGCTGAA
GCACCGGCCGTTGAGGAAAATGCAGCGGAAATCACTTTGGAAACGCCTGATTCC_AACACC
TCTGAGGCAGACGCTTTGCCCGACTTCCTGAAAGACGGCGAGGAGGAAA.CGGTAGATTGG
AGCATCTACCTCTCGGAAGAAAATATCCCAAATAATGCAGATACCAGTTTCCCTTCGGAA
10 TCTGTAGGTTCTGACGCGCCTTCCGAAGCGAAATACGACCTTGCCGAAATGTATCTCGAA
ATCGGCGACCGCGATGCCGCTGCCGAGACAGTGCAGAAATTGCTGGAAGAAGCGGAAGGC
GACGTACTCAAACGTGCCCAAGCATTGGCGCAGGAATTGGGTATTTGA
NBM0347 Protein sequence
15 MPAGRLPRRCPMMTKFTDCTRSNRIQPPTHRGYILKNNRQIKLIAASVAVAASFQAHAGL
GGLNIQSNLDEPFSGSITVTGEEAKALLGGGSVTVSEKGLTAKVHKLGDKAVIAVSSEQA
VRDPVLVFRIGAGAQVREYTAILDPVGYSPKTKSALSDGKTHRKTAPTAESQENQNAKAL
RKTDKKDSANAAVKPAYNGKTHTVRKGETVKQIAAAIRPKHLTLEQVADALLKANPNVSA
HGRLRAGSVLHIPNLNRIKAEQPKPQTAKPKAETASMPSEPSKQATVEKPVEKPEAKVAA
20 PEAKAEKPAVRPEPVPAANTAASETAAESAPQEAAASAIDTPTDETGNAVSEPVEQVSAE
EETESGLFDGLFGGSYTLLLAGGGAALIALLLLLRLAQSKRARRTEESVPEEEPDLDDAA
DDGIEITFAEVETPATPEPAPKNDVNDTLALDGESEEELSAKQTFDVETDTPSNRIDLDF
DSLAAAQNGILSGALTQDEETQKRADADWNAIESTDSVYEPETFNPYNPVEIVIDTPEPE
SVAQTAENKPETVDTDFSDNLPSNNHIGTEETASAKPASPSGLAGFLKASSPETILEKTV
AEVQTPEELHDFLKVYETDAVAETAPETPDFNAAADDLSALLQPAEAPSVEENITETVAE
TPDFNATADDLSALLQPSKVPAVEENAAETVADDLSALLQPAEAPAVEENVTETVAETPD
FNATADDLSALLQPSEAPAVEENAAETVADDLSALLQPAEAPAVEENAAEITLETPDSNT
SEADALPDFLKDGEEETVDWSIYLSEENIPNNADTSFPSESVGSDAPSEAKYDLAEMYLE
IGDRDAAAETVQKLLEEAEGDVLKRAQALAQELGI
NMB0338 DNA sequence
ATGGAAAGGAACGGTGTATTTGGTAAAATTGTCGGCAATCGCATACTCCGTATGTCGTCC
GAACACGCTGCCGCATCCTATCCGAAACCGTGCAAA.TCGTTTAAACTAGCGCAATCTTGG
TTCAGAGTGCGAAGCTGTCTGGGCGGCGTTTTTATTTACGGAGCAAACATGAAACTTATC
TATACCGTCATCAAAATCATTATCCTGCTGCTCTTCCTGCTGCTTGCCGTCATTAATACG
GATGCCGTTACCTTTTCCTACCTGCCGGGGCAAAAATTCGATTTGCCGCTGATTGTCGTA
TTGTTCGGCGCATTTGTAGTCGGTATTATTTTTGGAA.TGTTTGCCTTGTTCGGACGGTTG
TTGTCGTTACGTGGCGAGAACGGCAGGTTGCGTGCCGAAGTAAAGAAAAATGCGCGTTTG
ACGGGGAAGGAGCTGACCGCACCACCGGCGCAAAATGCGCCCGAATCTACCAAACAGCCT
TAA
NMB0338 Protein sequence
MERNGVFGKIVGNRILRMSSEHAAASYPKPCKSFKLAQSWFRVRSCLGGVFIYGANMKLI
YTVIKIIILLLFLLLAVINTDAVTFSYLPGQKFDLPLIVVLFGAFVVGIIFGMFALFGRL
LSLRGENGRLRAEVKKNARLTGKELTAPPAQNAPESTKQP
Analysis of the polypeptide indicates that it is predicted to have two
membrane
spanning domains, from residues 54 to 70 and 88 to 107. Thus, fragments from
the regions 1 to 53, and 108 to the end (C-term,;na1_) may be particularly
useful as
i-ininunogens.
NMB1345 DNA sequence
ATGAAAAAACCTTTGATTTCGGTTGCGGCAGCATTGCTCGGCGTTGCTTTGGGCACGCCT
TATTATTTGGGTGTCAAAGCCGAAGAAAGCTTGACGCAGCP.GCAAAA-~ATATTGCAGG_AA
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ACGGGCTTCTTGACCGTCGAATCGCACCAATATGAGCGCGGCTGGTTTACCTCTATGGAA
ACGACGGTCATCCGTCTGAAACCCGAGTTGCTGAATAATGCCCGAAAATACCTGCCGGAT
AACCTGAAAACAGTGTTGGAACAGCCGGTTACGCTGGTTAACCATATCACGCACGGCCCT
TTCGCCGGCGGATTCGGCACGCAGGCGTACATTGAAACCGAGTTCAAATACGCGCCTGAA
ACGGAAAAAGTTCTGGAACGCTTTTTTGGAAAACAAGTCCCGGCTTCCCTTGCCAATACC
GTTTATTTTAACGGCAGCGGT_AAAATGGAAGTCAGTGTTCCCGCCTTCGATTATGAAGAG
CTGTCGGGCATCAGGCTGCACTGGGAAGGCCTGACGGGAGAAACGGTTTATCAAAAAGGT
TTCAAAAGCTACCGGAACGGCTATGATGCCCCCTTGTTTAAAATCAAGCTGGCAGACAAA
GGCGATGCCGCGTTTGAAAAAGTGCATTTCGATTCGGAAACTTCAGACGGCATCAATCCG
CTTGCTTTGGGCAGCAGCAATCTGACCTTGGAAAAATTCTCCCTAGAATGGAAAGAGGGT
GTCGATTACAACGTCAAGTTAAACGAACTGGTCAATCTTGTTACCGATTTGCAGATTGGC
GCGTTTATCAATCCCAACGGCAGCATCGCACCTTCCAAAATCGAAGTCGGCAAACTGGCT
TTTTCAACCAAGACCGGGGAATCAGGCGCGTTTATCAACAGTGAAGGGCAGTTCCGTTTC
GATACACTGGTGTACGGCGATGAAAAATACGGCCCGCTGGACATCCATATCGCTGCCGAA
CACCTCGATGCTTCTGCCTTAACCGTATTGAAACGCAAGTTTGCACAAATTTCCGCCAAA
AAAATGACCGAGGAACAAATCCGCAATGATTTGATTGCCGCCGTCAAAGGAGAGGCTTCC
GGACTGTTCACCAACAATCCCGTATTGGACATTAAAACTTTCCGATTCACGCTGCCATCG
GGAAAAATCGATGTGGGCGGAAAAATCATGTTTAAAGACATGAAGAAGGAAGATTTGAAT
CAATTGGGTTTGATGCTGAAGAAAACCGAAGCCGACATCAGAATGAGTATTCCCCAAAAA
ATGCTGGAAGACTTGGCGGTCAGTCAAGCAGGCAATATTTTCAGCGTCAATGCCGAAGAT
GAGGCGGAAGGCAGGGCAAGTCTTGACGACATCAACGAGACCTTGCGCCTGATGGTGGAC
AGTACGGTTCAGAGTATGGCAAGGG.AAAAATATCTGACTTTGAACGGCGACCAGATTGAT
ACTGCCATTTCTCTGAAAAACAATCAGTTGAAATTGAACGGTAAAACGTTGCAAAACGAA
CCGGAGCCGGATTTTGATGAAGGCGGTATGGTTTCAGAGCCGCAGCAGTAA
NMB 1345 Protein sequence
MKKPLISVAAALLGVALGTPYYLGVKAEESLTQQQKILQETGFLTVESHQYERGWFTSME
TTVIRLKPELLNNARKYLPDNLKTVLEQPVTLVNHITHGPFAGGFGTQAYIETEFKYAPE
TEKVLERFFGKQVPASLANTVYFNGSGKMEVSVPAFDYEELSGIRLHWEGLTGETVYQKG
FKSYRNGYDAPLFKIKLADKGDAAFEKVHFDSETSDGINPLALGSSNLTLEKFSLEWKEG
VDYNVKLNELVNLVTDLQIGAFINPNGSIAPSKIEVGKLAFSTKTGESGAFINSEGQFRF
DTLVYGDEKYGPLDIHZAAEHLDASALTVLKRKFAQISAKKMTEEQIRNDLIAAVKGEAS
GLFTNNPVLDIKTFRFTLPSGKIDVGGKIMFKDMKKEDLNQLGLMLKKTEADIRMSIPQK
MLEDLAVSQAGNIFSVNAEDEAEGRASLDDINETLRLMVDSTVQSMAREKYLTLNGDQID
TAISLKNNQLKLNGKTLQNEPEPDFDEGGMVSEPQQ
Selection with vaccinees sera
Sera from the Meningococcal Reference Laboratory in Manchester has been made
available to us. This sera has come from a clinical trial of OMV immunisation
of
volunteers.
Mutants selected by vaccinee Cl sera (screened once)
The following sequences were isolated
NMB0338 (as above)
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NMB0738 DNA sequence
ATGAAGATCGTCCTGATTAGCGGCCTGTCCGGTTCGGGCAAGTCCGTCGCACTGCGCCAA
ATGGAAGATTCGGGTTATTTCTGCGTGGACAATTTGCCTTTGG_AAATGTTGCCCGCGCTG
GTGTCGTATCATATCGAACGTGCGGACGAAACCGAATTGGCGGTCAGCGTCGATGTGCGT
TCCGGCATTGACATCGGACAGGCGCGGGAACAGATTGCCTCTCTGCGCAGACTGGGGCAC
AGGGTTGAAGTTTTGTTTGTCGAGGCGG_AAGAAAGCGTGTTGGTCCGCCGGTTTTCCGAA
ACCAGGCGAGGACATCCTCTGAGCAATCAGGATATGACCTTGTTGGAAAGCTTAAAGAAA
G_AACGGGAATGGCTGTTCCCGCTTAAAGAAA.TCGCCTATTGTATCGACACTTCCAAGATG
AATGCCCAACAGCTCCGCCATGCAGTCCGGCAGTGGCTGAAGGTCGAACGTACCGGGCTG
CTGGTGATTTTGGAGTCCTTCGGGTTCAAATACGGTGTGCCGAACAACGCGGATTTTATG
TTCGATATGCGCAGCCTGCCCAACCCGTATTACGATCCCGAGTTGAGGCCTTACACCGGT
ATGGACAAGCCCGTTTGGGATTATTTGGACGGACAGCCGCTTGTGCAGGAAATGGTTGAC
GACATCG?D-7~.AGGTTTGTTACGCATTGGTTACCGCGTTTGGAGGATGAAAGCAGGAGCTAC
GTTACCGTCGCCATCGGTTGCACGGGAGGACAGCACCGTTCGGTCTATATTGTCGAP.AAA
CTCGCCCGAAGGTTGAAAGGGCGTTATGAATTGCTGATACGGCACAGACAGGCGCAAAAC
CTGTCAGACCGCTAA
NMB0738 Protein sequence
MKIVLISGLSGSGKSVALRQMEDSGYFCVDNLPLEMLPALVSYHIERADETELAVSVDVR
SGIDIGQAREQIASLRRLGHRVEVLFVEAEESVLVRRFSETRRGHPLSNQDMTLLESLKK
EREWLFPLKEIAYCIDTSKMNAQQLRHAVRQWLKVERTGLLVILESFGFKYGVPNNADFM
FDMRSLPNPYYDPELRPYTGMDKPVWDYLDGQPLVQEMVDDIERFVTHWLPRLEDESRSY
VTVAIGCTGGQHRSVYIVEKLARRLKGRYELLIRHRQAQNLSDR
NMB0792 NadC family (transporter) DNA sequence
ATGAACCTGCATGCAAAGGACAAAACCCAGCATCCCGAAAACGTCGAGCTGCTCAGTGCG
CAGAAGCCGATTACCGACTTTAAGGGCCTGCTGACCACCATTATTTCCGCCGTCGTCTGT
TTCGGCATTTACCACATCCTGCCTTACAGCCCCGATGCCAATAAAGGTATCGCGCTGCTG
ATTTTCGTTGCCGCACTTTGGTTTACCGAGGCCGTCCACATTACCGTAACCGCACTGP_TG
GTGCCGATTCTCGCCGTCGTACTCGGTTTCCCCGACATGGACATCAAAAAGGrGATGGCT
GATTTTTCCAACCCGATTATCTACATTTTTTTCGGCGGCTTCGCGCTTGCCACCGCCCTG
CATATGCAGCGGCTGGACCGTAAAATCGCCGTCAGCCTGTTGCGCCTGTCGCGCGGCAAT
ATGAAAGTGGCGGTTTTGATGTTGTTCCTCGTTACCGCCTTTCTGTCCATGTGGATCAGC
AACACCGCCACCGCCGCGATGATGCTGCCTCTAGCAATGGGTATGCTGAGCCACCTCGAC
CAGGAAAAAGAACACAAAACCTACGTCTTCCTCCTGCTCGGCATCGCCTATTGCGCCAGC
ATCGGCGGCTTGGGCACGCTCGTCGGCTCGCCGCCCAACCTGATTGCCGCCAAAGCCCTA
AATCTGGACTTCGTCGGCTGGATGAA.GCTCGGCCTGCCGATGATGCTGTTGATTCTGCCC
