JP2002524035A - Chlamydia antigens and corresponding DNA fragments and uses thereof - Google Patents

Abstract

  (57) [Summary] In summary of the present disclosure, the present invention relates to a nucleotide sequence encoding an omp protein of a Chlamydia pneumoniae strain and a promoter that results in expression of the omp gene in a host against diseases caused by infection of the Chlamydia strain, particularly Chlamydia pneumoniae. The present invention provides a method for immunizing a host (including human) with a nucleic acid (including DNA) using a vector containing the same. Modifications may be within the scope of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

(Related US Application) This patent application is filed with US Provisional Patent Application No. 60 / 094,203 (July 2, 1998).
And US Provisional Patent Application No. 60 / 122,045 (March 1, 1999).
Priority application).

FIELD OF THE INVENTION [0002] The present invention relates to Chlamydia antigens and corresponding DNA molecules, which can be used in methods for preventing and treating diseases caused by Chlamydia infection in mammals (eg, humans). .

BACKGROUND OF THE INVENTION [0003] Chlamydia is a prokaryote. They show morphological and structural similarities to Gram-negative bacteria, including the three-layer outer membrane. This trilayer outer membrane contains lipopolysaccharide and some membrane proteins. Chlamydia are distinguished from other bacteria by their morphology and by their unique developmental cycle. They are obligate intracellular parasites with a unique biphasic life cycle consisting of an extracellular stage that is metabolically inert but infectious and an intracellular stage that is replicable but non-infectious . This life cycle replication step occurs in membrane-bound inclusions that sequester bacteria from the cytoplasm of infected host cells.

[0004] Chlamydia have long been considered to be viruses because they replicate only in small and susceptible cells. However, they have many of the following features in common with other bacteria: (1) they contain both DNA and RNA, (2) they divide by dichotomy, (3) their cells Envelopes are similar to the cell envelopes of other Gram-negative bacteria. (4)
It contains ribosomes similar to the ribosomes of other bacteria, and (5) they are sensitive to various antibiotics. Chlamydia can be seen under a light microscope, and its genome is about 1 / the size of the Escherichia coli genome.
3.

[0005] Many different strains of Chlamydia have been isolated from birds, humans, and other mammals, and these strains are distinguished based on host range, virulence, pathogenicity, and antigenic composition. obtain. Although the DNA within each species is highly homologous, surprisingly, there is little homology between species. This suggests an evolutionary segregation that has been established over time.

C. Trachomatis has a high degree of host specificity, and its specificity is almost completely restricted to humans; Trachomatis causes a wide range of severe eye and urogenital infections. Symmetrically, C.I. ps
Ittaci strains, although rare in humans, are found in a wide range of birds and also in wildlife, livestock, and laboratory animals. Here, they replicate in cells of many organs.

C. pneumoniae is a common human pathogen, originally from C. aureus. p
Although described as a TWAR strain of sitaci, it was later recognized as a new species. C. pneumoniae are antigenically, genetically, and morphologically similar to other Chlamydia species (C. trachomatis, C. pecoru).
m and C.I. psittaci). C. trachomati
s or C.I. Less than 10% homology of DNA sequence with any of the psittaci is seen and so far appears to consist only of a single strain TWAR.

C. pneumoniae is a common cause of community-acquired pneumonia,
tococcus pneumoniae and Mycoplasma pne
less frequently than umoniae alone. Grayston et al. Infect
. Dis. 168: 1231 (1995); Campos et al., Invest. O
phthalmol. Vis. Sci. 36: 1477 (1995), each of which is incorporated herein by reference. It can also cause upper respiratory tract symptoms and diseases, including bronchitis and sinusitis. For example, Grayston et al.
J. Infect. Dis. 168: 1231 (1995); Campos et al.,
Invest. Ophthalmol. Vis. Sci. 36: 1477 (19
95); Grayston et al. Infect. Dis. 161: 618 (1
990); Marrie, Clin. Infect. Dis. 18: 501 (1
993). The majority (> 60%) of the adult population is C.I. pneumon
iae (Wang et al., Chlamydial Infec).
Tions, Cambridge University Press, Cam
bridge, p. 329 (1986)). This indicates that past infections were unrecognized or asymptomatic.

C. Pneumoniae infection is usually indicated as acute respiratory disease (ie, cough, pharyngitis, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, cough persists for 2-6 weeks and recovery is slow. In about 10% of these cases, bronchitis or pneumonia occurs after upper respiratory tract infection. Further, C.I. Subsequent co-infection with pneumococci during the pneumoniae epidemic was observed in about half of these pneumonia patients, especially frail or elderly patients. As described above, C.I. There is increasing evidence that pneumoniae infection is also associated with diseases other than respiratory infections.

[0010] The reservoir for infection of the organism is:
Probably a person. C. In contrast to psittaci infection, there is no reservoir of known bird or animal infection. Transmission has not been revealed. This can result from direct contact with secretions, from formses, or from airborne transmission. There is a long incubation period, which can last for several months. Based on trend analysis, C
. pneumoniae appears to propagate slowly throughout the population (the interval between cases is on average 30 days). This is because infected patients are inefficient transmitters of the organism. C. pneu
Susceptibility to moniae is universal. Reinfection occurs in adults, even after the initial infection as a child. C. pneumoniae is notable, 2
It appears to be a worldwide epidemic disease with overlapping incidence (epidemic) intervals lasting ~ 3 years. C. trachomatis infection is C. trachomatis infection. pneu
No cross immunity with Moniae is given. Infection is easily treated with oral antibiotics (tetracycline or erythromycin (2 g / day for at least 10-14 days)). Azithromycin, a recently developed drug, is very effective as a single dose treatment for chlamydial infections.

In most cases, C.I. pneumoniae infection is mild and without complications. And up to 90% of infections are subacute or unrecognized. Infections up to 5 years of age are thought to be rare among children in developed countries, but recent studies have shown that many children in this age group, despite being serum negative, It shows evidence of infection and is reported to predict a prevalence of 17-19% at the age of 2-4 years. Normann et al., Acta. Paedia
trica, 87: 23-27 (1998). In developing countries, C.I. pneumoniae antibody serum retention (seropreba
length) and C.I. It is suspected that pneumoniae may be a significant cause of acute lower respiratory illness and infant and child mortality in the tropics of the world.

[0012] From studies of serum prevalence and studies of local epidemics, the first C. pneumoni
ae infection usually occurs between the ages of 5 and 20 years. For example, in the United States, 30
Pneumonia in 2,000 children has C. pneumoniae. Infection can occur intensively among a group of children or adolescents (eg, school children or recruiters).

C. Pneumoniae is responsible for 10-25% of community lower respiratory tract infections (reported from Sweden, Italy, Finland and the United States). During the epidemic, C.I. pneumoniae infection can cause 50-60% of pneumonia. These periods are also defined by S.M. More manifestations of co-infection with pneumoniae have been reported.

Reinfection among adults is common; clinical presentation appears to be milder. Based on population serum retention studies, there is a tendency for detection to increase with age,
This is especially noticeable among people. According to some researchers, persistent asymptomatic C
. pneumoniae infection was presumed to be common.

In middle-aged or elderly adults, C.I. Pneumoniae infection can progress to chronic bronchitis and sinusitis. Studies in the United States have shown that C.I. The incidence of pneumonia caused by Pneumoniae is 1
It was found that the incidence of the disease increased three-fold in individuals over the age of 60, one in 1,000 cases per year. Except for patients who are already ill, C. No hospitalization for pneumoniae infection.

Of considerable importance is that atherosclerosis and C. pneumoniae infection. C. There are several epidemiological studies showing a correlation between pneumoniae and heart attack, coronary and carotid artery disease and previous infections. S
aikku et al., Lancet 2: 983 (1988); Thom et al., JAMA.
268: 68 (1992); Linnanmaki et al., Circulatio.
n 87: 1030 (1993); Saikku et al., Annals Int. M
ed. 116: 273 (1992); Melnick et al., Am. J. Med. 9
5: 499 (1993). In addition, the organism was detected in coronary, cervical, and peripheral arterial and aortic atheromas and fatty streaks. Sh
or et al., South Africa Med. J. 82: 158 (1992)
Kuo et al. Infect. Dis. 167: 841 (1993); Kuo.
Et al., Arteriosclerosis and Thrombosis 13
: 1500 (1993); Campbell et al. Infect. Dis. 1
72: 585 (1995); Chiu et al., Circulation 96:21.
44-2148 (1997). Live C. pneumoniae was recovered from the coronary and carotid arteries. Ramirez et al., Annals In.
t. Med. 125: 979 (1996); Jackson et al., Abst. K1
21, page 272, 36th ICAAC, New Orleans (1996)
). Further, C.I. pneumoniae has been shown to be capable of inducing atherosclerotic changes in a rabbit model. Fong et al., (1997) Journa.
See l of Clinical Microbiology 35:48. In summary, these results indicate that C.I. Although pneumoniae can cause atherosclerosis in humans, it is shown that the epidemiological significance of chlamydial atherosclerosis is likely to remain to be demonstrated.