TTGATGCTGCTCTCCCTGTACGTCATCCTCAAACCTAATTTGAACGAACGCGTGGAAATC
AAAGCCGAATCCATCCCTTGGACGCTGCACCGCGTGATCGCGCTGTTGATTTTCCTTGCC
ACAGCCGCCGCGTGGATATTCAGCTCCAAAATCAAAACCGCCTTCGGCATTTCCAATCCC
GACACCGTTATCGCCCTGAGTGCCGCCGTCGCCGTCGTCGTCTTCGGCGTGGCGCAATGG
AAGGAAGTCGCCCGCAATACCGACTGGGGCGTGTTGATGCTCTTCGGCGGCGGCATCAGC
CTGAGCACGCTGTTGAAAACATCCGGCGCGTCCGAAGCCTTGGGACAGCAGGTTGCCGCC
ACCTTTTCCGGCGCGCCCGCATTTTTGGTGATACTCATCGTCGCCGCCTTCATTATTTTT
CTGACCGAGTTCACCAGCAACACCGCCTCCGCCGCATTGCTTGTACCGATTTTCTCCGGC
AT CGC TATGCAGAT GGGGCTGCCCGAACAAG T CTTGGTATT CGT CAT CGGCAT CGGCGCA
TCTTGTGCCTTCATGCTGCCGGTTGCCACACCGCCTAACGCGATTGTGTTCGGCACGGGC
TTAATCAAGCAACGCGAAATGATGAA~1GTCGGCATACTGCTGAACATCCTCTGCGTAGTA
TTGGTTGCTCTGTGGGCTTATGCTGTACTGATGTAA
NIVMB0792 Protein sequence
MNLHAKDKTQHPENVELLSAQKPITDFKGLLTTIISAVVCFGIYHILPYSPDANKGIALL
IFVAALWFTEAVHrTVTALMVPILAVVLGFPDMDIKKAMADFSNPIIYIFFGGFALATAL
HMQRLDRKIAVSLLRLSRGNMKVAVLMLFLVTAFLSMWISNTATAAMMLPLAMGMLSHLD
QEKEHKTYVFLLLGIAYCASIGGLGTLVGSPPNLIAAKALNLDFVGWMKLGLPMMLLILP
LMLLSLYVILKPNLNERVEIKAESI PWTLHRVIALLI FLATAAAWI FS SKI KTAFGI SNP
DTVIALSAAVAVVVFGVAQWKEVARNTDWGVLMLFGGGISLSTLLKTSGASEALGQQVAA
TFSGAPAFLVILIVAAFIIFLTEFTSNTASAALLVPIFSGIAMQMGLPEQVLVFVIGIGA
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SCAFMLPVATPPNAIVFGTGLIKQREMMNVGILLNILCVVLVALWAYAVLM
NMB0279 DNA sequence
ATGCAACGACAAATCAAACTGA-kAAATTGGCTTCAGACCGTTTATCCCGAACGGGACTTC
GATCTGACTTTTGCGGCGGCGGATGCTGATTTCCGCCGCTATTTCCGTGCAACGTTTTCA
GACGGCAGCAGTGTCGTCTGCATGGATGCACCGCCCGACAAGATGAGTGTCGCACCTTAT
TTGAAAGTGCAGAAACTGTTTGACATGGTCAATGTGCCGCAGGTATTGCACGCGGACACG
GATCTGGGGTTTGTGGTATTGAACGACTTGGGCAATACGACGTTTTTGACCGCAATGCTT
CAGGAACAGGGCGAAACGGCGCACAAAGCCCTGCTTTTGGAGGCAATCGGCGAGTTGGTC
GAATTGCAGAAGGCGAGCCGTGAAGGGGTTTTGCCCGAATATGACCGTGAAACGATGTTG
CGCGAAATCAACCTGTTCCCGGAATGGTTTGTCGCAAAAGAATTGGGGCGCGAATTAACA
TTCAAACAACGCCAACTTTGGCAGCAAACCGTCGATACGCTGCTGCCGCCCCTGTTGGCG
CAGCCCAAAGTCTATGTGCACCGCGACTTTATCGTCCGCAACCTGATGCTGACGCGCGGC
AGGCCGGGCGTTTTAGACTTCCAAGACGCGCTTTACGGCCCGATTTCCTACGATTTGGTG
TCGCTGTTGCGCGATGCCTTTATCGAATGGGAAGAAGAATTTGTCTTGGACTTGGTTATC
CGCTACTGGGAAAAGGCGCGGGCTGCCGGCTTGCCCGTCCCCGAAGCGTTTGACGAGTTT
TACCGCTGGTTCGAATGGATGGGCGTGCAGCGGCACTTGAAGGTTGCAGGCATCTTCGCA
CGCCTGTACTACCGCGACGGCAAAGACAAATACCGTCCGGAAATCCCGCGTTTCTTAAAC
TATCTGCGCCGCGTATCGCGCCGTTATGCCGAACTCGCCCCGCTCTACGCGCTCTTGGTC
GAACTGGTCGGCGATGAAGAACTGGAAACGGGCTTTACGTTTTAA
NMB0279 Protein sequence
MQRQIKLKNWLQTVYPERDFDLTFAAADADFRRYFRATFSDGSSVVCMDAPPDKMSVAPY
LKVQKLFDMVNVPQVLHADTDLGFVVLNDLGNTTFLTAMLQEQGETAHKALLLEAIGELV
ELQKA.SREGVLPEYDRETMLREINLFPEWFVAKELGRELTFKQRQLWQQTVDTLLPPLLA
QPKVYVHRDFIVRNLMLTRGRPGVLDFQDALYGPISYDLVSLLRDAFIEWEEEFVLDLVI
RYWEKARAAGLPVPEAFDEFYRWFEWMGVQRHLKVAGIFARLYYRDGKDKYRPEIPRFLN
YLRRVSRRYAELAPLYALLVELVGDEELETGFTF
NMB2050 DNA sequence
ATGGAACTGATGACTGTTTTGCTGCCTTTGGCGGCGTTGGTGTCGGGCGTGTTGTTTACA
TGGTTGCTGATGAAGGGCCGGTTTCAGGGCGAGTTTGCCGGTTTGAACGCGCACCTGGCG
GAAAAGGCGGCAAGATGTGATTTTGTCGAACAGGCACACGGCAAAACCGTGTCGGAATTG
GCGGTGTTGGACGGGAAATACCGGCATTTGCAGGACGAAAATTATGCTTTGGGCAACCGT
TTTTCCGCAGCCGAAAAGCAGATTGCCCATTTGCAGGAAAAAGAGGCGGAGTCGGCGCGG
CTGAAGCAGTCGTATATCGAGTTGCAGGAAAAGGCACAGGGTTTGGCGGTTGAAAACGAA
CGTTTGGCAACGCAGCTCGGACAGGAACGGAAGGCGTTTGCCGACCAATATGCCTTGGAA
CGCCAAATCCGCCAAAGAATCGAAACCGATTTGGAAGAAAGCCGCCAAACTGTCCGCGAC
GTGCAAAACGACCTTTCCGATGTCGGCAACCGTTTTGCCGCAGCCGAAAAACAGATTGCC
CATTTGCAGGAAAAAGAGGCGGAAGCGGAGCGGTTGAGGCAGTCGCATACCGAGTTGCAG
GAAAAGGCACAGGGTTTGGCGGTTGAAAACGAACGTTTGGCAACGCAAATCGAACAGGAA
CGCCTTGCTTCTGAAGAGAAGCTGTCCTTGCTGGGCGAGGCGCGCAAAAGTTTGAGCGAT
CAGTTTCAAAA.TCTTGCCAACACGATTTTGGAAGAAAAAAGCCGCCGTTTTACCGAGCAG
AACCGCGAGCAGCTCCATCAGGTTTTGAACCCGCTAAACGAACGCATCCACGGTTTCGGC
GAGTTGGTCAAGCAAACCTATGATAAAGAATCGCGCGAGCGGCTGACGTTGGAAAACGAA
TTGAAACGGCTTCAGGGGTTGAACGCGCAGCTGCACAGCGAGGCAAAGGCCCTGACCAAC
GCGCTGACCGGTACGCAGAATAAGGTTCAGGGCAATTGGGGCGAGATGATTCTGGAAACG
GTTTTGGAAAATTCCGGCCTTCAGAAAGGGCGGGAATATGTGGTTCAGGCGGCATCCGTC
CGAAAAGAGGAAGACGGCGGCACGCGCCGCCTCCAGCCCGACGTTTTGGTCAACCTGCCC
GACAACAAGCAGATTGTGATTGATTCCAAGGTCTCGCTGACAGCTTATGTGCGCTACACG
CAGGCGGCGGATGCGGATACGGCGGCACGCGP??CTGGCGGCACACGTTGCCAGCATCCGT
GCACACATGAAAGGCTTGTCGCTGAAGGATTACACCGATTTGGAAGGTGTGAACACATTG
GATTTCGTCTTTATGTTTATCCCTGTCGAACCGGCCTACCTGTTGGCGTTGCAGAATGAC
GCGGGCTTGTTCCAAGAGTGTTTCGACAAACGGATTATGCTGGTCGGCCCCAGTACGCTG
CTGGCGACTTTGAGGACGGTGGCGAATATTTGGCGCAACGAACAGCAAAATCAGAACGCA
CTGGCGATTGCGGACGAAGGCGGCAAGCTGTACGACAAGTTTGTCGGCTTCGTACAGACG
CTCGAAAGCGTCGGCAAAGGCATCGATCAGGCGCAAAGCAGTTTTCAGACGGCATTCAAG
CAACTTGCCGAAGGGCGCGGGAATCTGGTCGGACGCGCCGAGAAACTGCGTCTGTTGGGC
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
24
GTGAAGGCAGGCAAACAACTTCAACGGGATTTGGTCGAGCGTTCCAATGAAAC-kACGr,CG
TTGTCGGAATCTTTGGAATACGCGGCAGAAGATGAAGCAGTCTGA
NMB2050 Protein sequence
MELMTVLLPLAALVSGVLFTWLLMKGRFQGEFAGLNAHLAEKAARCDFVEQAHGKTVSEL
AVLDGKYRHLQDENYALGNP.FSAAEKQIAHLQEKEAESARLKQSYIELQEKAQGLAVENE
RLATQLGQERKAFADQYALERQIRQRIETDLEESRQTVRDVQNDLSDVGNRFAAAEKQIA
HLQEKEAEAERLRQSHTELQEKAQGLAVENERLATQIEQERLASEEKLSLLGEARKSLSD
QFQNLANTILEEKSRRFTEQNREQLHQVLNPLNERIHGFGELVKQTYDKESRERLTLENE
LKRLQGLNAQLHSEAKALTNALTGTQNKVQGNWGEMILETVLENSGLQKGREYVVQAASV
RKEEDGGTRRLQPDVLVNLPDNKQIVIDSKVSLTAYVRYTQAADADTAARELAAHVASIR
AHMKGLSLKDYTDLEGVNTLDFVFMFIPVEPAYLLALQNDAGLFQECFDKRIMLVGPSTL
LATLRTVANIWRNEQQNQNALAIADEGGKLYDKFVGFVQTLESVGKGIDQAQSSFQTAFK
QLAEGRGNLVGRAEKLRLLGVKAGKQLQRDLVERSNETTALSESLEYAAEDEAV
NMB 13 3 5 CreA protein DNA sequence
ATGAACAGACTGCTACTGCTGTCTGCCGCCGTCCTGCTGACTGCCTGCGGCAGCGGCGAA
ACCGATAAAATCGGACGGGCAAGTACCGTTTTCAACATACTGGGCAAAAACGACCGTATC
GAAGTGGAAGGATTCGACGATCCCGACGTTCAAGGGGTTGCCTGTTATATTTCGTATGCA
AAAAAAGGCGGCTTGAAGGAAATGGTCAATTTGGAAGAGGACGCGTCCGACGCATCGGTT
TCGTGCGTTCAGACGGCATCTTCGATTTCTTTTGACGAAACCGCCGTGCGCAAACCGAAA
GAAGTTTTCAAACACGGTGCGAGCTTCGCGTTCAAGAGCCGGCAGATTGTCCGTTATTAC
GACCCCAAACGCAAAACCTTCGCCTATTTGGTGTACAGCGATAAAATCATCCAAGGCTCG
CCGAAAAATTCCTTAAGCGCGGTTTCCTGTTTCGGCGGCGGCATACCGCAAACCGATGGG
GTGCAAGCCGATACTTCCGGCAACCTGCTTGCCGGCGCCTGCATGATTTCCAACCCGATA
GAAAATCTCGACAA.ACGCTGA
NMB 13 3 5 Protein sequence
MNRLLLLSAAVLLTACGSGETDKIGRASTVFNILGKNDRIEVEGFDDPDVQGVACYISYA
KKGGLKEMVNLEEDASDASVSCVQTASSISFDETAVRKPKEVFKHGASFAFKSRQIVRYY
DPKRKTFAYLVYSDKIIQGSPKNSLSAVSCFGGGIPQTDGVQADTSGNLLAGACMISNPI
ENLDKR
NMB2035 DNA sequence
ATGACCGCCTTTGTCCACACCCTTTCAGACGGCATGGAACTGACCGTCGAAATCAAGCGC
CGTGCCAAGAAAAACCTGATTATCCGCCCCGCCGGCACACATACCGTCCGCATCAGCGTC
CCACCCTGCTTCTCCGTCTCCGCTCTAAACCGCTGGCTGTATGAAAACGAAGCCGTCCTG
CGGCAAACACTGGCGAAAACACCGCCGCCGCAAACTGCCGAAAACCGGCTGCCCGAATCC
ATCCTCTTCCACGGCAGACAGCTTGCCCTCACCGCCCATCAAGACACGCAAATCCTGCTG
ATGCCGTCTGAAATCCGTGTTCCCGAAGGCGCACCCGAAAAACAGCTTGCGCTGCTGCGG
GACTTTTTGGAACGGCAGGCGCACAGTTACCTGATTCCCCGCCTCGAACGCCACGCCCGC
ACCACACAACTGTTCCCCGCCTCCTCCTCGCTGACCTCTGCCAAAACCTTCTGGGGCGTG
TGCCGCAAAACCACAGGCATACGCTTCAACTGGCGGCTGGTCGGCGCACCGGAATACGTT
GCCGACTATGTCTGCATACACGAACTCTGCCACCTCGCCCATCCCGACCACAGCCCCGCC
TTTTGGGAACTGACCCGCCGCTTCGCCCCCTACACGCCCAAAGCGAAACAGTGGCTCAAA
ATCCACGGCAGGGAACTTTTCGCCTTAGGCTGA
NMB2035 Protein sequence
MTAFVHTLSDGMELTVEIKRRAKKNLIIRPAGTHTVRISVPPCFSVSALNRWLYENEAVL
RQTLAKTPPPQTAENRLPESILFHGRQLALTAHQDTQILLMPSEIRVPEGAPEKQLALLR
DFLERQAHSYLIPRLERHARTTQLFPASSSLTSAKTFWGVCRKTTGIRFNWRLVGAPEYV
ADYVCIHELCHLAHPDHSPAFWELTRRFAPYTPKAKQWLKIHGRELFALG
NMB 1351 Fmu and Fmv protein DNA sequence
ATGAACGCCGCACAACTCGACCATACCGCCAAAGTTTTGGCTGAAATGCTGACTTTCAAA
CAGCCTGCCGATGCCGTCCTCTCCGCCTATTTCCGCGAACACAAAAAGCTCGGCAGTCAA
GATCGCCACGAAATCGCCGAAACCGCCTTTGCCGCGCTGCGCCACTATCAAAAAATCAGT
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
ACCGCCCTACGCCGTCCGCACGCGCAGCCGCGCAAAGCCGCTCTCGCCGCACTGGTTCTC
GGCAGAAGCACCAACATCAGCCAAATCAAAGACCTGCTTGATGAAGAAGAAACAGCGTTC
CTCGGCPATTTG_AAAGCCCGTAAAACCGAGTTTTCAGACAGCCTGAATACCGCCGCAGAA
TTGCCGCAATGGCTGGTGGAACAACTGAAACAGCATTGGCGCGAAGAAGAAATCCTCGCT
5 TTCGGCCGCAGCATCA.ACCAGCCTGCCCCGCTCGACATCCGCGTCAACACTTTGAPAGGC
AAACGCGATAAAGTGCTGCCGCTGTTGCAAGCCGAAAGTGCCGATGCAGAGGrAACGCCT
TATTCGCCTTGGGGCATCCGCCTGAAAAACAAAATCGCGCTTAACAAACACGAACTGTTT
TTAGACGGCACACTGG_AAGTCC_AAGACGAAGGCAGCCAGCTGCTTGCCTTATTGGTGGGC
GCAAAACGAGGCGAAATCATTGTCGATTTCTGTGCCGGTGCCGGCGGT_AAAACCTTGGCT
10 GTCGGTGCGCAAATGGCGAACAAAGGCAGAATCTACGCCTTCGATATCGCCGAAAAACGC
CTTGCCAACCTCAAACCGCGTATGACCCGCGCCGGACTGACCAATATCCACCCCGAACGC
ATCGGCAGCGAACACGATGCCCGTATCGCCCGACTGGCAGGCAAAGCCGACCGTGTGTTG
GTGGACGCGCCCTGCTCCGGTTTGGGCACTTTACGCCGCAATCCCGACCTCAAATACCGC
CAATCCGCCGAAACCGTCGCCAACCTTTTGGAACAGCAACACAGCATCCTCGATGCCGCC
15 TCCAAACTGGTAAAACCGCAAGGACGTTTGGTGTACGCCACTTGCAGCATCCTGCCCGAA
GAAAACGAGCTGCAAGTCGAACGTTTCCTGTCCGAACATCCCGAATTTGAACCCGTCAAC
TGCGCCGAACTGCTTGCCGGTTTGAAAATCGATTTGGATACCGGCAAATACCTGCGCCTC
AACTCCGCCCGACACCAAACCGACGGCTTCTTCGCCGCCGTATTGCAACGCAAATAA
2o NMB 13 51 Protein sequence
MNAAQLDHTAKVLAEMLTFKQPADAVLSAYFREHKKLGSQDRHEIAETAFAALRHYQKIS
TALRRPHAQPRKAALAALVLGRSTNISQIKDLLDEEETAFLGNLKARKTEFSDSLNTAAE
LPQWLVEQLKQHWREEEILAFGRSINQPAPLDIRVNTLKGKRDKVLPLLQAESADAEATP
YSPWGIRLKNKIALNKHELFLDGTLEVQDEGSQLLALLVGAKRGEIIVDFCAGAGGKTLA
25 VGAQMANKGRIYAFDIAEKRLANLKPRMTRAGLTNIHPERIGSEHDARIARLAGKADRVL
VDAPCSGLGTLRRNPDLKYRQSAETVANLLEQQHSILDAASKLVKPQGRLVYATCSILPE
ENELQVERFLSEHPEFEPVNCAELLAGLKIDLDTGKYLRLNSARHQTDGFFAAVLQRK
NMB 15 74 IlvC DNA sequence
ATGCAAGTCTATTACGATAAAGATGCCGATCTGTCCCTAATCAAAGGCAAAACCGTTGCC
ATCATCGGTTACGGTTCGCAAGGTCATGCCCATGCCGCCAACCTGAAAGATTCGGGTGTA
AACGTGGTGATTGGTCTGCGCCAAGGTTCTTCTTGGAAAAAAGCCGAAGCAGCCGGTCAT
GTCGTCAAAACCGTTGCTGAAGCGACCAAAGAAGCCGATGTCGTTATGCTGCTGCTGCCT
GACGAAACCATGCCTGCCGTCTATCACGCCGAAGTTACAGCCAATTTGAAAGAAGGCGCA
ACGCTGGCATTTGCACACGGCTTCAACGTGCACTACAACCAAATCGTTCCGCGTGCCGAC
TTGGACGTGATTATGGTTGCCCCCAAAGGTCCGGGCCATACCGTACGCAGTGAATACAAA
CGCGGCGGCGGCGTGCCTTCTCTGATTGCCGTTTACCAAGACAATTCCGGCAAAGCCAAA
GACATCGCCCTGTCTTATGCGGCTGCCAACGGCGGCACCAAAGGCGGTGTGATTGAAACC
ACTTTCCGCGAAGAAACCGAAACCGATCTGTTCGGCGAACAAGCCGTATTGTGCGGCGGC
GTGGTCGAGTTGATC.AAGGCGGGTTTTGAAACCCTGACCGAAGCCGGTTACGCGCCTGAA
ATGGCTTACTTCGAATGTCTGCACGAAATGAAACTGATCGTTGACCTGATTTTCGAAGGC
GGTATTGCCAATATGAACTACTCCATTTCCAACAATGCGGAGTACGGCGAATACGTTACC
GGCCCTGAAGTGGTCAATGCTTCCAGCAAAGAAGCCATGCGCAATGCCCTGAAACGCATT
CAAACCGGCGAATACGCAAAAATGTTTATCCAAGAGGGTAATGTCAACTATGCGTCTATG
ACTGCCCGCCGCCGTCTGAATGCCGACCACCAAGTTGA.AAAAGTCGGCGCACAACTGCGT
GCCATGATGCCTTGGATTACTGCCAACAAATTGGTTGACCAAGACAAAAACTGA
NMB 1574 Protein sequence
MQVYYDKDADLSLIKGKTVAIIGYGSQGHAHAANLKDSGVNVVIGLRQGSSWKKAEAAGH
VVKTVAEATKEADVVMLLLPDETMPAVYHAEVTANLKEGATLAFAHGFNVHYNQIVPRAD
LDVIMVAPKGPGHTVRSEYKP.GGGVPSLIAVYQDNSGKAKDIALSYAPvNGGTKGGVIET
TFREETETDLFGEQAVLCGGVVELIKAGFETLTEAGYAPEMAYFECLHEMKLIVDLIFEG
GIANMNYSISNNAEYGEYVTGPEVVNASSKEAMRNALKRIQTGEYAKMFIQEGNVNYASM
TARRRLNADHQVEKVGAQLRAMMPWITANKLVDQDKN
NMB 1298 rsuA DNA sequence
ATGAAACTTATCAAP_TACCTGCAATATCAAGGCATAGGAAGCCGCPAGCAGTGCCAATGG
CTGATTGCCGGCGGTTATGTTTTCATCAACGGAACCTGCATGGACGACACCGATGCAGAC
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
26
ATCGATTCCTCATCCGTCGAAACGTTGGATATTGACGGGG_AAGCAGTAACCGTCGTTCCC
GAACCCTATTTCTACATCATGCTCAACAAGCCTGAAGATTACGAAACTTCGCACAAACCC
AAGCACTACCGCAGCGTATTCAGCCTGTTCCCCGACAATATGCGGAACATCGATATGCAG
GCGGTCGGCAGGCTGGATGCAGATACGACCGGCGTATTGCTGATTACCAACGACGGCAAA
CTGAACCACAGCCTGACTTCGCCGAGCAGAAAAATTCCCAAGCTGTACGAAGTAACGCTC
AAACACCCCACAGGAGAAACGCTCTGCGAAACCTTGAAAAACGGCGTGCTGCTCCACGAC
GAAAACGAAACCGTTTGTGCCGCCGATGCCGTTTTGAAAAACCCGACCACCCTGCTGCTG
ACCATTACCGAAGGAAAATACCACCAAGTCAAACGCATGATCGCCGCCGCCGGCAACCGC
GTGCAACACCTTCATCGCCGGCGATTCGCACATCTGGAAACAGAAAACCTCAAACCCGGG
G_AATGGAAA.TTTATCGAATGTCCAAAATTCTGA
NMB 1298 Protein sequence
MKLIKYLQYQGIGSRKQCQWLIAGGYVFINGTCMDDTDADIDSSSVETLDIDGEAVTWP
EPYFYIMLNKPEDYETSHKPKHYRSVFSLFPDNMRNIDMQAVGRLDADTTGVLLITNDGK
LNHSLTSPSRKIPKLYEVTLKHPTGETLCETLKNGVLLHDENETVCAADAVLKNPTTLLL
TITEGKYHQVKRMIAAAGNRVQHLHRRRFAHLETENLKPGEWKFIECPKF
NMB1856 Lys R family (transcription regulator) DNA sequence
ATGAAAACCAATTCAGAAGAACTGACCGTATTTGTTCAAGTGGTGGAAAGCGGCAGCTTC
AGCCGTGCGGCGGAGCAGTTGGCGATGGCAAATTCTGCCGTAAGCCGCATCGTCAAACGG
CTGGAGGAAAAGTTGGGTGTGAACCTGCTCAACCGCACCACGCGGCAACTCAGTCTGACG
GAAGAAGGCGCGCAATATTTCCGCCGCGCGCAGAGAATCCTGCAAGAAATGGCAGCGGCG
GAAACCGAAATGCTGGCAGTGCACGAAATACCGCAAGGCGTGTTGAGCGTGGATTCCGCG
ATGCCGATGGTGCTGCATCTGCTGGCGCCGCTGGCAGCAAAATTCAACGAACGCTATCCG
CATATCCGACTTTCGCTCGTTTCTTCCGAAGGCTATATCAATCTGATTGAACGCAAAGTC
GATATTGCCTTACGGGCCGGAGAATTGGACGATTCCGGGCTGCGTGCACGCCATCTGTTT
GACAGCCGCTTCCGCGTAATCGCCAGTCCTGAATACCTGGCAAAACACGGCACGCCGCAA
TCTACAGAAGAGCTTGCCGGCCACCAATGTTTAGGCTTCACCGAACCCGGTTCTCT_AAAT
ACATGGGCGGTTTTAGATGCGCAGGGAAATCCCTATAAGATTTCACCGCACTTTACCGCC
AGCAGCGGTGAAATCTTACGCTCGTTGTGCCTTTCAGGTTGCGGTATTGTTTGCTTATCA
GATTTTTTGGTTGACAACGACATCGCTGAAGGAAAGTTAATTCCCCTGCTCGCCGA_ACAA
ACCTCCGATAAAACACACCCCTTTAATGCTGTTTATTACAGCGATAAAGCCGTCAATCTC
CGCTTACGCGTATTTTTGGATTTTTTAGTGGAGGAACTGGGAAACAATCTCTGTGGATAA
NMB1856 Protein sequence
MKTNSEELTVFVQVVESGSFSRAAEQLAMANSAVSRIVKRLEEKLGVNLLNRTTRQLSLT
EEGAQYFRRAQRILQEMAAAETEMLAVHEIPQGVLSVDSAMPMVLHLLAPLAAKFNERYP
HIRLSLVSSEGYINLIERKVDIALRAGELDDSGLRARHLFDSRFRVIASPEYLAKHGTPQ
STEELAGHQCLGFTEPGSLNTWAVLDAQGNPYKISPHFTASSGEILRSLCLSGCGIVCLS
DFLVDNDIAEGKLIPLLAEQTSDKTHPFNAVYYSDKAVNLRLRVFLDFLVEELGNNLCG
NMB0119 DNA sequence
ATGATGAAGGATTTGAATTTGAGCAACAGCCTGTTCAAAGGCTACAACGACAAACATGGC
TTAATGATTTGTGGCTATGAATGGGGTTGGAGTAAAGCCGATGAGGCTGCTTATGTAGCA
GGTGAATACAAACTCCCTGAAAACAAAATCGACCATACATTTGCAAACAAATCCCTCTAT
TTCGGAGAGCAGGC AGTGGCGTTACGACAATACGATAAAAAATTGGTTTGAAATG
.TGGGGACACCCCTTAGACGAAAATGGATTGGGCGGTGCATTTGAAAAATCCCTGGTTCAA
ACCAACTGGGCTGCTACACAGGGCAACACTATCGACAATCCCGACAAGTTCACACAACCC
GAGCACATCGATAATTTTCTCTACCACATCGAAAAACTGCGTCCGAAAGTCATCCTCTTC
ATGGGCAGCAGGTTGGCGGATTTTCTGAACAACCAAAATGTACTGCCACGCTTCGAGCAG
T'TGGTCGGTAAGCAGACCAAACCGCTGGAGACGGTGCAAAAAGAATTTGACGGTACACGT
TTCAATGTCAAATTCCAATCGTTTGAAGATTGCGAAGTCGTCTGCTTTCCCCATCCCAGT
GCCAGTCGCGGTCTATCTTACGATTACATCGCCTTGTTTGCGCCTGAAATGAACCGGAT"'
TTATCGGACTTTAAAACAACACGCGGATTCAAATAA
NMB 0119 Protein sequence
MMKDLNLSNSLFKGYNDKHGLMICGYEWGWSKADEAAYVAGEYKLPENKIDHTFANKSLY
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
27
FGEQAKKWRYDNTIKNWFEMWGHPLDENGLGGAFEKSLVQTNWAATQGNTIDNPDKFTQP