[0017] Much of the recent work has also An association between pneumoniae infection and asthma was shown. Infection has been linked to wheezing, asthmatic bronchitis, adult-onset asthma and acute exacerbations of asthma in adults, and small studies show that long-term antibiotic treatment increases the severity of the disease in some individuals. It was shown to be effective in reducing. Hahn et al., Ann Allergy Asthma
Immunol. 80: 45-49 (1998); Hahn et al., Epidemi.
ol Infect. 117: 513-517 (1996); Bjornsso.
n et al., Scan J Infect Dis. 28: 63-69 (1996)
Hahn, J .; Fam. Pract. 41: 345-351 (1995); A
llegra et al., Eur. Respir. J. 7: 2165-2168 (199
4); Hahn et al., JAMA 266: 225-230 (1991).

In view of these results, C.I. Protective vaccines against diseases caused by pneumoniae infection are very important. Human C. pneumoni
There is still no effective vaccine against ae infection. Nevertheless, C.I. t
Rachomatis and C.I. Psittaci's research has shown that this is an attainable goal. For example, C.I. Mice recovered from pulmonary infection with trachomatis are protected from infertility induced by subsequent vaginal challenge. Pal et al., Infection and Immunity 64: 5.
341 (1996). Similarly, inactivated C.I. Sheep immunized with psittaci were protected from subsequent chlamydia-induced abortion and stillbirth. Jones et al.
Vaccine 13: 715 (1995). Protection against Chlamydia infection is T
It is associated with the h1 immune response, particularly the generation of INFγ-producing CD4 + T cells. Igi
etsemes et al., Immunology 5: 317 (1993). CD4 +
Adoptive transfer of cell lines or CD4 + clones to nude or SCID mice confers protection from challenge or eliminates chronic disease (Igietseme et al., Regional Immunology 5).
: 317 (1993); Magee et al., Regional Immunolog.
y 5: 305 (1993)) and depletion of CD4 + T cells in vivo exacerbated the disease after challenge (Landers et al., Infection & I).
mmunity 59: 3774 (1991); Magee et al., Infecti.
on & Immunity 63: 516 (1995)). However, the protective effect can also be exerted by the presence of a sufficiently high neutralizing antibody on the mucosal surface. Co
Tter et al., Infection and Immunity 63: 4704.
(1995).

Species C. The extent of antigenic variation within Pneumoniae has not been well characterized. C. Trachomatis subgroups are defined based on antigenic variations in the major outer membrane protein (MOMP), but are published in the context of C. trachomatis. The pneumoniae MOMP gene sequence shows no variation among several different isolates of this organism. Campbell et al., Infection and Immunity 58:93 (1990);
McCafferty et al., Infection and Immunity 6
3: 2387-9 (1995); Knudsen et al., Third Meetin.
go of European Society for Chlamydia
See Research, Vienna (1996). Other Chlamydia M
Regions of the protein known to be conserved in OMPs are described in pn
eumoniae. Campbell et al., Infect
ion and Immunity 58:93 (1990); McCaffe.
rty et al., Infection and Immunity 63: 2387-.
9 (1995). One study involved MOMPs larger than normal molecular weights.
Having C.I. One strain of Pneumoniae has been described, but its gene has not been sequenced. Grayston et al. Infect. Dis. 168:
1231 (1995). The partial sequence of outer membrane protein 2 from nine different isolates was also found to be unchanged. Ramirez et al., Annals In.
t. Med. 125: 979 (1996). The HSP60 and HSP70 genes show little change from other Chlamydia species, as expected. The gene encoding the 76 kDa antigen has a single C. pneumoniae strain. This gene has no significant similarity to other known chlamydia genes. Marrie, Clin. Infect. Dis. 18: 5
01 (1993).

C. Many antigens recognized by immune sera against Pneumoniae are
Conserved throughout all Chlamydia. However, 98 kDa, 76 kDa
And the 54 kDa protein were obtained from C. pneumoniae may be specific. Campos et al., Invest. Ophthalmol. Vis. Sci. 3
6: 1477 (1995); Marrie, Clin. Infect. Dis.
18: 501 (1993); Wiedmann-Al-Ahmad et al., Clin.
. Diagn. Lab. Immunol. 4: 700-704 (1997). Immunoblotting of the isolates with serum from the patient does show variations in the blotting pattern between the isolates. This is because C.I. pneumo
Indicates that niae serotype may be present. Grayston et al. Infec
t. Dis. 168: 1231 (1995); Ramirez et al., Annals.
Int. Med. 125: 979 (1996). However, this result can be confused by the infection status of the patient. This is because the immunoblot profile of patient sera changes with time after infection. Evaluation of the number and relative frequency of any serotype, as well as the defining antigen, is not yet possible.

Therefore, there is still a need for effective compositions for preventing, treating, and diagnosing Chlamydia infection.

SUMMARY OF THE INVENTION In one aspect, the present invention provides an outer membrane protein (omp) or CPN10
0314 SEQ ID NO: 1-the full length sequence and SEQ ID NO: 3-the coding sequence for the mature polypeptide), provided a purified and isolated DNA molecule encoding the Chlamydia polypeptide designated. The terms omp and CPN10
0314 is used interchangeably in this application. The encoded polypeptides can be used in methods for preventing, treating, and diagnosing Chlamydia infection, and include polypeptides having the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4 Is mentioned. One of skill in the art will understand that the present invention also includes DNA molecules that encode such variants, variants, and derivatives of such polypeptides. This results from the addition, deletion or substitution of non-essential amino acids described herein. The present invention also includes RNA molecules corresponding to the DNA molecules of the present invention.

[0023] In addition to DNA and RNA molecules, the invention includes the corresponding polypeptides and monospecific antibodies that specifically bind to such polypeptides.

The present invention has a wide range of applications and includes expression cassettes, vectors, and cells transformed or transfected with a polynucleotide of the present invention. Accordingly, the invention provides: (i) a method for producing a polypeptide of the invention in a recombinant host system, and related expression cassettes, vectors, and transformed or transfected. Cells; (ii) live vaccine vectors, such as, for example, live viral or bacterial live vaccine vectors, which comprise a poxvirus, alphavirus, Salmonella typhimurium, or Vibr comprising a polynucleotide of the present invention.
io cholerae vectors, such vaccine vectors are useful for preventing and treating Chlamydia infection, for example, in combination with diluents or carriers and associated pharmaceutical compositions, and associated therapeutic and / or prophylactic methods. (Iii) an RNA or DNA molecule of the present invention (
Administered in naked form, or formulated with a delivery vehicle, polypeptide or combination of polypeptides of the invention, or a monospecific antibody, and related pharmaceutical compositions). A method of treatment and / or prevention; (iv) a method of diagnosing the presence of Chlamydia in a biological sample, which may include the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention. And (v) a method for purifying the polypeptide of the present invention by antibody-based affinity chromatography.

(Detailed Description of the Invention) In the pneumoniae genome, an open reading frame (ORF) encoding a Chlamydia polypeptide was identified. These polypeptides include polypeptides that are found invariably in the bacterial membrane structure (polypeptides that are present near the outside of the bacterial membrane), and polypeptides that are found invariably in the inclusion body membrane structure (the inclusion body membrane). And polypeptides released into the cytoplasm of infected cells. These polypeptides have the C
hlamydia infection can be used in vaccination methods to prevent and treat.

According to a first aspect of the present invention there is provided an isolated polynucleotide encoding a precursor and mature form of a Chlamydia polypeptide.

[0027] An isolated polynucleotide of the invention encodes a polypeptide having an amino acid sequence that is homologous to a Chlamydia amino acid sequence. This Chlamy
The dia amino acid sequence is selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 2 or 4.

The term “isolated polynucleotide” is defined as a polynucleotide that has been removed from the environment in which it naturally occurs. For example, a naturally occurring DNA molecule that is present in the bacterial genome is not isolated, but the same molecule that has been separated from the rest of the bacterial genome, for example, as a result of a cloning event (amplification), Has been isolated. Typically, an isolated DNA molecule will not contain a DNA region immediately adjacent (eg, a coding region) at the 5 ′ and 3 ′ ends in a naturally occurring genome. Such an isolated polynucleotide may be part of a vector or composition, and still be isolated in that such a vector or composition is not part of its natural environment.

The polynucleotide of the present invention may be in the form of RNA or DNA (eg, cDNA, genomic DNA, or synthetic DNA) or a modification or combination thereof. The DNA can be double-stranded or single-stranded, and if single-stranded,
It can be the coding strand or the non-coding strand (antisense). The sequences encoding the polypeptides of the invention shown in SEQ ID NO: 2 and SEQ ID NO: 4 are: (a) the coding sequence shown in SEQ ID NO: 1 or 3; (b) the ribonucleotide sequence induced by transcription of (a) Or (c) a different coding sequence; this latter encodes, as a result of the degeneracy or degeneracy of the genetic code, the same polypeptide as the DNA molecule (the nucleotide sequence of which is set forth in SEQ ID NOS: 1 and 3).

By “homologous amino acid sequence”, only one or more conservative amino acid substitutions, or one or more non-conservative amino acid substitutions, placed in a position that does not destroy the specific antigenicity of the polypeptide An amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 2 or 4 only by deletion, addition, or the like.