EHIDNFLYHIEKLRPKVILFMGSRLADFLNNQNVLPRFEQLVGKQTKPLETVQKEFDGTR
FNVKFQSFEDCEVVCFPHPSASRGLSYDYIALFAPEMNRILSDFKT!i'RGFK
NMB 1705 rfaK DNA sequence
ATGGAAAAAGAATTCAGGATATTAAATATCGTATCGGCCAAGATTTGGGGTGGAGGCGAA
CAATATGTCTATGATGTTTCAAAAGCATTGGGGCTTCGGGGCTGCACPATGTTTACCGCC
GTCAATP.,AAAATAATGAATTGATGCACAGGCGATTTTCCG_AAGTTTCTTCCGTTTTCACA
ACGCGCCTTCACACGCTCAACGGGCTGTTTTCGCTCTACGCACTTACCCGCTTTATCCGG
AAAAACCGCATTTCCCACCTGATGATACACACCGGCAAAATTGCCGCCTTATCCATACTT
TTGAAAAAACTGACCGGGGTGCGCCTGATATTTGTCAAACATAATGTCGTCGCCAACAAA
ACCGATTTTTACCACCGCCTGATACAGAAA.AACACAGACCGCTTTATTTGCGTTTCCCGT
CTGGTTTACGATGTGCAAACCGCCGACAATCCCTTTAAAGAAAAATACCGGATTGTTCAT
AACGGTATCGATACCGGCCGTTTCCCTCCCTCTCAAGAAAAACCCGACAGCCGTTTTTTT
ACCGTCGCCTACGCCGGCAGGATCAGTCCAGAAAAAGGATTGG.AAAACCTGATTGAAGCC
TGTGTGATACTGCATCGGAAATATCCTC.AAATCAGGCTCAAATTGGCAGGGGACGGACAT
CCGGATTATATGTGCCGCCTGAAGCGGGACGTATCTGCTTCAGGAGCAGAACCATTTGTT
TCTTTTGAAGGGTTTACCGAAAAACTTGCTTCGTTTTACCGCCAAAGCGATGTCGTGGTT
TTGCCCAGCCTCGTCCCGGAGGCATTCGGTTTGTCATTATGCGAGGCGATGTACTGCCGA
ACGGCGGTGATTTCCAATACTTTGGGGGCGCAAA.AGGAAATTGTCGAACATCATCAATCG
GGGATTCTGCTGGACAGGCTGACACCTGAATCTTTGGCGGACGAAATCGAACGCCTCGTC
TTGAACCCTGAAACGAAAAACGCACTGGCAACGGCAGCTCATCAATGCGTCGCCGCCCGT
TTTACCATCAACCATACCGCCGACAAATTATTGGATGCAATATAA
NMB 1705 Protein sequence
MEKEFRILNIVSAKIWGGGEQYVYDVSKALGLRGCTMFTAVNKNNELMHRRFSEVSSVFT
TRLHTLNGLFSLYALTRFIRKNRISHLMIHTGKIAALSILLKKLTGVRLIFVKHNVVANK
TDFYHRLIQKNTDRFICVSRLVYDVQTADNPFKEKYRIVHNGIDTGRFPPSQEKPDSRFF
TVAYAGRISPEKGLENLIEACVILHRKYPQIRLKLAGDGHPDYMCRLKRDVSASGAEPFV
SFEGFTEKLASFYRQSDVWLPSLVPEAFGLSLCEAMYCRTAVISNTLGAQKEIVEHHQS
GILLDRLTPESLADEIERLVLNPETKNALATAAHQCVAARFTINHTADKLLDAI
NMB2065 Hernk protein DNA sequeiice
ATGCAGGAACAGAATCGGAAACCAAGTTTTCCCATAGTGATGTTGCTGGTGTCGGTTGCC
CTGTGGATAGCGTCTTTATCCAATGTTGCATTTTATTTGGGCAATCATGGAAGCATGGAG
GGTTTGACCGTTTTGATTTTGGGGTCGATATTTGCTTCTTTGGATATCAGGTATTGTGCG
GTCTATGCGAA.TTATGTTTGGTTGGCGGCCATTGTTTTGCTGGCGTTGCGGAAGAAGGTC
GTGCCTGTCCATGCGGCACTTTGGGGCTTGGCGTTGGTGGCTTTCAGTGTGAAAGCCGTA
TACGTCGATGAAGCAGGGAATACATCGGATATTGTGCGCTACGGTGCAGGATTTTATTTG
TGGTATGCCGCATTTGCGGTTGCCACCATCGGTACGTTTGCCGGAAAGAATAAGGAAAGA
AAAGCCGCATCAGCGGCAGACGGGATAAAAATGACGTTTGATAAATGGTTGGGCTTGTCA
AAACTGCCTAAAAATGAAGC.AAGAATGCTGCTACAATATGTTTCGGAATATACGCGCGTG
CAGTTGTTGACGCGGGGCGGGGAAGAAATGCCGGACGAAGTCCGACAGCGGGCGGACAGG
CTGGCGCAACGCCGTCTGAACGGCGAGCCGGTTGCCTATATTTTAGGTGTGCGCGAATTT
TATGGCAGACGCTTTACAGTCAATCCGAGCGTGCTGATTCCGCGCCCCGAAACCGAACAT
TTGGTCGAAGCCGTATTGGCGCGCCTGCCCGAAA.ACGGGCGCGTGTGGGATTTGGGGACG
GGCAGCGGCGCGGTTGCCGTAACCGTCGCGCTCGAACGCCCCGATGCGTTTGTGCGCGCA
TCCGACATCAGCCCGCCCGCCCTTGAAACGGCGCGGAAAAATGCGGCGGATTTGGGCGCG
CGGGTCGAATTTGCACACGGTTCGTGGTTCGACACCGATATGCCGTCTGAAGGGAAATGG
GACATCATCGTGTCCAACCCGCCCTATATCG_AAAACGGCGATAAACATTTGTTGC.AAGGC
GATTTGCGGTTTGAGCCGCAAATCGCGCTGACCGACTTTTCAGACGGCCTAAGCTGCATC
CGCACCTTGGCGCAAGGCGCGCCCGACCGTTTGGCGGAAGGCGGTTTTTTATTGCTGGAA
CACGGTTTCGATCAGGGCGCGGCGGTGCGCGGCGTGTTGGCGGAGAATGGTTTTTCAGGA
GTGGAAACCCTGCCGGATTTGGCGGGTTTGGACAGGGTTACGCTGGGGAAGTATATGAAG
CATTTGAAATAA
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
28
NMB2065 Protein sequence
MQEQNRKPSFPIVMLLVSVALWIASLSNVAFYLGNHGSMEGLTVLILGSIFASLDIRYCA
VYANYVWLAAIVLLALRKKVVPVHAALWGLALVAFSVKAVYVDEAGNTSDIVRYGAGFYL
WYAAFAVATIGTFAGKNKERKAASAADGIKMTFDKWLGLSKLPKNEARMLLQYVSEYTRV
QLLTRGGEEMPDEVRQRADRLAQRRLNGEPVAYILGVREFYGRRFTVNPSVLIPRPETEH
LVEAVLARLPENGRVWDLGTGSGAVAVTVALERPDAFVRASDISPPALETARKNAADLGA
RVEFAHGSWFDTDMPSEGKWDIIVSNPPYIENGDKHLLQGDLRFEPQIALTDFSDGLSCI
RTLAQGAPDRLAEGGFLLLEHGFDQGAAVRGVLAENGFSGVETLPDLAGLDRVTLGKYMK
HLK
Mutants selected by vacinee's 17 D sera (Screened once oiily)
NMB0339 DNA sequence
ATGGACAACGAATTGTGGATTATCCTGCTGCCGATTATCCTTTTGCCCGTCTTCTTCGCG
ATGGGCTGGTTTGCCGCCCGCGTGGATATGAAAACCGTATTGAAGCAGGCAAAAAGCATC
CCTTCGGGATTTTAT A.GCTTGGACGCTTTGGTCGACCGCAACAGCGGGCGCGCGGCA
AGGGAGTTGGCGGAAGTCGTCGACGGCCGGCCGCAATCGTATGATTTGAACCTCACCCTC
GGCAAACTTTACCGCCAGCGTGGCGAAA.A.CGACAAAGCCATCAACATACACCGGACAATG
CTCGATTCTCCCGATACGGTCGGCGAAAAGCGCGCGCGCGTCCTGTTTGAATTGGCGCAA
AACTACCAAAGTGCGGGGTTGGTCGATCGTGCCGAACAGATTTTTTTGGGGCTGCAAGAC
GGTAAAATGGCGCGTGAAGCCAGACAGCACCTGCTCAATATCTACCAA.CAGGACAGGGAT
TGGGAAAA.AGCGGTTGAAACCGCCCGGCTGCTCAGCCATGACGATCAGACCTATCAGTTT
GAAATCGCCCAGTTTTATTGCGAACTTGCCCAAGCCGCGCTGTTCAAGTCCAATTTCGAT
GTCGCGCGTTTCAATGTCGGCAAGGCACTCGAAGCCAACAAAAAATGCACCCGCGCCAAC
ATGATTTTGGGCGACATCGAACACCGACAAGGCAATTTCCCTGCCGCCGTCG_AAGCCTAT
GCCGCCATCGAGCAGCAAAACCATGCATACTTGAGCATGGTCGGCGAGAAGCTTTACGAA
GCCTATGCCGCGCAGGGAAAACCTGAAGAAGGCTTGAACCGTCTGACAGGATATATGCAG
ACGTTTCCCGAACTTGACCTGATCAATGTCGTGTACGAG.AAATCCCTGCTGCTTAAGTGC
GAGAAAGAAGCCGCGCAAACCGCCGTCGAGCTTGTCCGCCGCAAGCCCGACCTTAACGGC
GTGTACCGCCTGCTCGGTTTGAAACTCAGCGATATGAATCCGGCTTGGAAAGCCGATGCC
GACATGATGCGTTCGGTTATCGGACGGCAGCTACAGCGCAGCGTGATGTACCGTTGCCGC
AACTGCCACTTCAAATCCCAA.GTCTTTTTCTGGCACTGCCCCGCCTGCAACAAATGGCAG
ACGTTTACCCCGAATAAAATCGAAGTTTAA
NMB0339 Protein sequence
MDNELWIILLPIILLPVFFAMGWFAARVDMKTVLKQAKSIPSGFYKSLDALVDRNSGRAA
RELAEVVDGRPQSYDLNLTLGKLYRQRGENDKAINIHRTMLDSPDTVGEKRARVLFELAQ
NYQSAGLVDRAEQIFLGLQDGIIHAREARQHLLNIYQQDRDWEKAVETARLLSHDDQTYQF
EIAQFYCELAQAALFKSNFDVARFNVGKALEANKKCTRANMILGDIEHRQGNFPAAVEAY
AAIEQQNHAYLSMVGEKLYEAYAAQGKPEEGLNRLTGYMQTFPELDLINVVYEKSLLLKC
EKEAAQTAVELVRRKPDLNGVYRLLGLKLSDMNPAWKADADMMRSVIGRQLQRSVMYRCR
NCHFKSQVFFWHCPACNKWQTFTPNKIEV
Selection with patient's sera
We have a collection of acute and convalescent sera available to us for
screening.
This is from individuals infected with different serogroup of N.
naeningitidis.
Screens have been performed with acute (A) or convalescent (C) sera. The
period
between the acute infection and collection of sera was from 2 weeks to 3
months.
NMB0401 putA DNA sequence
ATGTTTCATTTTGCATTTCCGGCACAAACTGCCCTGCGCCAAGCGATAACCGATGCCTAC
CGCCGTAATGAAATCGAAGCCGTACAGGATATGTTGCAACGTGCACAGATGAGCGACGAA
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
29
GAGCGCAACGCCGCCTCCGAGCTTGCCCGCCGTTTGGTTACCC_AAGTCCGCGCCGGCCGC
ACCAAAGCCGGCGGCGTGGATGCGCTGATGCACGAGTTTTCACTCTCCAGCGAAGAAGGC
ATCGCGCTGATGTGTCTGGCAGAAGCCCTGCTGCGTATCCCCGACAACGCCACGCGCGAC
CGCCTGATTGCCGACAAGATTTCAGACGGCAACTGGAAAAGCCATTTGAACAACAGCCCT
TCCCTCTTCGTCAATGCTGCCGCCTGGGGCCTGCTGATTACCGGCAAACTGACCGCCACA
AACGACAAACAAATGAGTTCCGCACTCAGCCGCCTGATCAGCAAAGGCGGCGCACCGCTC
ATCCGCCAAGGCGTAAATTACGCCATGCGGCTTCTGGGC_AAACAGTTCGTAACCGGACAG
ACCATTGAAGAAGCCCTGCAAAACGGCAAAGAACGCGAAAP,AATGGGCTACCGCTTCTCC
TTCGATATGTTGGGCGAAGCCGCCTACACCCAAGCCGATGCCGACCGCTACTACCGCGAC
TATGTCGAAGCCATCCACGCCATCGGCAAAGATGCGGCAGGACAAGGCGTTTACGAAGGT
AACGGTATTTCCGTCAAACTTTCCGCCATCCATCCGCGCTACTCGCGCACCCAACACGGC
CGCGTGATGGGCGAACTGTTGCCGCGCCTGAAAGAGCTGTTCCTTTTGGGTAA.AAAATAC
GATATCGGTATCAACATCGATGCCGAAGAAGCCAACCGTCTGGAGCTGTCTTTGGATTTG
ATGGAGGCTTTGGTTTCAGACCCTGACTTGGCTGGCTACAAAGGTATCGGTTTCGTTGTC
CAAGCCTACCAAAAACGTTGTCCGTTCGTTATCGACTACCTGATCGACCTTGCCCGCCGC
AACAACCAAAAACTAATGATCCGCCTCGTCAAAGGCGCGTATTGGGACAGCGAAATCAAA
TGGGCGCAAGTGGACGGCTTGAACGGCTATCCGACCTACACCCGCAAAGTCCACACCGAC
ATCTCCTACCTCGCCTGCGCGCGCAAACTGCTTTCCGCGCAAGACGCGGTATTCCCGCAA
TTTGCCACCCACAACGCCTACACTTTGGGCGCAATCTACCAAATGGGTAAAGGCAAAGAT
TTTGAACACCAATGCCTGCACGGTATGGGCGAAACCCTGTACGACCAAGTCGTCGGCCCG
CAAAACTTAGGCCGCCGCGTGCGCGTGTACGCCCCAGTCGGCACACACGAAACCCTGCTC
GCCTACTTGGTGCGCCGCCTGTTGGAAAACGGCGCGAACTCGTCTTTCGTCAACCAAATC
GTCGATGAA.AACATCAGCATCGACACGCTCATCCGCAGCCCGTTCGACACCATCGCCGAA
C_AAGGCATCCACCTGCACAACGCCCTGCCGCTGCCGCGCGATTTGTACGGCAAATGCCGT
CTGAACTCGCAAGGCGTGGACTTGAGCAACGAAAA.CGTATTGCAGCAGCTTCAAG_AACAG
ATGAACAAAGCCGCCGCGCAAGACTTCCACGCCGCATCCATCGTCAACGGCAAAGCCCGC
GATGTCGGCGAA.GCGCAACCGATTAAAAACCCTGCCGACCACGACGACATCGTCGGCACA
GTCAGCTTTGCCGATGCCGCGCTTGCCCAAGAAGCGGTTGGCGCAGCCGTTGCCGCGTTC
CCCGAATGGAGTGCGACACCTGCCGCCGAACGCGCCGCCTGCCTGCGCCGTTTTGCCGAT
TTGCTGGAGCAGCACACCCCAGCACTGATGATGCTTGCCGTGCGCGAAGCAGGCAAAACG
CTGAACAACGCCATTGCCGAAGTGCGCGAAGCCGTCGATTTCTGCCGCTACTACGCAAAC
GAAGCCGAACATACCCTGCCTCAAGACGCAAAAGCCGTCGGCGCGATTGTCGCCATCAGC
CCGTGGAACTTCCCGCTCGCCATCTTTACCGGCGAAGTCGTTTCCGCATTGGCGGCAGGC
AACACCGTCATCGCCAAACCCGCCGAACAAACCAGCCTGATTGCCGGTTATGCCGTTTCC
CTCATGCACGAAGCCGGCATCCCGACTTCCGCCCTGCAACTCGTCCTCGGCGCAGGCGAC
GTGGGTGCGGCATTGACCAACGATGCCCGCATCGGCGGCGTGATTTTCACCGGCTCGACC
GAAGTGGCGCGCCTGATCAACAAAGCCCTTGCCAAACGCGGCGACAATCCCGTCCTGATT
GCCGAAACCGGCGGACAAAACGCCATGATTGTCGATTCCACCGCACTTGCCGAGCAAGTC
TGCGCCGACGTATTGAACTCCGCCTTCGACAGCGCGGGACAACGCTGCTCCGCCCTGCGC
ATTTTGTGCGTCCAAGAAGACGTTGCCGACCGTATGCTCGACATGATCAAAGGCGCTATG
GACGAACTCGTCGTCGGCAAACCGATTCAGCTCACTACCGATGTCGGCCCCGTCATCGAT
GCCGAAGCACAGCAAAACCTGTTGAACCACATCAACAAAATGAAAGGTGTTGCCAAGTCC
TACCACGAAGTCAA.AACCGCCGCCGATGTCGATTCCAAAAAATCCACGTTCGTTCGCCCC
ATCCTGTTTGAATTGAACAACCTCAACGAACTGCAACGCGAAGTCTTCGGTCCCGTCCTG
CACGTCGTCCGCTACCGCGCCGACGAACTCGACAACGTCATCGACCAAATCAACAGCAAA
GGCTACGCCCTGACCCACGGCGTACACAGCCGCATCGAAGGCACGGTACGCCACATCCGC
AGCCGCATCGAAGCCGGCAACGTTTACGTCAACCGCAACATCGTCGGCGCAGTCGTCGGC
GTACAGCCCTTCGGCGGACACGGTCTGTCCGGCACAGGCCCCAAAGCAGGCGGTTCGTTC
TACCTGCAAAAACTGACCCGCGCCGGCGAATGGGTTGCCCCGACCTGAGCCAAATCGGA
CAGGCGGACGAAGCCGCACTCAAACGCCTCGAAGCACTGGTTCACAAACTACCGTTCAAC
GCCGAAGAGAAAAA.AGCCGCAGCGGCCGCTTTGGGACACGCCCGCATCCGCACCCTGCGC
CGTGCCGAAACCGTCCTTACCGGACCGACCGGCGAGCGCAACAGCATCTCATGGCACGCG
CCCAAACGCGTTTGGATACACGGCGGCAGCACGGTTCAAGCCTTTGCCGCACTGACCGAA
CTTGCCGCCTCCGGCATACAGGCAGTGGTCGAACCCGACAGCCCCTTGGCTTCCTACACT
GCCGACTTGGAAGGTCTGCTGCTGGTCAACGGCAAACCCGAAACCGCCGGCATCAGCCAC
GTTGCCGCCCTGTCGCCTTTGGACAGCGCGCGCAAACAGGAACTTGCCGCCCACGACGGC
GCACTCATCCGCATCCTCCCTTCGGAAAACGGA.CTCGACATCCTGCAAGTGTTTGAAGAA
ATCTCTTGCAGCGTCAACACCACAGCCGCCGGCGGCAACGCCAGCCTGATGGCGGTCGCC
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
GACTGA
NMB0401 Protein sequence
MFHFAFPAQTALRQAITDAYRRNEIEAVQDMLQRAQMSDEERNAASELARRLVTQVRkGR
5 TKAGGVDALMHEFSLSSEEGIALMCLAEALLRZPDNATRDRLIADKISDGNWKSI?LNNSP
SLFVNAAAWGLLITGKLTATNDKQMSSALSRLISKGGAPLIRQGVNYAMRLLGKQFVTGQ
TIEEALQNGKEREKMGYRFSFDMLGEAAYTQADADRYYRDYVEAIHAIGKD_ziAGQGVYEG
NGISVKLSAIHPRYSRTQHGRVMGELLPRLKELFLLGKKYDIGINIDAEEANRLELSLDL
MEALVSDPDLAGYKGIGFVVQAYQKRCPFVIDYLIDLARRNNQKLMIRLVKGAYWDSEIK
10 WAQVDGLNGYPTYTRKVHTDISYLACARKLLSAQDAVFPQFATHNAYTLGAIYQMGKGKD
FEHQCLHGMGETLYDQVVGPQNLGRRVRVYAPVGTHETLLAYLVRRLLENGANSSFVNQI
VDENISIDTLIRSPFDTIAEQGIHLHNALPLPRDLYGKCRLNSQGVDLSNENVLQQLQEQ
MNKAAAQDFHAASIVNGKARDVGEAQPIKNPADHDDIVGTVSFADAALAQEAVGAAVAAF
PEWSATPAAERAACLRRFADLLEQHTPALMMLAVREAGKTLNNAIAEVREAVDFCRYYAN
15 EAEHTLPQDAKAVGAIVAISPWNFPLAIFTGEVVSALAAGNTVIAKPAEQTSLIAGYAVS
LMHEAGIPTSALQLVLGAGDVGAALTNDARIGGVIFTGSTEVARLINKALAKRGDNPVLI
AETGGQN.AMIVDSTALAEQVCADVLNSAFDSAGQRCSALRILCVQEDVADRMLDMIKGAM
DELVVGKPIQLTTDVGPVIDAEAQQNLLNHINKMKGVAKSYHEVKTAADVDSKKSTFVRP
ILFELNNLNELQREVFGPVLHVVRYRADELDNVIDQINSKGYALTHGVHSRIEGTVRHIR
20 SRIEAGNVYVNRNIVGAVVGVQPFGGHGLSGTGPKAGGSFYLQKLTRAGEWVAPTLSQIG
QADEAALKRLEALVHKLPFNAEEKKAA.AAALGHARIRTLRRAETVLTGPTGERNSISWHA
PKRVWIHGGSTVQAFAALTELAASGIQAVVEPDSPLASYTADLEGLLLVNGKPETAGISH
VAALSPLDSARKQELAAHDGALIRILPSENGLDILQVFEEISCSVNTTAAGGNASLMAVA
D
NMB 1335 CreA
DNA and Protein sequences given above
NMB 1467 PPX DNA sequen.ce
ATGACCACCACCCCCGCAAACGTCCTCGCCTCCGTCGATTTGGGTTCCAACAGTTTCCGC
CTCCAGATTTGCGAAAACAACAACGGACAATTAAAAGTCATCGATTCGTTCAA.ACAGATG
GTGCGCTTCGCCGCCGGACTGGACGAACAGAAAAATCTGAGTGCCGCTTCCCAAGAACAG
GCTTTGGACTGTCTGGCAAAATTCGGCGAACGCCTGCGCGGCTTCCGCCCTGAACAGGTA
CGCGCCGTGGCAACCAACACATTCCGCGTTGCCAAAAACATCGCAGATTTCCTTCCCAAA
GCCGAAGCGGCATTGGGTTTCCCCATCGAA.ATCATCGCCGGGCGCGAAGAGGCGCGGCTG
ATTTATACCGGCGTGATCCACACCCTCCCCCCGGGCGGCGGCAAAA.TGCTGGTTATCGAC
ATCGGCGGCGGTTCGACAGAATTTGTCATCGGCTCGACGCTGAATCCCGACATTACCGAA
AGCCTGCCCTTGGGCTGCGTAACCTACAGCCTGCGCTTCTTCCAAAACAAAATCACCGCC
AAAGACTTCCAATCTGCCATTTCCGCCGCCCGCAACGAAATCCAGCGTATCAGCAAPAAT
ATGAGGCGCGAAGGTTGGGATTTCGCCGTCGGCACATCGGGTTCGGCAAAATCCATCCGC
GACGTGCTTGCCGCCGAAATGCCCCAAGAGGCGGACATTACCTACAAAGGCATGCGCGCC
CTCGCCGAACGCATCATCGAAGCCGGTTCGGTCAAAAAAGCCAAATTTGAAAACCTGAAA
CCGGAACGCATCGAAGTTTTTGCCGGCGGACTTGCCGTGATGATGGCGGCGTTTGAGGAA
ATGAAACTCGACAGGATGACCGTAACCGAAGCCGCCCTGCGCGACGGCGTGTTTTACGAT
TTGATCGGGCGCGGTTTAAACGAAGATATGCGCGGACAAACGGTTGCCGAGTTCCAACAC
CGCTACCACGTCAGCCTCAATCAGGCGAAACGCACCGCCGAGACCGCGCAAACCTTTATG
GACAGCCTCTGCCACGCTAAAAACGTTACAGTTCAAGAGCTTGCCTTGTGGCAACAGTAT
CTCGGACGCGCCGCCGCGCTGCACGAAATCGGTTTGGACATCGCCCACACCGGCTATCAC
AAGCATTCCGCCTACATCCTCGAAAACGCCGATATGCCGGGTTTCTCACGCA.AAGAACAG
ACCATACTTGCCCAACTGGTCATCGGTCATCGCGGCGATATGAAAAAAATGAGCGGCATC
ATCGGCACCAACGAAATGTTGTGGTATGCCGTTTTGTCCCTGCGCCTTGCCGCACTGTTC
TGCCGTTCGCGCCAAGACCTGTCTTTCCCGAAAAATATGCAGTTGCGCACGGATACGGAA
AGCTGCGGCTTCATCCTGCGTATTGACAGGGAATGGCTGGAACGCCATCCCCTGATTGCC
GACGCATTGGAATATGAAAGCGTCCAATGGCAAP.AAATCAATATGCCGTTCAA.AGTCGAG
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
31
GCCGTCTGA
NMB 1467 Protein sequence
MTTTPANVLASVDLGSNSFRLQICENNNGQLKVIDSFKQMVRFAAGLDEQKNLSAASQEQ
ALDCLAKFGERLRGFRPEQVRAVATNTFRVAKNIADFLPKAEAALGFPIEIIAGREEARL
IYTGVIHTLPPGGGKMLVIDIGGGSTEFV"IGSTLNPDITESLPLGCVTYSLRFFQNKITA
KDFQSAISAARNEIQRISKNMRREGWDFAVGTSGSAKSIRDVLAAEMPQEADITYKGMRA
.LAERIIEAGSVKKAKFENLKPERIEVFAGGLAVMMAAFEEMKLDRMTVTEAALRDGVFYD
LIGRGLNEDMRGQTVAEFQHRYHVSLNQAKRTAETAQTFMDSLCHAKNVTVQELALWQQY
LGRAAALHEIGLDIAHTGYHKHSAYILENADMPGFSRKEQTILAQLVIGHRGDMKKMSGI
IGTNEMLWYAVLSLRLAALFCRSRQDLSFPKNMQLRTDTESCGFILRIDREWLERHPLIA
DALEYESVQWQKINMPFKVEAV
NMB2056 HemK
ATGAACGGTAAATACTACTACGGCACAGGCCGCCGCAAAAGTTCAGTGGCTCGTGTATTC
CTGATTAAAGGTACAGGTC_.AATCATCGTAAACGGTCGTCCCGTTGACGAATTCTTCGCA
CGGGAAACCAGCCGAATGGTTGTTCGCCAACCCTTGGTTCTGACTGAAAACGCCGAATCT
TTCGACATCAAAGTCAATGTTGTTGGCGGCGGCGAAACCGGCCAGTCCGGCGCAATCCGC
CACGGCATTACCCGTGCCCTGATCGACTTCGATGCCGCGTTGAAACCCGCCTTGTCTCAA
GCTGGTTTTGTTACCCGCGATGCCCGCGAAGTCGAACGTAAP.,AAACCGGGTCTGCGCAAA
GCACGCCGTGCAAAACAATTCTCCAAACGTTAA
NMB2056 Protein sequence
MNGKYYYGTGRRKSSVARVFLIKGTGQIIVNGRPVDEFFARETSRMVVRQPLVLTENAES
FDIKVNVVGGGETGQSGAIRHGITRALIDFDAALKPALSQAGFVTRDAREVERKKPGLRK
ARRAKQFSKR
NMB0808 DNA sequence
ATGTCCGCCCTCCTCCCCATCATC_AACCGCCTGATTCTGCAAAGCCCGGACAGCCGCTCG
GAACTTGCCGCCTTTGCAGGCAAA.ACACTGACCCTGAACATTGCCGGGCTGAAACTGGCG
GGACGCATCACGGAAGACGGTTTGCTCTCGGCGGGAAACGGCTTTGCAGACACCGAAATT
ACCTTCCGCAACAGCGCGGTACAGAAAATCCTCCAAGGAGGCGAACCCGGGGCGGGCGAC