Preferably, such a sequence is at least 75%, more preferably 80%, and most preferably 9%, relative to the amino acid sequence set forth in SEQ ID NO: 2 or 4.
0% identical.

The homologous amino acid sequences are identical to the amino acid sequences shown in SEQ ID NOs: 2 and 4,
Or sequences that are substantially identical. By "substantially identical amino acid sequence" is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to the reference amino acid sequence, and preferably
A sequence that differs from the reference sequence by most, if any, conservative amino acid substitutions is meant.

[0033] Conservative amino acid substitutions typically involve substitutions within amino acids of the same class. These classes include, for example: (a) amino acids with uncharged polar side chains (eg, asparagine, glutamine, serine,
(B) amino acids having a basic side chain (e.g., threonine, and tyrosine);
(C) amino acids with acidic side chains (lysine, arginine, and histidine);
And (d) amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine).

Homology is typically determined by sequence analysis software (eg, Sequence).
Analysis Software Package of the Gen
etics Computer Group, University of W
isconsin Biotechnology Center, 1710 U
measured by the University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the greatest degree of homology (ie, identity). For this purpose, it may be necessary to introduce gaps in the sequence. Once the optimal sequence has been set, the degree of homology (ie,
Identity) is established by recording all the positions where the amino acids of both sequences are identical, relative to the total number of positions.

Alternatively, homology is described, for example, in Needleman et al. Mol. Biol
. 48: 443 (1970) and A
Aligning candidate and reference sequences using an alignment tool such as light Program (a commercially available software package manufactured by DNAstar, Inc.), the teachings of which are incorporated herein by reference. Can be determined by After the initial alignment is made, it can be refined by comparison to multiple sequence alignments of a family of related proteins. Once the alignment between the candidate sequence and the reference sequence is made and corrected, a percent homology score is calculated. The individual amino acids of each sequence are sequentially compared according to their similarity to each other.

Similarity factors include similar sizes, shapes, and charges. One particularly preferred method of determining amino acid similarity is described in Dayhoff et al., 5 Atl.
as of Protein Sequence And Structure
345-352 (1978 and augmented), which is a PAM250 matrix as described herein by reference. First, a similarity score is calculated as the sum of the amino acid similarity scores of the aligned pairs. Insertions and deletions are ignored for purposes of percent homology and percent identity.
Therefore, gap penalties are not used in this calculation. The raw score is then normalized by dividing it by the geometric mean of the scores of the candidate compound and the reference sequence. This geometric mean is the square root of the product of these scores.
This normalized raw score is the percent homology.

Preferably, the homologous sequence is (i) the coding sequence of SEQ ID NO: 1, or (ii)
A sequence that is at least 45%, more preferably 60%, and most preferably 85% identical to the coding sequence of SEQ ID NO: 3.

Polypeptides having sequences homologous to one of the sequences set forth in SEQ ID NOs: 2 and 4 include naturally occurring allelic variants, and variants and variants, or the sequences set forth in SEQ ID NOs: 2 or 4. Includes any other non-naturally occurring variants that are similar in antigenicity to the polypeptide having the sequence.

An allelic variant is an alternative form of a polypeptide that is characterized as having one or more amino acid substitutions, deletions, or additions that do not substantially alter the biological function of the polypeptide. . By "biological function" is meant the function of a polypeptide in a cell in which the polypeptide is naturally occurring, even if the function is not essential for the growth or survival of the cell. For example, the biological function of porin is to allow entry of a compound present in the extracellular medium into a cell. This biological function is distinguished from antigenic function. A polypeptide may have more than one biological function.

[0040] Allelic variants are very common in nature. For example, bacterial species (eg, C
. pneumoniae) are usually represented by different strains that differ from each other by insignificant allelic changes. In fact, polypeptides that fulfill the same biological function in different strains may have amino acid sequences that are not identical in each of the strains. Such allelic changes can be equally reflected at the polynucleotide level.

Support for the use of allelic variants of a polypeptide antigen can be found, for example, in Chl
amydial MOMP antigen. Even if interstrain antibody binding and neutralization of infectivity occur, the MOMP amino acid sequence varies from strain to strain,
When MOMP is used as an immunogen, it indicates that it is tolerant of amino acid changes.

A polynucleotide, eg, a DNA molecule, encoding an allelic variant can be readily recovered by polymerase chain reaction (PCR) amplification of bacterial genomic DNA extracted by conventional methods. This means that the 5 ′ end and 3
'Including the use of synthetic oligonucleotide primers that match upstream and downstream of the terminus. Suitable primers can be designed according to the nucleotide sequence information provided in SEQ ID NOs: 1 and 3. Typically, a primer may consist of 10 to 40, preferably 15 to 25 nucleotides. A sufficient ratio of C nucleotides and G nucleotides to ensure efficient hybridization (eg, at least 40%, preferably 50%, of the C nucleotides and G nucleotides
It may also be advantageous to select primers containing (amount of nucleotides).

Useful non-naturally occurring homologs can be designed using known methods to identify antigenic regions that appear to be tolerant of amino acid sequence changes and / or deletions. For example, antigen sequences from different species can be compared to identify conserved sequences.

[0044] Polypeptide derivatives encoded by the polynucleotides of the present invention include, for example, fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.

[0045] The polypeptide fragment of the present invention may have any of the sequences set forth in SEQ ID NOs: 1 and 3 to the extent that the fragment retains the desired substantial antigenicity (specific antigenicity) of the parent polypeptide. Derived from a polypeptide having a sequence homologous to Polypeptide derivatives can also be constructed by large internal deletions that remove a significant portion of the parent polypeptide, but retain the desired specific antigenicity. In general,
A polypeptide derivative should be at least about 12 amino acids in length to retain antigenicity. Advantageously, the polypeptide derivative may be at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.

Useful polypeptide derivatives (eg, polypeptide fragments) use computer-assisted analysis of amino acid sequences to identify sites in protein antigens that have potential as surface exposed antigenic regions. Can be designed. Hu
ghes et al., Infect. Immun. 60: 3497 (1992).

[0047] Polypeptide fragments and polypeptides with large internal deletions are otherwise covered by the parent polypeptide and identify epitopes that may be important, for example, to induce a protective T cell-dependent immune response. Can be used to indicate. Deletions can also remove highly dominant immunodominant regions between strains.

The use of fragments and variants of protein immunogens as vaccines and immunogens is an accepted practice in the field of immunology. Because
This is because all of this is required to elicit an immune response against the protein, which may be a small (eg, 8-10 amino acid) region of the protein. This has been done for a number of vaccines against pathogens other than Chlamydia. For example, a peptide containing 11 amino acids of mouse mammary tumor virus (Dion et al., Virology 179: 474-477 (1990)); a peptide containing 16 amino acids of Semliki Forest virus (Snijders et al., J. Am.
Gen. Virol. 72: 557-565 (1991)); and two overlapping peptides each containing 15 amino acids (Langev) of canine parvovirus.
Eld et al., Vaccine 12: 1473-1480 (1994))) indicate that short synthetic peptides corresponding to antigens exposed on the surface of pathogens are effective vaccine antigens against their respective pathogens. Have been.

[0049] Polypeptide fragments and polynucleotides encoding polypeptides with large internal deletions can be prepared using standard methods (eg, Ausubel et al., CURR).
ENT PROTOCOLS IN MOLECULAR BIOLOGY, J
ohn Wiley & Sons Inc. (1994); for example, by PCR, including inverse PCR, by restriction enzyme treatment of cloned DNA molecules, or by Kunkel et al. (Proc. Natl. Acad. Sci. USA 8
2: 448 (1985)); biological materials available at Stratagene).

[0050] Polypeptide derivatives can also be produced as fusion polypeptides to any protein polypeptide, including, for example, a polypeptide or polypeptide derivative of the invention fused at the N-terminus or C-terminus. For construction of DNA encoding the amino acid sequence corresponding to the hybrid fusion protein, CPN10020
A first DNA encoding an amino acid sequence corresponding to a portion of a two nucleotide sequence (SEQ ID NO: 1 or 3) is described, for example, in US Pat. No. 5,844,095, which is incorporated herein by reference. And ligated to the second DNA using the method described.
The product can then be easily obtained by translation of the gene fusion. Vectors for expressing the fusion polypeptide are commercially available, for example, pMal from New England Biolabs, where the fusion peptide is a maltose binding protein.
-C2 or pMal-p2 system, Pharmacia glutathione-S-transferase system, or His-Tag system available from Novagen. These and other expression systems provide a convenient means for further purification of the polypeptides and derivatives of the present invention.

Another specific example of a fusion polypeptide included in the invention is a polypeptide having adjuvant activity (eg, subunit B of either cholera toxin or E. coli heat labile toxin). And the polypeptide or polypeptide derivative of the present invention. Several possibilities can be used to achieve the fusion. First, the polypeptide of the invention can be fused to the N-terminus or, preferably, the C-terminus of the polypeptide having adjuvant activity. Second, a polypeptide fragment of the invention can be fused within the amino acid sequence of a polypeptide having adjuvant activity.