ATCGGGCTCGAAGGCGACCTCATCCTCGGCATCGCGGTACTGTCCCTGCTCGGCAGCCTG
CGTTCCCGCGCATCGGACGAATTGGCACGGATTTTCGGCACGCAGGCAGACATCGGCAGC
CGTGCCGCCGACATCGGACACGGCATCAAACAAATCGGCAGGAACATCGCCGAACAAATC
GGCGGATTTTCCCGCGAATCCGAGTCCGCAAACATCGGCAACGAAGCCCTTGCCGACTGC
CTCGACGAAATAAGCAGACTGCGCGACGGCGTGGAACGCCTCAACGAACGCCTCGACCGG
CTCGAACGCGACATTTGGATAGACTAA
NIVIBO808 Protein sequence
MSALLPIINRLILQSPDSRSELAAFAGKTLTLNIAGLKLAGRITEDGLLSAGNGFADTEI
TFRNSAVQKILQGGEPGAGDIGLEGDLILGIAVLSLLGSLRSRASDELARIFGTQADIGS
RAADIGHGIKQIGRNIAEQIGGFSRESESANIGNEALADCLDEISRLRDGVERLNERLDR
LERDIWID
NMB0774 upp DNA sequence
ATGAACGTTAATGTTATCAACCATCCGCTCGTCCGCCACAAATTAACCCTGATGAGGGAG
GCGGATTGCAGCACCTACAAATTCCGGACGCTTGCCACCGAGCTGGCGCGCCTGATGGCA
TACGAGGCAAGCCGTGATTTTGAAATCGAAAAATACCTTATCGACGGATGGTGCGGTCAG
ATTGAAGGCGACCGCATCAAGGGCAAAACATTGACCGTCGTTCCCATACTGCGTGCAGGT
TTGGGTATGCTTGACGGTGTGCTCGACCTGATTCCGACTGCCAAAATCAGTGTAGTCGGA
CTGCAGCGCGACGAAGAAACGCTG_AAGCCTATTTCCTATTTTGAGAPATTTGTGGACAGT
ATGGACGAACGTCCGGCTTTGATTATCGATCCTATGCTGGCGACAGGCGGTTCGATGGTT
GCCACCATCGACCTTTTGAAAGCCAAGGGCTGCAAAAATATCAAGGCACTGGTGCTGGTT
GCCGCGCCCGAGGGTGTGAAGGCGGTCAACGACGCGCACCCTGACGTTACGATTTACACC
GCCGCGCTCGACAGCCACTTGAACGAGAACGGCTACATCATCCCCGGCTTGGGCGATGCG
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
32
GGCGACAAGATTTTCGGCACGCGCTAA
NMB0774 Protein sequence
MNVNVINHPLVRHKLTLMREADCSTYKFRTLATELARLMAYEASRDFEIEKYLIDGWCGQ
IEGDRIKGKTLTWPILRAGLGMLDGVLDLIPTAKISVVGLQRDEETLKPISYFEKFVDS
MDERPALIIDPMLATGGSMVATZDLLKAKGCKNIKALVLVAAPEGVKAVNDAHPDVTIYT
.AALDSHLNENGYIIPGLGDAGDKIFGTR
NMA0078 putative integral membrance protein DNA sequence
TTGGCGTTTACTTTAATGCGTCGCGCCATGATACGTAAAATGCCCTATACGGAAGATATG
CGCCCAGGCGATACCGCTAATCCTTATGGTGCGTCCAAAGCGATGGTGGAACGGATGTTA
ACCGACATCCAAAAAGCCGATCCGCGCTGGAGCATGATTTTGTTGCGTTATTTCAATCCG
ATTGGCGCGCATGAAAGCGGCTTGATTGGCGAGCAGCCAAACGGCATCCCGAATAATTTG
TTGCCTTATATCTGCC_AAGTGGCGGCAGGCAAACTGCCGCAATTGGCGGTATTTGGCGAT
GACTACCCTACCCCCGACGGCACGGGGATGCGTGACTATATTCATGTGATGGATTTGGCA
GAAGGCCATGTCGCGGCTATGCAGGCAAAAAGTAATGTAGCAGGCACGCATTTGCTGAAC
TTAGGCTCCGGCCGCGCTTCTTCGGTGTTGGAAATCATCCGCGCATTTGAAGCAGCTTCG
GGTTTGACGATTCCGTATGAAGTCAAACCGCGCCGTGCCGGTGATTTGGCGTGCTTCTAT
GCCGACCCTTCCTATACAAAGGCGCAAATCGGCTGGCAAACCCAGCGTGATTTAACCCAA
ATGATGGAAGACTCATGGCGCTGGGTGAGTAATAATCCGAATGGCTACGACGATTAA
NMA0078 Protein sequence
MAFTLMRR.AMIRKMPYTEDMRPGDTANPYGASKAMVERMLTDIQKADPRWSMILLRYFNP
IGAHESGLIGEQPNGIPNNLLPYICQVAAGKLPQLAVFGDDYPTPDGTGMRDYIHVMDLA
EGHVAAMQAKSNVAGTHLLNLGSGRASSVLEIIRAFEAASGLTIPYEVKPRRAGDLACFY
ADPSYTKAQIGWQTQRDLTQMMEDSWRWVSNNPNGYDD
NMB0337 Branched-chain amino acid aininotransferase DNA sequence
ATGAGCAGACCCGTACCCGCCGTATTCGGCAGCGTTTTTCACAGTCAAATGCCCGTCCTC
GCCTACCGCGAAGGCAAATGGCAGCCGACCGAATGGCAATCTTCCCAAGACCTCTCCCTC
GCACCGGGCGCGCACGCCCTGCACTACGGCAGCGAATGTTTCGAGGGACTGAAAGCCTTC
CGTCAGGCAGACGGCAAAATCGTGCTGTTCCGTCCGACTGCCAATATCGCGCGTATGCGG
CAAAGTGCGGACATTTTGCACCTGCCGCGCCCCG.AAACCGAAGCTTATCTTGACGCGCTA
ATCAAATTGGTCAAACGTGCCGCCGATGAAATTCCCGATGCGCCTGCCGCCCTGTACCTG
CGTCCGACCTTAATCGGTACCGATCCCGTTATCGGCAAGGCCGGTTCTCCTTCCGAAACC
GCCCTGCTGTATATTTTGGCTTCCCCCGTCGGCGACTATTTCAAAGTCGGATCGCCCGTC
AAAATTTTGGTGGAAACCGAACACATCCGCTGCGCCCCGCATATGGGCCGCGTCAAATGC
GGCGGCAACTACGCTTCCGCCATGCACTGGGTGCTGAAGGCGAAAGCCGAATATGGCGCA
AATCAAGTCCTGTTCTGCCCGAACGGCGACGTGCAGGAAACCGGCGCGTCCAACTTTATC
CTGATTAACGGCGATGAAATCATTACCAAACCGCTGACCGACGAGTTTTTGCACGGCGTA
ACCCGCGATTCCGTACTGACGGTTGCCAAAGATTTGGGCTATACCGTCAGCGAACGCAA.T
TTCACGGTTGACGAACTCAAAGCTGCGGTGGAAAACGGTGCGGAAGCCATTTTGACCGGT
ACGGCAGCCGTCATCTCGCCCGTTACTTCCTTCGTCATCGGCGGCAAAGAAATCGAAGTG
AAAAGCCAAGAACGCGGCTATGCCATCCGTAAGGCGATTACCGACATCCAGTATGGTTTG
GCGGAAGACAAATACGGCTGGCTGGTTGAAGTGTGCTGA
NMB0337 Protein sequence
MSRPVPAVFGSVFHSQMPVLAYREGKGdQPTEWQSSQDLSLAPGAHALHYGSECFEGLKAF
RQADGKIVLFRPTANIARMRQSADILHLPRPETEAYLDALIKLVKRAADEIPDAPAALYL
RPTLIGTDPVIGKAGSPSETALLYILASPVGDYFKVGSPVKILVETEHIRCAPHMGRVKC
GGNYASAMHWVLKAKAEYGANQVLFCPNGDVQETGASNFILINGDEIITKPLTDEFLHGV
TRDSVLTVAKDLGYTVSERNFTVDELKAAVENGAEAILTGTAAVISPVTSFVIGGKEIEV
KSQERGYAIRKAITDIQYGLAEDKYGWLVEVC
NMB0191 ParA family protein DNA sequence
ATGAGTGCGAACATCCTTGCCATCGCCAATCAGAAGGGCGGTGTGGGCAAAACGACGACG
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
33
ACGGTAAATTTGGCGGCTTCGCTGGCATCGCGCGGCAAACGCGTGCTGGTGGTCGATTTG
GATCCGCAGGGCAATGCGACGACGGGCAGCGGCATCGACAAGGCGGGTTTGCAGTCCGGC
GTTTATCAGGTCTTATTGGGCGATGCGGACGTGCAGTCGGCGGCGGTACGCAGCAAAGAG
GGCGGATACGCTGTGTTGGGTGCGAACCGCGCGCTGGCCGGCGCGGAAATCGAACTGGTG
CAGGAAATCGCCCGGGAAGTGCGTTTG_AAAAACGCGCTCAAGGCAGTGGAAGAAGATTAC
GACTTTATCCTGATCGACTGCCCGCCTTCGCTGACGCTGTTGACGCTfi_AACGGGCTGGTG
GCGGCGGGCGGCGTGATTGTGCCGATGTTGTGCGAATATTACGCGCTGGAAGGGATTTCC
GATTTGATTGCGACCGTGCGCAAAATCCGTCAGGCGGTCAATCCCGATTTGGACATCACG
GGCATCGTGCGCACGATGTACGACAGCCGCAGCAGGCTGGTTGCCGAAGTCAGCG_AACA
TTGCGCAGCCATTTCGGGGATTTGCTTTTTGAAACCGTCATCCCGCGCAA.TATCCGCCTT
GCGGAAGCGCCGAGCCACGGTATGCCGGTGATGGCTTACGACGCGCAGGCAAAGGGTACC
AAGGCGTATCTTGCCTTGGCGGACGAGCTGGCGGCGAGGGTGTCGGGGAAATAG
NMB0191 Protein sequence
MSANILAIANQKGGVGKTTTTVNLAASLASRGKRVLVVDLDPQGNATTGSGIDKAGLQSG
VYQVLLGDADVQSAAVRSKEGGYAVLGANRALAGAEIELVQEIAREVRLKNALKAVEEDY
DFILIDCPPSLTLLTLNGLVAAGGVIVPMLCEYYALEGISDLIATVRKIRQAVNPDLDIT
GIVRTMYDSRSRLVAEVSEQLRSHFGDLLFETVIPRNIRLAEAPSHGMPVMAYDAQAKGT
KAYLALADELAARVSGK
NMB 1710 Glutamate dehydrogenase(gdhA) DNA sequence
ATGACTGACCTGAACACCCTGTTTGCCAACCTCAAACAACGCAATCCCAATCAGGAGCCG
TTCCATCAGGCGGTTGAAGAAGTCTTCATGAGTCTCGATCCGTTTTTGGCAAAAAz1TCCG
AAATACACCCAGCAAAGCCTGCTGGAACGCATCGTCGAACCCGAACGCGTCGTGATGTTC
CGCGTAACCTGGCAGGACGATAAAGGGCAAGTCCAAGTCAACCGGGGCTACCGCGTGCAA
ATGAGTTCCGCCATCGGTCCTTACAAAGGCGGCCTGCGCTTCCATCCGACCGTCGATTTG
GGCGTATTGAAATTCCTCGCTTTTGAACAAGTGTTCAAAAACGCCTTGACCACCCTGCCT
ATGGGCGGCGGCAAAGGCGGTTCCGACTTCGACCCCAAAGGCAAATCCGATGCCGAAGTA
ATGCGCTTCTGCCAAGCCTTTATGACCGAACTCTACCGCCACATCGGCGCGGACACCGAT
GTTCCGGCCGGCGACATCGGCGTAGGCGGGCGCGAAATCGGCTACCTGTTCGGACAATAC
AP,AAAAATCCGCAACGAGTTTTCTTCCGTCCTGACCGGCAAAGGTTTGGAATGGGGCGGC
AGCCTCATCCGTCCCGAAGCGACCGGCTACGGCTGCGTCTATTTCGCCCAAGCGATGCTG
CAAACCCGCAACGATAGTTTTGAAGGCAAA.CGCGTCCTGATTTCCGGCTCCGGCAATGTG
GCGCAATACr,CCGCCGA.AAAAGCCATCCAA.CTGGGTGCGAAAGTACTGACCGTTTCCGAC
TCCAACGGCTTCGTCCTCTTCCCCGACAGCGGTATGACCGAAGCGCAACTCGCCGCCTTG
ATCGAATTGAAAGAAGTCCGCCGCGAACGCGTTGCCACCTACGCCAAAGAGCAAGGTCTG
CAATACTTTGAAAAACAAAAACCGTGGGGCGTCGCCGCCGAAATCGCCCTGCCCTGCGCG
ACCCAGAACGAATTGGACGAAGAAGCCGCCAAAACCCTGTTGGCAAACGGCTGCTACGTC
GTTGCCGAAGGTGCGAATATGCCGTCGACTTTGGGCGCGGTCGAGCAATTTATCAA.AGCC
GGCATCCTCTACGCCCCGGGAAAAGCCTCCAATGCCGGCGGCGTGGCAACTTCAGGTTTG
GAAATGAGCCAAAACGCCATCCGCCTGTCTTGGACTCGTGAAGAAGTCGACCAACGCCTG
TTCGGCATCATGCAAAGCATCCACGAATCCTGTCTGAAATACGGCAAAGTCGGCGACACA
GTAAACTACGTCAATGGTGCGAACATTGCCGGTTTCGTCAAAGTTGCCGATGCGATGCTG
GCGCAAGGCTTCTAA
NMB 1710 Protein sequence
MTDLNTLFANLKQRNPNQEPFHQAVEEVFMSLDPFLAKNPKYTQQSLLERIVEPERVVMF
RVTWQDDKGQVQVNRGYRVQMSSAIGPYKGGLRFHPTVDLGVLKFLAFEQVFKNALTTLP
MGGGKGGSDFDPKGKSDAEVMRFCQAFMTELYRHIGADTDVPAGDIGVGGREIGYLFGQY
KKIRNEFSSVLTGKGLEWGGSLIRPEATGYGCVYFAQAMLQTRNDSFEGKRVLISGSGNV
AQYAAEKAIQLGAI,'VLTVSDSNGFVLFPDSGMTEAQLAALIELKEVF.F.ERVATYAKEQGL
QYFEKQKPWGVAAEIALPCATQNELDEEAAKTLLANGCYVVAEGANMPSTLGAVEQFIKA
GILYAPGKASNAGGVATSGLEMSQNAIRLSWTREEVDQRLFGIMQSIHESCLKYGKVGDT
VNYVNGANIAGFVKVADAMLAQGF
NMB0062 Glucose-l-phosphate thyinidylytransferase(rfbA-1) DNA sequence
ATGAAAGGCATCATACTGGCAGGCGGCAGCGGCACGCGCCTCTACCCCATCACGCGCGGC
GTATCCAAACAGCTCCTGCCCGTGTACGACAAACCGATGATTTATTACCCCTTGTCGGTT
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
34
TTGATGCTGGCGGGAATCCGCGATATTTTGGTGATTACCGCGCCTGAAGACAACGCCTCT
TTCAAACGCCTGCTTGGCGACGGCAGCGATTTCGGCATTTCCATCAGTTATGCCGTGC_AA
CCCAGTCCGGACGGCTTGGCACAGGCATTTATCATCGGCGAAGAATTTATCGGCAACGAC
AATGTTTGCTTGGTTTTGGGCGAC_AATATTTTTTACGGTCAGTCGTTTACGCAAACATTG
AAACAGGCGGCAGCGCAAACGCACGGCGCAACCGTGTTTGCTTATCAGGTCAAA-AACCCC
GAACGTTTCGGCGTGGTTGAATTTAACGAAAACTTCCGCGCCGTTTCCATCGAAGAAAAA
CCGCAACGGCCCAAATCCGATTGGGCGGTAACCGGCTTGTATTTCTACGACAACCGCGCC
GTCGAGTTCGCCAAACAGCTCAAACCGTCCGCACGCGGCGAATTGGAAATTACCGACCTC
AACCGGATGTATTTGGAAGACGGCTCGCTCTCCGTTCAAATATTGGGACGCGGTTTCGCG
TGGCTGGACACCGGCACCCACGAGAGCCTGCACGAAGCCGCTTCATTCGTCCAAACCGTG
C_AAAATATCCAAAACCTGCACATCGCCTGCCTCGAAGAAATCGCTTGGCGCAACGGTTGG
CTTTCCGATGAAAAACTGGAAGAATTGGCGCGCCCGATGGCGAAAAACCAATACGGCCAA
TATTTGCTGCGCCTGTTGAAAAAATAA
NMB0062 Protein sequence
MKGIILAGGSGTRLYPITRGVSKQLLPVYDKPMIYYPLSVLMLAIRDILVITAPEDNAS
FKRLLGDGSDFGISISYAVQPSPDGLAQAFIIGEEFIGNDNVCLVLGDNIFYGQSFTQTL
KQAAAQTHGATVFAYQVKNPERFGVVEFNENFRAVSIEEKPQRPKSDWAVTGLYFYDNRA
VEFAKQLKPSARGELEITDLNRMYLEDGSLSVQILGRGFAWLDTGTHESLHEAASFVQTV
QNIQNLHIACLEEIAWRNGWLSDEKLEELARPMAKNQYGQYLLRLLKK
NMB1583 Imidazoleglycerol-phosphate dehydratase(hisB) DNA sequence
ATGAATTTGACTAAAACACAACGCCAACTGCACAACTTTCTGACCCTCGCCCAAGAAGCA
GGTTCGCTGTCCAAGCTCGCCAAACTCTGCGGCTACCGTACCCCCGTCGCACTCTACAAA
CTCAAACAACGCCTTGAAAAGCAGGCAGAAGACCCAGATGCACGCGGCATCCGTCCCAGC
CTGATGGCP.AAACTCGAAAAACACACCGGCAAACCCAAAGGCTGGCTCGACAG_AAAACAC
CGCGAACGCACTGTCCCCGAAACCGCCGCAGAAAGCACCGGAACTGCCGAAACCCAAATT
GCCGAAACCGCATCTGCTGCCGGCTGCCGCAGCGTTACCGTCAACCGCAATACCTGCGAA
ACCCAAATCACCGTCTCCATCAACCTCGACGGCAGCGGCAAA~AGCAGGCTGGATACCGGC
GTACCCTTCCTCGAACACATGATCGATCAAATCGCCCGCCACGGCATGATTGACATCGAC
ATCAGCTGCAAAGGCGACCTGCACATCGACGACCACCACACCGCCGAAGACATCGGCATC
ACACTCGGACAAGCAATCCGGCAGGCACTCGGCGACAAAAAAGGCATCCGCCGTTACGGA
CATTCCTACGTCCCGCTCGACGAAGCCCTCAGCCGCGTCGTCATCGACCTTTCCGGCCGC
CCCGGACTCGTGTACAACATCGAATTTACCCGCGCACTAATCGGACGTTTCGATGTCGAT
TTGTTTGAAGAATTTTTCCACGGCATCGTCAACCACAGTATGATGACCCTGCACATCGAC
AACCTCAGCGGCAPAFiACGCCCACCATCAGGCGGAAACCGTATTCAAAGCCTTCGGGCGC
GCCCTGCGTATGGCAGTCGAACACGACCCGCGCATGGCAGGACAGACCCCCTCGACCAAA
GGCACGCTGACCGCATAA
NMB1583 Protein sequence
MNLTKTQRQLHNFLTLAQEAGSLSKLAKLCGYRTPVALYKLKQRLEKQAEDPDARGIRPS
LMAKLEKHTGKPKGWLDRKHRERTVPETAAESTGTAETQIAETASAAGCRSVTVNRNTCE
TQITVSINLDGSGKSRLDTGVPFLEHMIDQIARHGMIDIDISCKGDLHIDDHHTAEDIGI
TLGQAIRQALGDKKGIRRYGHSYVPLDEALSRVVIDLSGRPGLVYNIEFTRALIGRFDVD
LFEEFFHGIVNHSMMTLHIDNLSGKNAHHQAETVFKAFGRALRMAVEHDPRMAGQTPSTK
GTLTA
The following additional antigens were identified using essentially _the
xiictl'iou.oiogy described av~vVe:
NMB1333 Nucleic acid sequence
ATGCGCTACAAACCCCTTCTGCTTGCCCTGATGCTCGTTTTTTCCACGCCCGCCGTTGCC
GCCCACGACGCGGCACACAACCGTTCCGCCGAAGTGAAAAAACAGACGAAGAACAAAAAA
GAACAGCCCGAAGCGGCGGAAGGCP.AAAAAGAAAAAGGCAA.P,AATGGCGCAGTGAAAGAT
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
,AAAAAACAGGCGGCAAAGAGGCGGCAAAAGAGGGCAAAGAGTCCAAAAAAACCGCCAAA
AACCGCAAAGAAGCAGAG_AAGGAGGCGACATCCAGGCAGTCTGCGCGCAAAGGACGCGAA
GGGGATAAGAAATCGAAGGCGGAACACAAAAAGGCACATGGCAAGCCCGTGTCCGGATCC
AAAGAAAAAAACGCAAAAACACAGCCTGAAAACAAACAAGGCAAAAAAGAGGCAAAAGGA
5 CAGGGC_AATCCGCGC_AAGGGCGGCAAGGCGGAAAAAGACACTGTTTCTGCAAATAAAAAA
GTCCGTTCCGACAA.GAACGGCAAAGCAGTGAAACAGGArAAAAAATACAGGGAAGAGAAA
AATGCC_AAAACCGATTCCGACGAA.TTGAAAGCCGCCGTTGCCGCTGCCACCAATGATGTC
GAAA7ICAAAAAAGCCCTGCTCAAACAAAGCGAAGGAATGCTGCTTCATGTCAGCAATTCC
CTCAAACAGCTTCAGGAAGAGCGTATCCGCCAAGAGCGTATCCGTCAGGCGCGCGGCAAC
10 CTTGCTTCCGTCAACCGCAAACAGCGCGAGGCTTGGGACAAGTTCCAAAAACTCAATACC
GAGCTGAACCGTTTGAAAACGGAAGTCGCCGCTACGAAAGCGCAGATTTCCCGTTTCGTA
TCGGGGAACTATAAAAACAGCCAGCCGAATGCGGTTGCCCTGTTCCTGAAAAACGCCGAA
CCGGGTCAGAAAAACCGCTTTTTGCGTTATACGCGTTATGTAAACGCCTCCAATCGGGAA
GTTGTCAAGGATTTGGAAAAACAGCAGAAGGCTTTGGCGGTACAAGAGCAGAAAATCAAC
15 AATGAGCTTGCCCGTTTGAAG.AAAA.TTCAGGCAAACGTGCAATCTCTGCTGP.AAAAACAG
GGTGTAACCGATGCGGCGGAACAGACGGAAAGCCGCAGACAGAATGCCAAAATCGCCAAA
GATGCCCGAAAACTGCTGGAACAGAAAGGGAACGAGCAGCAGCTGAACAAGCTCTTGAGC
AATTTGGAGAAGAAAAAGGCCGAACACCGCATTCAGGATGCGGAAGCAAAAAGAAAATTG
GCTGAAGCCAGACTGGCGGCAGCCGAAAAAGCCAGAAAAGAAGCGGCGCAGCAGAAGGCT
20 GAAGCACGACGTGCGGAAATGTCCAACCTGACCGCCGAAGACAGGAACATCCAAGCGCCT
TCGGTTATGGGTATCGGCAGTGCCGACGGTTTCAGCCGCATGCAAGGACGTTTGAAAAAA
CCGGTTGACGGTGTGCCGACCGGACTTTTCGGGCAGAACCGGAGCGGCGGCGATATTTGG
AAAGGCGTGTTCTATTCCACTGCACCGGCAACGGTTGAAAGCATTGCGCCGGGAACGGTA
AGCTATGCGGACGAGTTGGACGGCTACGGCAAAGTGGTCGTGGTCGATCACGGCGAG.A.AC
25 TACATCAGCATCTATGCCGGTTTGAGCGAAA.TTTCCGTCGGCAAGGGTTATATGGTCGCG
GCAGGAAGCAAAATCGGCTCGAGCGGGTCGCTGCCGGACGGGGAAGAGGGGCTTTACCTG
CAAATACGTTATCAAGGTCAGGTATTGAACCCTTCGAGCTGGATACGTTGA
NMB1333 Aiizino acid sequence
30 MP.YKPLLLALMLVFSTPAVAAHDAAHNRSAEVKKQTKNKKEQPEAAEGKKEKGKNGAVKD
KKTGGKEAAKEGKESKKTAKNRKEAEKEATSRQSARKGREGDKKSKAEHKKAHGKPVSGS
KEKNAKTQPENKQGKKEAKGQGNPRKGGKAEKDTVSANKKVRSDKNGKAVKQDKKYREEK
NAKTDSDELKAAVAAATNDVENKKALLKQSEGMLLHVSNSLKQLQEERIRQERIRQARGN
LASVNRKQREAWDKFQKLNTELNRLKTEVAATKAQISRFVSGNYKNSQPNAVALFLKNAE
35 PGQKNRFLRYTRYVNASNREVVKDLEKQQKALAVQEQKINNELARLKKIQANVQSLLKKQ
GVTDAAEQTESRRQNAKIAKDARKLLEQKGNEQQLNKLLSNLEKKKAEHRIQDAEAKRKL
AEARLAAAEKARKEAAQQKAEARRAEMSNLTAEDRNIQAPSVMGIGSADGFSRMQGRLKK
PVDGVPTGLFGQNRSGGDIWKGVFYSTAPATVESIAPGTVSYADELDGYGKVVVVDHGEN
YISIYAGLSEISVGKGYMVAAGSKIGSSGSLPDGEEGLYLQIRYQGQVLNPSSWIR
NMB0377 Nucleic acid sequence
ATGGCGTTTTGCACCAGTTTGGGAGTGATGATGGAAACACAGCTTTACATCGGCATCATG
TCGGGAACCAGCATGGACGGGGCGGATGCCGTACTGATACGGATGGACGGCGGCAAATGG
CTGGGCGCGGAAGGGCACGCCTTTACCCCCTACCCCGGCAGGTTACGCCGCCAATTGCTG
GATTTGCAGGACACAGGCGCAGACGAACTGCACCGCAGCAGGATTTTGTCGCAAGAACTC
AGCCGCCTATATGCGCAAACCGCCGCCGAACTGCTGTGCAGTCAAAACCTCGCACCGTCC
GACATTACCGCCCTCGGCTGCCACGGGCAAACCGTCCGACACGCGCCGGAACACGGTTAC
AGCATACAGCTTGCCGATTTGCCGCTGCTGGCGGAACGGACGCGGATTTTTACCGTCGGC
GACTTCCGCAGCCGCGACCTTGCGGCCGGCGGACAAGGCGCGCCACTCGTCCCCGCCTTT
CACGAAGCCCTGTTCCGCGACAACAGGGAAACACGCGCGGTACTGAACATCGGCGGGATT
GCCAACATCAGCGTACTCCCCCCCGACGCACCCGCCTTCGGCTTCGACACAGGGCCGGGC
AATATGCTGATGGACGCGTGGACGCAGGCACACTGGCAGCTTCCTTACGACAAAAACGGT
GCAAAGGCGGCACAAGGCAACATATTGCCGCAACTGCTCGACAGGCTGCTCGrCCACCCG
TATTTCGCACAACCCCACCCTAAAAGCACGGGGCGCGAACTGTTTGCCCTAAATTGGCTC
GAAACCTACCTTGACGGCGGCGAAAACCGATACGACGTATTGCGGACGCTTTCCCGTTTT
ACCGCGCAAACCGTTTGCGACGCCGTCTCACACGCAGCGGCAGATGCCCGTCAAATGTAC
ATTTGCGGCGGCGGCATCCGCAATCCTGTTTTAATGGCGGATTTGGCAGAATGTTTCGGC
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
36
ACACGCGTTTCCCTGCACAGCACCGCCGACCTG_AACCTCGATCCGCAATGGGTGGAAGCC
GCCGCATTTGCGTGGTTGGCGGCGTGTTGGATT_AATCGCATTCCCGGTAGTCCGCACAAA
GCAACCGGCGCATCCAAACCGTGTATTCTGGGCGCGGGATATTATTAT't'GP'.