As mentioned above, the polynucleotides of the present invention may be in the precursor or mature form of C
encodes a hlamydia polypeptide. They may also encode hybrid precursors containing a heterologous signal peptide that can mature to the polypeptides of the invention. By "heterologous signal peptide" is meant a signal peptide that is not found in the naturally occurring precursor of a polypeptide of the invention.

A polynucleotide of the invention having a homologous coding sequence hybridizes, preferably under stringent conditions, to a polynucleotide having the sequence shown in SEQ ID NOs: 1 and 3. Hybridization procedures are described, for example, in Ausubel et al., CURRENT PROT, each of which is incorporated herein by reference.
OCOLS IN MOLECULAR BIOLOGY, John Wile
y & Sons Inc. (1994); Silhavy et al., EXPERIM.
ENTS WITH GENE FUSIONS, Cold Spring H
arbor Laboratory Press (1984); Davis et al.,
A MANUAL FOR GENETIC ENGINEERING: ADV
ANCED BACTERIAL GENETICS, Cold Spring
Harbor Laboratory Press (1980). An important parameter that can be considered for optimizing hybridization conditions is reflected in the equation above, which allows the calculation of the melting temperature, the critical value at which two complementary DNA strands separate from each other. Casey and Davidson,
Nucl. Acid Res. 4: 1539 (1977). The formula is as follows: Tm = 81.5 + 0.5 × (% G + C) +1.6 log (cation concentration) −0
. 6 × (% formamide) Under appropriate stringency conditions, hybridization temperature (Th)
Is lower than the calculated Tm, about 20-40 ° C, 20-25 ° C, or preferably 30-40 ° C. One skilled in the art will understand that optimal temperature and salt conditions can be readily determined empirically in preliminary experiments using conventional procedures.

For example, stringent conditions can be achieved for both prehybridizing and hybridizing incubations in the following: (i) 4 times at 42 ° C. in 6 × SSC with 50% formamide.
１６16 h, or (ii) 6 × SSC aqueous solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0) at 65 ° C. for 4-16 h).

For polynucleotides containing 30-600 nucleotides, the above formula is used and then corrected by subtracting (600 / polynucleotide size in base pairs). Stringency conditions are defined by a Th that is 5-10 ° C below the Tm.

Hybridization conditions for oligonucleotides shorter than 20-30 bases do not exactly follow the above rules. In such a case, the formula for calculating the Tm is as follows: Tm = 4 × (G + C) +2 (A + T) For example, a 50% G + C 18 nucleotide fragment has an approximate Tm of 54 ° C.

The polynucleotide molecules of the present invention (including RNA, DNA, or variants or combinations thereof) may have a variety of applications. For example, DNA molecules may be used in: (i) a process for producing the encoded polypeptide in a recombinant host system, (ii) preventing and / or preventing Chlamydia infection.
Or construction of a vaccine vector, such as a poxvirus, for further use in methods and compositions for treating, (iii) vaccine agents (and RNA molecules) formulated in a naked form or delivery vehicle, and (iv) Construction of an attenuated Chlamydia strain capable of overexpressing a polynucleotide of the invention or, where appropriate, expressing it in a modified mutant form (eg, a non-toxic form).
With respect to vaccine compositions and uses of the proteins and peptides and encoding nucleotides of the invention for protection against diseases caused by Chlamydia, the use of naked DNA encoding the protein or peptide and the use of these nucleotides intranasally or intramuscularly Is not preferred. For these proteins, the encoding nucleic acid can be transferred to other pathways (eg, intradermally).
And / or encoding nucleic acid (such as to improve the immune response)
Or adjuvant). It is also preferred to include the encoding nucleic acid as part of a recombinant live vector (eg, a viral or bacterial vector) for use as an immunizing agent. It is also preferred to immunize with a vaccine formulation comprising the protein or peptide of the invention itself. These vaccine formulations may involve the use of adjuvants.

According to a second aspect of the present invention there is therefore provided: (i) placed under the control of an element required for expression, in particular under the control of a suitable promoter (Ii) an expression vector comprising the expression cassette of the invention; (iii) a prokaryotic cell or eukaryote transformed or transfected with the expression cassette and / or vector of the invention. Culturing the cells, and (iv) prokaryotic or eukaryotic cells transformed or transfected with the expression cassette and / or vector of the invention under conditions that allow expression of the DNA molecules of the invention. And the step of recovering the encoded polypeptide or polypeptide derivative from the cell culture. A process for producing a polypeptide or polypeptide derivative encoded by a reotide.

[0059] Recombinant expression systems can be selected from prokaryotic and eukaryotic hosts. Eukaryotic hosts include yeast cells (eg, Saccharomyces cerev).
siaea or Pichia pastoris), mammalian cells (eg,
COS1 cells, NIH3T3 cells or JEG3 cells), arthropod cells (eg, Spodoptera frugiperda (SF9) cells) and plant cells. Preferably, a prokaryotic host (eg, E. coli) is used. Bacterial and eukaryotic cells can be obtained from a number of different sources (eg, the American Type Culture Collection (
ATCC; Rockville, Maryland)).

The choice of the expression system depends on the characteristics desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or in any other form.

The choice of the expression cassette will depend on the host system chosen as well as the characteristics desired for the polypeptide to be expressed. Typically, the expression cassette will be functional in the host system of choice and a promoter that may be constitutive or inducible;
Ribosome binding site; initiation codon (ATG), if necessary, a region encoding a signal peptide (eg, a lipidated signal peptide); a DNA molecule of the invention;
A stop codon; and, optionally, a 3'-terminal region (translation terminator and / or transcription terminator). The region encoding the signal peptide is adjacent to the polynucleotide of the invention and is placed in the appropriate reading frame. The region encoding the signal peptide can be homologous or heterologous to the DNA molecule encoding the mature polypeptide, and can be specific for the secretory apparatus of the host used for expression. The open reading frame constructed by the DNA molecule of the present invention, alone or with a signal peptide, is placed under the control of a promoter such that transcription and translation occur in a host system. The region encoding the promoter, signal peptide, is widely known and available to those of skill in the art, and is inducible by, for example, arabinose; Salmonel Functional in Gram-Negative Bacteria such as E. coli
la typhimurium promoter (and derivative) (promoter araB) (US Pat. No. 5,028,530 and Cagnon (Cagno)
n et al., Protein Engineering 4: 843 (1991)); a number of E. coli expressing T7 polymerase. promoter of the gene of bacteriophage T7, which encodes an RNA polymerase that is functional in E. coli strains (described in US Pat. No. 4,952,496); OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takas
e. Bact. 169: 5692 (1987)).

[0062] The expression cassette is typically part of an expression vector, and the expression vector is selected for its ability to replicate in the chosen expression system. Expression vectors (eg, plasmids or viral vectors) are available from Pouwels et al. (CLONING).
VECTORS: LABORATORY MANUAL, 85, Addendum 1987
)). These can be purchased from various commercial sources.

Methods for transforming / transfecting host cells with expression vectors are described in
ussubel et al., CURRENT PROTOCOLS IN MOLECULL
AR BIOLOGY, John Wiley & Sons Inc. (19
94) depends on the host system chosen.

Upon expression, a recombinant polypeptide (or polypeptide derivative) of the invention is produced and remains in the intracellular compartment, is secreted / excreted into the extracellular medium or periplasmic space, or Embedded in the cell membrane. The polypeptide can then be recovered from the cell extract in substantially pure form or from the supernatant following centrifugation of the recombinant cell culture. Typically, recombinant polypeptides can be purified by antibody-based affinity purification or by any other method that can be readily applied by those skilled in the art, such as by gene fusion to a small affinity binding domain. . Antibody-based affinity purification methods are also available for purifying polypeptides of the invention extracted from Chlamydia strains. Antibodies useful for purifying polypeptides of the invention by immunoaffinity can be obtained as described below.

The polynucleotides of the present invention may also be useful in the field of vaccines, for example to achieve DNA vaccination. There are two main possibilities, either using a viral or bacterial host as a gene delivery vehicle (live vaccine vector) or administering the gene in free form (eg, inserted into a plasmid). Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be assessed as described below.

Thus, in a third aspect of the invention, there is provided: (i) a vaccine, such as a poxvirus, comprising a DNA molecule of the invention arranged under the control of elements required for expression. (Ii) a composition comprising a vaccine vector of the invention together with a diluent or carrier; specifically (iii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a vaccine vector of the invention; iv) administering an immunologically effective amount of a vaccine vector of the invention to a mammal to produce an immune response (eg,
A method for inducing an immune response against Chlamydia in a mammal (eg, a human) comprising the step of eliciting a protective or therapeutic immune response against Chlamydia (or a method comprising administering to an animal (eg, a cat or bird)
And veterinary applications for treating or preventing Chlamydia infection of the present invention); and specifically (v) administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an individual in need thereof. Including, Chlamydia (
For example, C.I. trachomatis, C.I. psittaci, C.I. pneum
onia, C.I. pecorum) A method for preventing and / or treating infection. Further, a third aspect of the present invention includes the use of a vaccine vector of the present invention in the preparation of a medicament for preventing and / or treating Chlamydia infection.