NMB0377 Amino acid sequence
MAFCTSLGVMMETQLYIGIMSGTSMDGADAVLIRMDGGI,a7LGAEGHAFTPYPGRLRRQLL
DLQDTGADELHRSRILSQELSRLYATAAELLCSQNLAPSDITALGCHGQTVRHAPEHGY
SIQLADLPLLAERTRIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDNRETRAVLNIGGI
ANISVLPPDAPAFGFDTGPGNMLMDAWTQAHWQLPYDKNGA.KAAQGNILPQLLDRLLAHP
YFAQPHPKSTGRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHAAA.DARQMY
ICGGGIRNPVLMADLAECFGTRVSLHSTADLNLDPQWVEAAAFAWLAACWINRIPGSPHK
ATGASKPCILGAGYYY
NMB0264 Nucleic acid sequence
ATGTTGAACAAAATATTTTCCTGGTTCGAGTCCCGAATCGACCCTTATCCCGAAGCCGCC
CCGAAAACGCCAGAAAAAGGCTTGTGGCGGTTTGTCTGGAGCAGCATGGCCGGCGTGCGG
AAATGGATAGCCGCCCTGGCTGCGCTGACCGCCGGCATCGGCATTATGGAAGCCCTGGTT
TTTCAATTTATGGGCAAAATCGTGGAGTGGCTCGGCAAATACGCGCCCGCCGAACTGTTT
GCCGAAAAAAGTTGGGAACTGGCGGCAATGGCGGCGATGATGGTATTTTCGGTTGCGTGG
GCGTTTGCCGCGTCCAACGTGCGCCTGCAAACCCTTCAGGGCGTGTTCCCCATGCGCCTG
CGCTGGAACTTCCACCGCCTGATGCTGAACCAAAGCCTCGGTTTTTATCAGGACGAATTT
GCCGGACGCGTGTCCGCCAAAGTCATGCAGACCGCGCTGGCGTTGCGCGACGCGGTGATG
ACGGTTGCCGATATGGTCGTTTATGTGTCGGTGTATTTCATTACCTCCGGCGTGATTCTC
GCCTCGCTCGACTCATGGCTGCTGCTGCCCTTTATCGGCTGGATTGTCGGTTTCGCTTCG
GTGATGCGCCTGCTGATTCCCAAATTGGGGCAAACCGCCGCATGGCAGGCGGATGCCCGC
TCGCTGATGACCGGCCGCATTACCGATGCCTATTCCAATATCGCCACCGTCAAACTCTTC
TCCCACGGCGCGCGTGAAGCCGCCTATGCCAAGCAGTCGATGGAAGAATTTATGGTTACG
GTGCGCGCCCAAATGCGGCTGGCGACGCTGCTGCATTCGTGCAGCTTCATCGTCAACACC
TCCCTGACCCTCTCCACCGCCGCACTGGGCATCTGGCTCTGGCACAACGGGCAGGTCGGC
GTGGGCGCGGTTGCTACAGCCACCGCCATGGCGTTGCGCGTCAACGGTTTGTCGCAATAC
ATTATGTGGGAATCCGCGCGGCTGTTTGAAAACATCGGCACCGTCGGCGACGGCATGGCA
ACCCTGTCC_AAACCGCACACCATCCTCGACAAGCCCCGGGCACTGCCGCTGAACGTGCCG
CAAGGCGCAATCAAATTTGAACACGTCGATTTCTCCTACGAAGCGGGCAAACCGCTGCTC
AACGGCTTCAA.CCTCACCATCCGCCCGGGCGAAAAAGTCGGCTTGATCGGACGCAGCGGC
GCGGGCAAATCCACCATCGTCAACCTGCTTTTGCGCTTCTACGAACCGCAAAGCGGCACG
GTTTCGATCGACGGGCAGGACATAAGCGGCGTTACCCAAGAATCTTTACGCGCCCAAATC
GGTTTGGTCACGCAAGATACCTCGCTGCTGCACCGTTCCGTGCGCGACAACATTATTTAC
GGCCGCCCCGACGCGACCGATGCCGAAATGGTTTCTGCCGCCGAACGCGCCGAAGCCGCC
GGCT T CAT CCCCGACCTT TCCGATGCCAAAGGGCGGCGCGGC TAC GACGCACAC GT CGGC
GAACGCGGCGTGAAACTCTCCGGCGGGCAACGCCAGCGCATCGCCATCGCCCGCGTGATG
CTCAAAGACGCACCGATTCTTCTTTTGGACGAAGCCACCAGCGCGCTCGATTCCGAAGTC
GAAGCCGCCATCCAA.GAA.AGCCTCGACAAA.ATGATGGACGGCAAAACCGTCATCGCCATC
GCCCACCGCCTCTCCACCATCGCCGCAATGGACAGGCTCGTCGTCCTCGACAAAGGCCGC
ATCATCGAAGAAGGCACACACGCCGAACTCCTCGAAAAACGCGGGCTTTACGCCAAACTC
45= TGGGCGCACCAGAGCGGCGGCTTCCTCAACGAACACGTCGAGTGGCAGCACGACTGA
NMB0264 Amino acid sequence
MLNKIFSWFESRIDPYPEAAPKTPEKGLWRFVWSSMAGVRKWIAALAALTAGIGIMEALV
FQFMGKIVEWLGKYAPAELFAEKSWELAAMAAMMVFSVAWAFAASNVRLQTLQGVFPMRL
RWNFHRLMLNQSLGFYQDEFAGRVSAKVMQTALALRDAVMTVADMVVYVSVYFITSGVIL
ASLDSWLLLPFIGWIVGFASVMRLLIPKLGQTAAWQADARSLMTGP.ITDAYSNIATVKLF
SHGAREAAYAKQSMEEFMVTVRAQMRLATLLHSCSFIVNTSLTLSTAALGIWLWHNGQVG
VGAVATATAMALRVNGLSQYIMWESARLFENIGTVGDGMATLSKPHTILDKPRALPLNVP
QGAIKFEHVDFSYEAGKPLLNGFNLTIRPGEKVGLIGRSGAGKSTIVNLLLRFYEPQSGT
VSIDGQDISGVTQESLRAQIGLVTQDTSLLHRSVRDNIIYGRPDATDAEMVSAAERAEAA
GFIPDLSDAKGRRGYDAHVGERGVKLSGGQRQRIAIARVMLKDAPILLLDEATSALDSEV
EAAIQESLDKMMDGKTVIAIAHRLSTIAAMDRLVVLDKGRIIEEGTHAELLEI<RGLYAKL
WAHQSGGFLNEHVEWQHD
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
37
NMB 103 6 Nucleic acid sequence
ATGACAGCACAAACCCTCTACGACAAACTTTGGAACAGCCACGTCGTCCGCGAAGAAGAA
GACGGCACCGTCCTGCTCTACATCGACCGCCATTTGGTGCACGAAGTTACCAGCCCTCAG
GCATTTGAAGGCTTGAAAATGGCGGGGCGCAAGCTr,TGGCGCATCGACAGCGTCGTCTCC
ACCGCCGACCACAACACCCCGACCGGCGATTGGGACAAAGGCATCCAAGACCCGATTTCC
AAGCTGCAAGTCGATACTTTGGACAAAAACATTAAAGAGTTTGGCGCACTCGCCTATTTT
CCGTTTATGGACAAAGGTCAGGGCACGTACACGTTATGGGCCCCGAACAAGGCGCGACC
CTGCCCGGTATGACCGTCGTCTGCGGCGACTCGCACACTTCCACCCACGGCGCATTCGGC
GCACTGGCGCACGGCATCGGCACTTCCGAAGTCGAGCACACCATGGCGACCCAATGTATT
ACCGCGAA.AA.AATCCAAATCCATGCTGA TTCCGTTGACGGCAAATTAAAAGCGGGCGTT
ACCGCCAAAGACGTGGCGCTCTACATCATCGGGCAAATCGGCACGGCAGGCGGTACAGGC
TACGCCATCGAGTTTGGCGGCGAAGCCATCCGCAGCCTTTCTATGGAAAGCCGCATGACT
TTATGCAA.TATGGCGATTGAGGCAGGCGCGCGCTCAGGCATGGTTGCCGTCGACCAAACC
ACCATCGACTACGTAAAAGATAAACCCTTCGCACCCGAAGGCGAAGCGTGGGACAAAGCC
GTCGAGTACTGGCGTACGCTGGTGTCTGACGAAGGTGCGGTATTCGACA.AAGAATACCGT
TTCAACGCCGAAGACATCGAACCGCAAGTCACTTGGGGTACCTCGCCTGAAATGGTTTTA
GACATCAGCAGCAAAGTGCCGAATCCTGCCGAAGAAACCGATCCGGTCAAACGCAGCGGT
ATGGAACGCGCCCTTGAATACATGGGCTTGGAAGCCGGTACGCCATTAAACGAAATCCCC
GTCGACATCGTATTCATCGGCTCTTGCACCAACAGCCGCATCGAAGACTTGCGCGAAGCC
GCCGCCATCGCCAAAGACCGC_AAAAAAGCCGCCAACGTACAGCGCGTGTTAATCGTCCCC
GGCTCCGGTTTGGTTAAAGAACAAGCCGAAAAAGAAGGCTTGGACAAA.ATTTTCATCGAA
GCCGGTTTTGAATGGCGCGAACCGGGCTGTTCGATGTGTCTCGCCATGAACGCCGACCGC
CTGACCCCGGGGCAACGCTGCGCCTCCACCTCCAACCGTAACTTTGAAGGCCGTCAAGGC
AACGGCGGACGTACCCACCTCGTCAGCCCCGCTATGGCAGCAGCCGCCGCCGTTACCGGC
CGCTTTACCGACATCCGCATGATGGCGTAA
NNIB1036 Amino acid sequence
MTAQTLYDKLWNSHVVREEEDGTVLLYIDRHLVHEVTSPQAFEGLKMAGRKLWRIDSVVS
TADHNTPTGDWDKGIQDPISKLQVDTLDKNIKEFGALAYFPFMDKGQGIVHVMGPEQGAT
LPGMTVVCGDSHTSTHGAFGALAHGIGTSEVEHTMATQCITAKKSKSMLISVDGKLKAGV
TAKDVALYIIGQIGTAGGTGYAIEFGGEAIRSLSMESRMTLCNMAIEAGARSGMVAVDQT
TIDYVKDKPFAPEGEAWDKAVEYWRTLVSDEGAVFDKEYRFNAEDIEPQVTWGTSPEMVL
DISSKVPNPAEETDPVKRSGMERALEYMGLEAGTPLNEIPVDIVFIGSCTNSRIEDLREA
AAIAKDRKKAANVQRVLIVPGSGLVKEQAEKEGLDKIFIEAGFEWREPGCSMCLAMNADR
LTPGQRCASTSNRNFEGRQGNGGRTHLVSPAMAAAAAVTGRFTDIRMMA
NMB 1176 Nucleic acid sequence
ATGAAAGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT
TTTTTCAAGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC
GGCTCGAAGGTCAAAA.ACAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC
AATTCCCTTCCCTATAA.AATCAAGGTTAAAGGTTTGAACCACCAACGCCGCCCGGCATCC
GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA
CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC
GACCGCCGCCAACTGGACAGGCTGAAAA.AAGGAGACTGGTAA
NMB1176 Amino acid sequence
MKDKHDSSAMRLDKWLWAARFFKTRSLAQKHIELGRVQVNGSKVKNSKTIDIGDIIDLTL
NSLPYKIKVKGLNHQRRPASEARLLYEEDAKTATLREERKQLDQFSRITSAYPDGRPTKR
DRRQLDRLKKGDW
NMB 13 59 Nucleic acid sequence
ATGAACCACACCGTTACCCTGCCCGACCAAACCACCTTTGCCGCCAACGACGGCGAAACC
GTTTTGACCGCTGCCGCCCGTCAAAACCTCAACCTGCCCCATTCCTGCAAAAGCGGTGTC
TGCGGACAATGCAA.AGCCGAACTGGTCAGCGGCGATATTCAAATGGGCGGACACTCGGAA
CAGGCTTTATCCGAAGCAGAAAAAGCGCAAGGCAAGATTTTGATGTGCTGCACCACTGCG
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
3P)
CAAAGCGATATCAACATCAACATCCCCGGCTACAAAGCCGATGCCCTACCCGTCCGCACC
CTGCCCGCACGCATCGAAAGTATTATTTTCAAACACGATGTCGCCCTCCTG_AAACTTGCC
CTGCCC_AAAGCCCCGCCGTTTGCCTTCTACGCCGGGCAATACATTGATTTACTGCTGCCG
GGCAACGTCAGCCGCAGCTACTCCATCGCCAATTTACCCGACCAAGAAGGCATTTTGGAA
CTGCACATCCGCAGGCACGAAAACGGTGTCTGCTCGGAAATGATTTTCGGCAGCG_AACCC
AAAGTCAAAGAAA.AAGGCATCGTCCGCGTTAAAGGCCCGCTCGGTTCGTTTACCTTGCAG
GAAGACAGCGGCAAACCCGTCATCCTGCTGGCAACCGGCACAGGCTACGCCCCCATCCGC
AGCATCCTGCTCGACCTTATCCGCCAAGGCAGC_AACCGCGCCGTCCATTTCTACTGGGGC
GCGCGTCATCAGGATGATTTGTATGCCCTCGAAGAAGCACAAGGGTTGGCATGCCGTCTG
AAAAACGCCTGCTTCACCCCCGTATTGTCCCGCCCCGGAGAGGGCTGGCAGGGAAGAAAT
GGTCACGTACAAGACATCGCGGCACAAGACCACCCCGACCTGTCGGAATACGAAGTATTT
GCCTGCGGTTCTCCGGCCATGACCG.AACAAACAAAGAATCTGTTTGTGCAACAGCATAAG
CTGCCGGAAAACTTGTTTTTCTCCGACGCATTCACGCCGTCCGCATCATAA
NMB 1359 Amino acid sequence
MNHTVTLPDQTTFAANDGETVLTAAARQNLNLPHSCKSGVCGQCKAELVSGDIQMGGHSE
QALSEAEKAQGKILMCCTTAQSDININIPGYKADALPVRTLPARIESIIFKHDVALLKLA
LPKAPPFAFYAGQYIDLLLPGNVSRSYSIANLPDQEGILELHIRRHENGVCSEMIFGSEP
KVKEKGIVRVKGPLGSFTLQEDSGKPVILLATGTGYAPIRSILLDLIRQGSNRAVHFYWG
ARHQDDLYALEEAQGLACRLKNACFTPVLSRPGEGWQGRNGHVQDIAAQDHPDLSEYEVF
ACGSPAMTEQTKNLFVQQHKLPENLFFSDAFTPSAS
NMB 113 8 Nucleic acid sequence
ATGAAAGACAA.GCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT
TTTTTCAAGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC
GGCTCGAAGGTCAAAAACAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC
AATTCCCTTCCCTATAAAATCAAGGTTAAAGGTTTGAACCACCAACGCCGCCCGGCATCC
GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA
CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC
GACCGCCGCCAACTGGACAGGCTGAAAA.AAGGAGACTGGTAA
NMB1138 Amino acid sequence
MKDKHDSSAMRLDKWLWAARFFKTRSLAQKHIELGRVQVNGSKVKNSKTIDIGDIIDLTL
NSLPYKIKVKGLNHQRRPASEARLLYEEDAKTATLREERKQLDQFSRITSAYPDGRPTKR
DRRQLDRLKKGDW
Schedule of SEQ ID Nos
SEQ ID No Sequence
1 NMB0341 DNA
2 NMB0341 Protein
3 NMB1583 DNA
4 NMB1583 Protein
5 NMB1345 DNA
6 NMB1345 Protein
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
39
7 NMB0738 DNA
8 NMB 073 8 Protein
9 NMB0792 DNA
NMB0792 Protein
11 NMB0279 DNA
12 NMB0279 Protein
13 NMB2050 DNA
14 NMB2050 Protein
NMB1335 DNA
16 NMB 13 3 5 Protein
17 NMB2035 DNA
18 NMB203 5 Protein
19 NMB 13 51 DNA
NMB1351 Protein
21 NMB 1574 DNA
22 NMB 1574 Protein
23 NMB 1298 DNA
24 NMB1298 Protein
NMB1856 DNA
26 NMB1856 Protein
27 NMB0119 DNA
28 NMB0119 Protein
29 NMB 1705 DNA
NMB1705 Protein
31 NMB2065 DNA
32 NMB2065 Protein
33 NMB0339 DNA
34 NMB0339 Protein
NMB0401 DNA
36 NMB0401 Protein
37 NMB1467 DNA
38 NMB 1467 Protein
CA 02592156 2007-06-22
WO 2006/067518 PCT/GB2005/005113
39 NMB2056 DNA
40 NMB2056 Protein
41 NMB0808 DNA
42 NMB0808 Protein
43 NMB0774 DNA
44 NMB0774 Protein
NMA0078 DNA
46 NMA0078 Protein
47 NMB0337 DNA
48 NMB0337 Protein
49 NMB 0191 DNA
NMB0191 Protein
51 NMB 1710 DNA
52 NMB 1710 Protein
53 NMB 0062 DNA
54 NMB0062 Protein
NMB1333 DNA
56 NMB1333 Protein
57 NMB0377 DNA
58 NMB0377 Protein
59 NMB0264 DNA
NMB0264 Protein
61 NMB 103 6 DNA
62 NMB 1036 Protein
63 NMB 1176 DNA
64 NMB 1176 Protein
NMB1359 DNA
66 NMB 1359 Protein
67 NMB 113 8 DNA
68 NMB 113 8 Protein