A vaccine vector of the invention may express one or several polypeptides or derivatives of the invention, as well as at least one additional Chlamydia antigen, fragment, homolog, variant or derivative thereof. Furthermore, the vaccine vector of the present invention enhances the immune response (adjuvant effect),
For example, interleukin-2 (IL-2) or interleukin-12 (I
L-21)). Thus, a vaccine vector may include additional DNA sequences, eg, encoding a chlamydia antigen or cytokine, placed under the control of elements required for expression in mammalian cells.

Alternatively, a composition of the invention may comprise several vaccine vectors, each of which may express a polypeptide or derivative of the invention. The composition may also comprise an additional Chlamydia antigen, or subunit, fragment, homolog, variant or derivative thereof; or IL-2 or IL-1
And a vaccine vector capable of expressing a cytokine such as 2.

In a vaccination method for treating or preventing an infection in a mammal, the vaccine vector of the invention may be administered by any conventional route used in the field of vaccines, especially by mucosal (eg, ocular, nasal) Inside, oral, stomach, lung, intestine, rectum,
It may be administered to the vaginal or urinary tract surface or via a parenteral (subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal) route. The preferred route depends on the choice of vaccine vector. This administration can be accomplished in a single dose or repeatedly at intervals. Suitable dosages will vary according to various parameters understood by those skilled in the art, such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (
Weight, age, etc.)).

The live vaccine vectors available in the art include viral vectors (eg, adenovirus, alphavirus and poxvirus) and bacterial vectors (eg, Shigella, Salmonella, Vibrio)
cholerae, Lactobacillus, Bacille bilie
de Calmette-Guerin (BCG) and Streptoco
ccus).

Examples of adenovirus vectors, as well as methods for constructing adenovirus vectors capable of expressing a DNA molecule of the present invention, are described in US Pat. No. 4,920,209. Poxvirus vectors that can be used include, for example, the vaccinia and canary pox viruses described in U.S. Patent Nos. 4,722,848 and 5,364,773, respectively (e.g., vaccinia virus vectors). For a description of Tartaglia et al., Virolo
gy 188: 217 (1992); and Ta for reference to canarypox.
ylor et al., Vaccine 13: 539 (1995)).
Poxvirus vectors capable of expressing the polynucleotides of the present invention are described in Kiney et al., Nature 312: 163, such that the polynucleotides of the present invention are inserted into the viral genome under conditions suitable for expression in mammalian cells. (
1984). Generally, the dose of the vaccine viral vector for therapeutic or prophylactic use will be from about 1 × 10 ⁴ to about 1 × 10 ¹¹ per kilogram, advantageously from about 1 × 10 ⁷ to about 1 × 10 ¹⁰ , preferably There may be an amount of about 1 × 10 ⁷ to about 1 × 10 ⁹ plaque forming units. Preferably, the viral vector is administered parenterally; for example, three doses, four weeks apart. One of skill in the art will recognize that it is preferable to avoid adding a chemical adjuvant to a composition of the invention comprising a viral vector, thereby minimizing the immune response to the viral vector itself.

Non-Toxinogenic Vibrio cholera Useful as a Live Oral Vaccine
The e mutant is described in Mekalanos et al., Nature 306: 551 (1983).
) And U.S. Patent No. 4,882,278 (a strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted such that a functional cholera toxin is not produced); WO 92/11354. (A strain in which the irgA locus has been inactivated by a mutation; this mutation can be combined in a single strain with a ctxA mutation); and WO 94/1533 (functional ctxA and attRS1 DN
Deletion mutant lacking A sequence). These strains are described in WO 94/1948.
2, can be genetically engineered to express a heterologous antigen. Effective vaccine doses of Vibrio cholerae strains that can express a polypeptide or polypeptide derivative encoded by a DNA molecule of the present invention include, for example,
About 1 × 10 ⁵ to about 1 × 10 ⁹ , preferably about 1 × 10 ⁶ to about 1 × 10 ⁸ live bacteria,
It can be included in the appropriate volume for the chosen route of administration. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.

Attenuated Salmonella typhimurium strains, genetically engineered or not for recombinant expression of heterologous antigens, and their use as oral vaccines are described in Nakayama et al., Bio / Technology.
6: 693 (1988) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.

Other bacterial strains useful as vaccine vectors include High et al., EMBO 11:
1991 (1992); Sizemore et al., Science 270: 299.
(1995) (Shigella flexneri); Medaglini et al., Proc. Natl. Acad. Sci. USA 92: 6868 (1995)
) (Streptococcus gordonii); and Flynn, C
ell. Mol. Biol. 40:31 (1994), WO 88/6626,
WO 90/05594, WO 91/13157, WO 92/1796, and WO 92/21376 (Bacille Calmette Guerin)
).

[0075] In bacterial vectors, the polynucleotides of the present invention can be inserted into the bacterial genome, or can be carried on a plasmid and kept free.

An adjuvant can also be added to the composition containing the vaccine bacterial vector.
Numerous adjuvants are known to those skilled in the art. Preferred adjuvants may be selected from the list provided below.

According to a fourth aspect of the invention, there is also provided: (i) a composition comprising a polynucleotide of the invention together with a diluent or carrier; (ii) therapeutically or prophylactically effective A pharmaceutical composition comprising an effective amount of a polynucleotide of the present invention; (iii)
A) administering an immunologically effective amount of a polynucleotide of the present invention to a mammal to elicit an immune response (eg, a protective immune response to Chlamydia) to induce an immune response to Chlamydia in the mammal; By administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an individual in need thereof, such as C. trachomatis, C. psittaci, C. pneumon
iae, or C.I. pecorum) A method for preventing and / or treating infection. Further, a fourth aspect of the present invention is to prevent and / or prevent Chlamydia infection.
Or the use of a polynucleotide of the invention in the preparation of a medicament for treatment. A fourth aspect of the invention is preferably located under conditions for expression in mammalian cells, for example, on a plasmid that cannot replicate in a mammalian cell and cannot be substantially integrated into the mammalian genome. Including the use of DNA molecules.

The polynucleotides (DNA or RNA) of the present invention can also be themselves administered to a mammal for a vaccine, for example, for therapeutic or prophylactic purposes. When a DNA molecule of the present invention is used, it may be in the form of a plasmid that cannot replicate in mammalian cells and cannot be integrated into the mammalian genome. Typically, the DNA molecule is under the control of a promoter appropriate for expression in mammalian cells. This promoter can function universally or tissue-specific. Examples of non-tissue specific promoters include the cytomegalovirus (CMV) early promoter (described in US Pat. No. 4,168,062) and the Rous sarcoma virus promoter (Norton and Coffin, Molec. C).
ell Biol. 5: 281 (1985)). Desmin promoter (Li et al., Gene 78: 243 (1989), Li and Paulin, J. Biol. Chem. 266: 6562 (1991) and Li and Paulin, J. Biol. Chem. 268: 10403 (1).
993)) is tissue-specific and drives expression in muscle cells. More generally, useful vectors are, inter alia, WO 94/21797 and Hartikk.
a, et al., Human Gene Therapy 7: 1205 (1996).

For DNA / RNA vaccination, the polynucleotides of the invention may encode the precursor form or the mature form. Where the polynucleotide of the invention encodes a precursor form, the precursor form may be homologous or heterologous. In the latter case,
Eukaryotic leader sequences such as the leader sequence of tissue-type plasminogen factor (tPA) can be used.

The compositions of the present invention may include one or several polynucleotides of the present invention. A composition of the invention may also comprise at least one additional polynucleotide encoding another Chlamydia antigen or a fragment, derivative, variant or analog thereof. Cytokines (eg, interleukin-2 (I
A polynucleotide encoding L-2) or interleukin-12 (IL-12)) can also be added to the composition so that the immune response is enhanced.
These additional polynucleotides are placed under appropriate control for expression. Advantageously, a DNA molecule of the invention and / or an additional D to be included in the same composition
The NA molecule can be carried on the same plasmid.

Standard techniques of molecular biology for preparing and purifying polynucleotides can be used in preparing the polynucleotide therapeutics of the present invention. For use as a vaccine, the polynucleotides of the invention can be formulated according to various methods.

First, the polynucleotide may be in a naked form in any delivery vehicle (eg,
It can be used without anionic liposomes, cationic lipids, microparticles (eg, gold microparticles), precipitants (eg, calcium phosphate), or any other transfection facilitating agent. In this case, the polynucleotide may simply be diluted in a physiologically acceptable solution with or without a carrier, such as sterile saline or sterile buffered saline. When present, the carrier is preferably isotonic, hypotonic, or slightly hypertonic, and has a relatively low ionic strength, such as that provided by a sucrose solution (eg, a solution containing 20% sucrose). Having.

[0083] Alternatively, the polynucleotide may be associated with an agent that aids cellular uptake. This may in particular be (i) supplemented with chemical agents that modify the permeability of cells, such as bupivacaine (see, for example, WO 94/16737), or (ii) in liposomes. Or (iii)
It can be associated with cationic lipids or silica, gold or tungsten microparticles.

Anionic liposomes and neutral liposomes are well known in the art (eg, for a detailed description of how to make liposomes, see LIPOSOMES:
A PRACTICAL APPROACH, RPC New Ed, IRL
press (1990)), and are useful for delivering a wide range of products, including polynucleotides.

[0085] Cationic lipids are also known in the art and are commonly used for gene delivery. As such a lipid, DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N, -trimethylammonium chloride)
Also known as Lipofectin ^™ , DOTAP (1,2-bis (oleyloxy) -3- (trimethylammonio) propane), DDAB (dimethyldioctadecyl ammonium bromide), DOGS (dioctadecylamide logrisylspermine) ( dioctadecylamidologlycyl spe
rmine) and a cholesterol derivative (DC-Chol (3β- (N- (N
', N'-dimethylaminomethane) -carbamoyl) cholesterol)). Descriptions of these cationic lipids can be found in EP 187,702, WO 90.
/ 11092, U.S. Patent No. 5,283,185, WO 91/15501, W
O 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably, for example, WO 90/1.
DOPE (dioleyl phosphatidylethanolamine) described in 1092
Used in connection with neutral lipids such as

[0086] Other transfection facilitating compounds may be added to the formulation containing the cationic liposomes. A number of transfection facilitating compounds are described, for example, in WO 9
3/18759, WO 93/19768, WO 94/25608 and WO
95/2397. These include, inter alia, spermine derivatives (eg, WO 93/18759) which are useful for promoting the transport of DNA across the nuclear membrane.
And membrane permeabilizing compounds (eg, GALA, gramicidin S and cationic bile salts (see, eg, WO 93/19768)).

The gold fine particles or tungsten fine particles can also be used in WO 91/359, WO 93 /
17706, and Tang et al. (Nature 356: 152 (1992)).
Can be used for gene delivery as described in In this case, the microparticles coated with the polynucleotide form a needleless injection device ("gene gun").
(E.g., U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580)
And the injection device described in WO 94/24263).

The amount of DNA to be used in a vaccine recipient can be, for example,
The strength of the promoter used in the construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (eg, the weight, age and general health of the mammal), the mode of administration, and the formulation Depends on the type of Generally, about 1 μg
To about 1 mg, preferably about 10 μg to about 800 μg, and more preferably about 2 μg.
An effective therapeutically or prophylactic dose of 5 μg to about 250 μg can be administered to an adult human. Administration can be accomplished in a single dose or repeatedly at intervals.

The route of administration can be any of the conventional routes used in the field of vaccines. As general guidance, the polynucleotides of the invention can be administered to mucosal surfaces (eg, ocular, nasal, pulmonary, oral, intestinal, rectal, vaginal and urinary tract); or parenteral routes (eg, intravenous, (Subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route). The choice of the route of administration will depend, for example, on the formulation chosen. Polynucleotides formulated with bupivacaine are advantageously administered to muscle. Neutral or anionic liposomes or cationic lipids (eg, DOT
When MA or DC-Chol) is used, the formulation can be administered intravenously, intranasally (
(Aerosol application), may be advantageously injected via the intramuscular, intradermal, and subcutaneous routes. Naked forms of the polynucleotides may be advantageously administered via intramuscular, intradermal or subcutaneous routes.

[0090] Although not absolutely required, such compositions may also include an adjuvant. In such cases, systemic adjuvants that do not require co-administration to exhibit an adjuvant effect (eg, QS21 described in US Pat. No. 5,057,546) are preferred.

The sequence information provided in the present application allows for the design of specific nucleotide probes and primers that may be useful in diagnosis. Thus, in a fifth aspect of the present invention there is provided a nucleotide probe or primer having a sequence found in the sequence set forth in SEQ ID NOs: 1 and 3 or derived from this sequence by degeneracy of the genetic code.

As used herein, the term “probe” refers to a nucleic acid molecule having a sequence homologous to the sequence set forth in SEQ ID NOs: 1 and 3 or a complementary sequence under stringent conditions as described above. Alternatively, it refers to a DNA molecule (preferably single-stranded) or RNA molecule (or a variant or combination thereof) that hybridizes to an antisense sequence. Generally, the probes have SEQ ID NOs: 1 and 3
For example, they may include about 5 to about 100, preferably about 10 to about 80 nucleotides. In particular, the probe is a sequence that is at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of the sequence shown in SEQ ID NOs: 1 and 3, or is complementary to such a sequence. Has an array. Probes can include modified bases, such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphate residues can also be modified or substituted. For example, a deoxyribose residue may be replaced by a polyamide (Nielsen et al., Science 254: 149).
7 (1991)), and phosphate residues can be substituted by ester groups such as diphosphate esters, alkyl esters, aryl phosphonate esters and phosphorothioate esters. In addition, the 2 'on the ribonucleotide
The hydroxyl group can be modified, for example, by including an alkyl group;

The probes of the present invention can be used in any conventional hybridization technique, such as: dot blot (Maniatis et al., MOLECUL
AR CLONING: A LABORATORY MANUAL (1982)
Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, Southern Blot (Southern, J. Mol. Biol. 98: 503 (1975)), Northern Blot (identical to Southern Blot, except that RNA is used as a target). ) Or sandwich technology (Dunn et al., Cell 12).
: 23 (1977)). The latter technique involves the use of specific capture probes and / or specific detection probes that have nucleotide sequences that are at least partially different from each other.

Primers are typically probes of about 10 to about 40 nucleotides that are used to initiate enzymatic polymerization of DNA in an amplification process (eg, PCR), an extension process, or a reverse transcription method. Is done. In diagnostic methods involving PCR, the primers can be labeled.

Thus, the present invention also includes: (i) Chl in biological material
a reagent comprising a probe of the invention for detecting and / or identifying the presence of Amydia; (ii) a method for detecting and / or identifying the presence of Chlamydia in a biological material, wherein the method comprises the steps of: a) a sample is recovered from or derived from this biological material, and (b) DNA or RN
A is extracted from this material and denatured, and (c) a probe of the invention (
(Eg, a capture probe or a detection probe, or both) under stringent hybridization conditions so that hybridization is detected; and (iii) Chlamydi in biological material.
A method for detecting and / or identifying the presence of a, wherein (a) a sample is recovered or derived from the biological material,
b) DNA is extracted from this material, and (c) the extracted DNA is at least 1
One and preferably two primers of the invention are primed and amplified by the polymerase chain reaction, and (d) an amplified DNA fragment is produced.

As mentioned above, polypeptides that can be generated upon expression of a newly identified open reading frame are useful as vaccine agents.

Accordingly, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.

A “substantially purified polypeptide” is a polypeptide that has been separated from the environment in which the polypeptide naturally occurs and / or the polypeptide present in the environment in which the polypeptide was synthesized. Defined as a polypeptide that does not contain a large number. For example, a substantially purified polypeptide is free of cytoplasmic polypeptide. One skilled in the art will appreciate that the polypeptides of the present invention can be obtained from natural sources (ie, Chl).
(Amydia strain) or can be produced by recombinant means.

A homologous polypeptide or polypeptide derivative encoded by a polynucleotide of the present invention may be used in combination with an antiserum raised against a reference polypeptide having an amino acid sequence as set forth in SEQ ID NOs: 2 and 4. By testing for cross-reactivity, one can screen for specific antigenicity. Briefly, a monospecific hyperimmune antiserum can be raised against the purified reference polypeptide itself, or a fusion polypeptide (eg, an expression product of the MBP, GST or His tag system, or (Synthetic peptides predicted to be antigenic) can be raised against a purified reference polypeptide. Homologous polypeptides or polypeptide derivatives screened for specific antigenicity can be produced per se or as fusion polypeptides. In this latter case, and where the antiserum is also raised against a fusion polypeptide, two different fusion systems are used. Specific antigenicity can be determined by a number of methods (Western blot (Toowb
in et al., Proc. Natl. Acad. Sci. USA 76: 4350 (1
979)), including dot blots and ELISAs).

In a Western blot assay, the product to be screened (either as a purified preparation or a total E. coli extract) was labeled with Laemm.
li, Nature 227: 680 (1970).
It is subjected to DS-Page electrophoresis. After transfer to a nitrocellulose membrane, the material (nitrocellulose membrane) is diluted to a monospecific dilution ranging from about 1: 5 to about 1: 5000, preferably from about 1: 100 to about 1: 500. Further incubation with hyperimmune antiserum. Specific antigenicity is indicated once the band corresponding to this product shows reactivity at any of the above range of dilutions.

In an ELISA assay, the product to be screened is preferably used as a coating antigen. Although purified preparations are preferred, whole cell extracts can also be used. Briefly, about 100 μl of a preparation of about 10 μg protein / ml is dispensed into the wells of a 96-well polycarbonate ELISA plate. The plate is incubated at 37 ° C. for 2 hours and then at 4 ° C. overnight. The plate is washed with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS / Tween buffer). The wells are saturated with 250 μl of PBS containing 1% bovine serum albumin (BSA) to prevent non-specific antibody binding. After 1 hour incubation at 37 ° C., the plates are washed with PBS / Tween buffer. The antiserum was 0.5% BSA
Is serially diluted in PBS / Tween buffer containing 100 per well
μl of the dilution is added. The plate is incubated at 37 ° C. for 90 minutes, washed, and evaluated according to standard procedures. For example, if a specific antibody is raised in a rabbit, goat anti-rabbit peroxidase conjugate is added to the well. Incubation is performed at 37 ° C. for 90 minutes, and the plate is washed. The reaction is developed using an appropriate substrate, and the reaction is measured by colorimetry (absorbance is measured by a spectrophotometer). Under the above experimental conditions, a positive reaction is greater than the non-immune control serum.
. D. Indicated by value.

In a dot blot assay, purified products are preferred, but whole cell extracts can also be used. Briefly, a solution of the product at about 100 μg / ml
Serial two-fold dilutions in 0 mM Tris-HCl (pH 7.5). 100 μl of each dilution is applied to a 0.45 μm nitrocellulose membrane set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying a vacuum to the system. Wells containing 50 mM Tris-
It is washed by the addition of HCl (pH 7.5) and the membrane is air-dried. This membrane was treated with a blocking buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM).
15M NaCl, 10 g / L skim milk) and from about 1:50 to about 1
: 5000, preferably about 1: 500 antiserum dilution. The reaction is shown according to standard procedures. For example, if a rabbit antibody is used, a goat anti-rabbit peroxidase conjugate is added to the well. Incubation is performed at 37 ° C. for 90 minutes, and the blot is washed. The reaction is developed with a suitable substrate and stopped. The reaction is measured visually by the appearance of a colored spot, for example by colorimetry. Under the experimental conditions described above, a positive reaction is indicated once the spot to be colored has been combined with a dilution of at least about 1: 5, preferably at least about 1: 500.

[0103] The therapeutic or prophylactic efficacy of a polypeptide or derivative of the present invention can be evaluated as follows.

According to a seventh aspect of the present invention there is provided: (i) a composition comprising a polypeptide of the present invention together with a diluent or carrier; in particular, (ii) a therapeutically effective amount or a prophylactically effective A pharmaceutical composition comprising an amount of a polypeptide of the invention; (iii)
A method for inducing an immune response against Chlamydia in a mammal, comprising administering to the mammal an immunologically effective amount of a polypeptide of the invention, such as a protective immune response against Chlamydia. )); And, in particular, (iv) Chlamydia (eg, C. tra
chomatis, C.I. psittaci, C.I. pneumoniae or C
. pecorum) is a method for preventing and / or treating an infection by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an individual in need thereof. Further, a seventh aspect of the present invention is a method for preparing
a Use of the polypeptide of the present invention in the preparation of a medicament for preventing and / or treating infection.

The immunogenic compositions of the present invention may be administered by any conventional route of use in the field of vaccines, particularly mucosal (eg, ocular, nasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or Urinary tract)
It may be administered to the surface or via a parenteral (eg, subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of the route of administration will depend on a number of parameters, such as the adjuvant associated with the polypeptide. For example, if a mucosal adjuvant is used, the nasal or oral route is preferred, and if a lipid formulation or an aluminum compound is used, the parenteral route is preferred. In the latter case, the subcutaneous or intramuscular route is most preferred. This choice may also depend on the nature of the vaccine drug. For example, polypeptides of the invention fused to CTB or LTB are best administered to mucosal surfaces.

A composition of the invention may comprise one or several polypeptides or derivatives of the invention. The composition also comprises at least one additional Chlamydi
a antigen or a subunit, fragment, homolog, mutant or derivative thereof.

For use in the compositions of the present invention, the polypeptide or derivative thereof is incorporated into liposomes (preferably neutral or anionic liposomes), microspheres, ISCOMS, or virus-like particles (VLPs). Or may be formulated with them to facilitate delivery and / or enhance their immune response. These compounds are readily available to those skilled in the art;
See LIPOSOMES: A PRACTICAL APPROACH (supra).

Adjuvants other than liposomes and the like can also be used and are known in the art. Suitable selections can be made conventionally by a person skilled in the art, for example from the list provided below.

Administration can be accomplished in a single dose or, if necessary, repeated at intervals that can be determined by one skilled in the art. For example, a starting dose may be followed by three boost doses at weekly or monthly intervals. The appropriate dose can be determined by one of skill in the art,
Various parameters (recipient (eg, adult or infant), specific vaccine antigen, route and frequency of administration, presence / absence or type of adjuvant, and desired effect (eg, protection and / or treatment) A)). Generally, the vaccine antigens of the invention will range from about 10 μg to about 5 μg, depending on the mucosal route.
It may be administered in an amount of 00 mg, preferably about 1 mg to about 200 mg. For parenteral administration, the dose should generally not exceed about 1 mg, preferably 100 μg.

When used as a vaccine agent, the polynucleotides and polypeptides of the invention can be used sequentially as part of a multi-step immune process. For example,
A mammal can be primed first with a vaccine vector of the invention, such as a poxvirus, for example, via the parenteral route, and then with a polypeptide encoded by the vaccine vector, e.g., Via mucosal route, 2
Can be boosted times. In another example, liposomes conjugated to a polypeptide or derivative of the invention can also be used for priming, and a soluble polypeptide or derivative of the invention can be used in combination with a mucosal adjuvant (eg, LT) to provide mucosal Is boosted.

The polypeptide derivatives of the present invention are also useful as diagnostic reagents for detecting the presence of anti-Chlamydia antibodies, for example, in blood samples. Such polypeptides are about 5 to about 80 amino acids in length, preferably about 10 to about 50 amino acids in length, and can be labeled or unlabeled. This depends on the diagnostic method. Diagnostic methods involving such reagents are described below.

Upon expression of a DNA molecule of the present invention, a polypeptide or polypeptide derivative may be produced and purified using known laboratory techniques. For example, a polypeptide or polypeptide derivative can be produced as a fusion protein that contains a fusion tail that facilitates purification. The fusion product can be used to immunize a small mammal (eg, a mouse or rabbit) to raise antibodies (monospecific antibodies) to the polypeptide or polypeptide derivative. The eighth aspect of the present invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the present invention.

“Monospecific antibody” refers to an antibody that can react with a unique, naturally occurring Chlamydia polypeptide. The antibodies of the present invention can be polyclonal or monoclonal. Monospecific antibodies can be recombinant, eg, chimeric (eg, composed of a variable region of mouse origin linked to a human constant region), humanized (eg, of a hypervariable region of animal (eg, mouse) origin). The associated human immunoglobulin constant skeleton), and / or a single chain. Both polyclonal and monospecific antibodies also contain immunoglobulin fragments (eg, F
(Ab) '2 fragment or Fab fragment). Antibodies of the invention can be of any isotype (eg, IgG or IgA), and polyclonal antibodies can be of a single isotype or can comprise a mixture of isotypes.

Antibodies of the invention, which are raised against a polypeptide or polypeptide derivative of the invention, can be generated and standard immunological assays (eg, Western blot analysis, dot blot assay, Or ELISA (eg, C
Oligan et al., CURRENT PROTOCOLS IN IMMUNOL
OGY (1994) John Wiley & Sons, Inc. , New Yo
rk, NY). This antibody is used in diagnostic methods to extract Chlamydi in a sample (eg, a biological sample).
The presence of the a antigen can be detected. This antibody can also be used in affinity chromatography methods to purify the polypeptide or polypeptide derivative of the present invention. As discussed further below, such antibodies can be used in prophylactic and therapeutic passive immunization methods.

Thus, in a ninth aspect of the present invention there is provided: (i) for detecting the presence of Chlamydia in a biological sample comprising an antibody, polypeptide or polypeptide derivative of the present invention. A reagent; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, said method comprising contacting said biological sample with an antibody, polypeptide or polypeptide derivative of the invention. Resulting in an immune complex being formed, and detecting such a complex to indicate the presence of Chlamydia in the sample or to indicate the presence of Chlamidia in the organism from which the sample is derived. .

One of skill in the art will appreciate that this immune complex is formed between the components of the sample and the antibody, polypeptide or polypeptide derivative of the invention, whichever is used, and any non- It is understood that the binding material can also be removed before detecting the complex. As can be readily appreciated, the polypeptide reagent is a sample (
While useful for detecting the presence of anti-Chlamydia antibodies in blood samples (eg, blood samples), the antibodies of the present invention allow a sample (eg, a gastric extract or biopsy) to
It can be used to screen for the presence of a hlamydia polypeptide.

For use in diagnostic applications, the reagents (ie, antibodies, polypeptides, or polypeptide derivatives of the invention) may be in the free state or on a solid support (eg, tubes, beads, or (Any other conventional support used in the art). Anchoring may be achieved using direct or indirect means. Direct means include passive adsorption (non-covalent bonding) or covalent bonding between the support and the reagent. By "indirect means" is meant that the anti-reagent compound that interacts with the reagent is first attached to the solid support. For example, if a polypeptide reagent is used, the antibody that binds to the reagent can act as an anti-reagent, provided that the reagent binds to an epitope that is not involved in recognizing the antibody in a biological sample. Indirect means may also employ ligand-receptor systems, for example, molecules such as vitamins may be fused onto the polypeptide reagent, and the corresponding receptor may be immobilized on the solid phase. This is exemplified by the biotin-streptavidin system. Alternatively, indirect means include adding a peptide tail to this reagent, for example, by chemical or genetic manipulation, and fusion (
Grafted or fused products can be used by immobilization by passive adsorption or covalent attachment of the peptide tail.

According to a tenth aspect of the present invention, there is provided a process for purifying a polypeptide or polypeptide derivative of the present invention from a biological sample. The process involves performing an antibody-based affinity chromatography using the biological sample, wherein the antibody is a monospecific antibody of the invention.

For use in the purification process of the present invention, the antibodies may be polyclonal or monospecific, and preferably are of the IgG type. Purified Ig
G can be prepared from antisera using standard methods (eg, Colligan)
See above). Conventional chromatographic supports, as well as standard methods for fusing antibodies, are described, for example, in ANTIBODIES: A LABORA.
TOLY MANUAL, D. Lane, E.L. Harlow (1988).

[0120] Briefly, a biological sample (eg, a C. pneumoniae extract), preferably in a buffered solution, is provided with a chromatographic material, preferably for diluting the biological sample. (Equilibrated with the buffer used) so that the polypeptide or polypeptide derivative of the invention (e.g.
That is, the antigen) is adsorbed on this material. The chromatographic material (eg, a gel or resin bound to an antibody of the invention) can be in batch form or can be in a column. Unbound components are washed away and then
The antigen may be in a suitable elution buffer (eg, glycine buffer or a chaotropic agent (eg, guanidine hydrochloride) or a high salt concentration (eg, 3M MgCl ₂ ).
(A buffer containing). The eluted fractions are collected and the presence of the antigen is detected, for example, by measuring the absorbance at 280 nm.

The antibodies of the present invention can be screened for therapeutic efficacy as described below. According to an eleventh aspect of the invention, there is provided: (i) a composition comprising a monospecific antibody of the invention together with a diluent or carrier; (ii)
A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention,
And (iii) Chlamydia (eg, C. trachomatis,
C. psittaci, C.I. pneumoniae or C.I. pecorum)
A method for treating or preventing an infection, the method comprising administering to the individual in need a therapeutic or prophylactic amount of a monospecific antibody of the invention. Alternatively, the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing a Chlamydia infection.

For this purpose, the monospecific antibody may be polyclonal or monoclonal, and is preferably of the IgA isotype (preferentially). In passive immunization, the antibody can be administered to the mucosal surface of a mammal, such as the gastric mucosa, for example orally or intragastrically, advantageously in the presence of a bicarbonate buffer. Alternatively, systemic administration, which does not require a bicarbonate buffer, can be performed. The monospecific antibodies of the present invention can be administered as a single active ingredient or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide. The amount of antibody used and the particular regimen can be readily determined by one skilled in the art. For example, administration of about 100-1,000 mg of antibody daily for one week, or three doses of about 100-1,000 mg antibody per day for 2-3 days, is an effective regimen for most purposes. Can be

Therapeutic or prophylactic efficacy can be determined using methods standard in the art, for example, to induce a mucosal immune response or to induce a prophylactic and / or therapeutic immunity, eg, a C
. It can be assessed by measuring using the pneumoniae mouse model. One skilled in the art will appreciate the C.I. pneumoniae strain is another Chla
It is understood that it can be replaced with a mydia strain. For example, C.I. pneumonia
The potency of DNA molecules and polypeptides derived from C. e. pneum
Evaluated in a mouse model using the S. oniae strain. Defense is Chlam
The extent of ydia infection can be determined by comparing the extent of the control group. Protection is indicated if the infection is reduced compared to this control group. Such evaluations can be made for the polynucleotides, vaccine vectors, polypeptides and derivatives thereof, and antibodies of the present invention.

Adjuvants useful in any of the above vaccine compositions are as follows.

Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate. The antigen can be precipitated with or adsorbed on the aluminum compound according to standard protocols. Other adjuvants (eg, RIBI (ImmunoChem, Ha)
(milton, MT) can be used for parenteral administration.

Adjuvants for mucosal administration include bacterial toxins such as cholera toxin (C
T), E. E. coli heat-labile toxin (LT), Cl
Ostrichium difficile toxin A, and pertussis toxin (PT), or a combination, subunit, toxoid or variant thereof). For example, a purified preparation of natural cholera toxin B subunit (CTB) may be useful. Fragments, homologs, derivatives and fusions of any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, variants with reduced toxicity are used. Suitable mutants are, for example, WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly P
T variants). Additional LT variants that can be used in the methods and compositions of the present invention include, for example, Ser-63-Lys variants, Ala
-69-Gly mutant, Glu-110-Asp mutant, and Glu-112
-Asp mutant. Other adjuvants (eg, E. coli, Sa
lmonella minnesota, Salmonella typhim
urium, or Shigella flexneri bacterial monophosphoryl lipid A (MPLA); saponin, or polylactide glycolide (glycol).
ide) (PLGA) microspheres) can also be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral administration include polyphosphazenes (WO 95/2415), DC-
chol (3b- (N- (N ′, N′-dimethylaminomethane) -carbamoyl) cholesterol) (U.S. Pat. No. 5,283,185 and WO 96/14)
831) and QS-21 (WO 88/9336).

[0128] Any pharmaceutical composition of the present invention (polynucleotide, polypeptide,
Polypeptide derivatives, or antibodies) can also be produced in conventional manner. In particular, the pharmaceutical compositions of the present invention can be formulated with a pharmaceutically acceptable diluent or carrier, for example, water or saline solution, such as phosphate buffered saline. In general, the diluent or carrier may be chosen based on the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as the pharmaceutical necessities for their use in pharmaceutical formulations, are:
It is described in Remington's Pharmaceutical Science, standard reference documents in this field, and USP / NF.

The present invention also includes a method wherein a Chlamydia infection is treated by oral administration of a Chlamydia polypeptide of the present invention and a mucosal adjuvant with an antibiotic, antacid, sucralfate, or a combination thereof. Examples of such compounds that can be administered with the vaccine antigen and the adjuvant are, for example, antibiotics, including macrolides, tetracyclines, and derivatives thereof (particular examples of antibiotics that can be used are azithromycin or doxycycline Or immunomodulatory agents (eg, including cytokines or steroids). In addition, compounds that contain more than one of the above-listed components in combination may be used. The present invention also provides compositions for performing these methods (ie, a Chlamydia antigen of the present invention, an adjuvant and one or more of the above-listed compounds) in a pharmaceutically acceptable carrier or diluent. ).

The amounts of the above-listed compounds used in the methods and compositions of the present invention
It can be easily determined by one skilled in the art. In addition, one skilled in the art can easily design a treatment / immunization schedule. For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen plus adjuvant can be administered on days 7, 14, 21, and 28.

The above disclosure generally describes the present invention. A more complete understanding may be obtained by reference to the following specific examples. These examples are described for illustrative purposes only, and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated where circumstances may suggest or favor expedience. Although specific terms are used herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Example 1 Preparation of Plasmid Vector pCAI314 Containing the omp Gene This example illustrates the preparation of a plasmid vector pCAI314 containing the omp gene.

[0133] The omp gene was replaced with Chlamydia pneumoniae genomic DNA.
Was amplified by the polymerase chain reaction (PCR). In this PCR, the following 5 'primers:

[0134]

Embedded image

(SEQ ID NO: 5) (including the sequence encoding the Not I restriction site, ribosome binding site, initiation codon, and N-terminal sequence of omp), and the following 3 ′ primers:

[0136]

Embedded image

(SEQ ID NO: 6) (including the sequence encoding the C-terminal sequence of omp and a Bam HI restriction site) was used. The stop codon was removed and additional nucleotides were inserted to give an in-frame C-terminal fusion with a histidine tag.

After amplification, the PCR fragment was purified using the QIAquick ^™ PCR purification kit (
(Qiagen), then digested with Not I and Bam HI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 3). This vector transcribes under the control of the human CMV promoter.

Example 2 Preparation of Eukaryotic Expression Vector pCA / Myc-His This example illustrates the preparation of eukaryotic expression vector pCA / Myc-His.

The plasmid pcDNA3.1 (−) Myc-His C (Invitroge)
n) was restricted with Spe I and Bam HI to remove its CMV promoter and the remaining vector fragment was isolated. Plasmid VR-1
012 (Vical), the CMV promoter and intron A were replaced with Spe
Isolated on the I / Bam HI fragment. These fragments were ligated together to generate plasmid pCA / Myc-His. N containing the omp gene
The otI / BamHI restricted PCR fragment was ligated into NotI and BamHI restricted plasmid pCA / Myc-His to generate plasmid pCAI314 (FIG. 3).

The resulting plasmid pCAI314 was transformed into E. coli. coli XL-1 blue (St
ratagene) by electroporation. col
i was grown in LB broth containing 50 μg / ml carbenicillin. This plasmid was used as an Endo Free Plasmid Giga Kit ^™ (Qia
gen) isolated by a large-scale DNA purification system. DNA concentration was determined by absorbance at 260 nm, and the plasmid was confirmed after gel electrophoresis and ethidium bromide staining and comparison with molecular weight standards. The 5 'and 3' ends of this gene were confirmed by sequencing using a LiCor model 4000 L DNA sequencer and IRD-800 labeled primers.

Example 3 Protection Against Intranasal C. pneumoniae 2 illustrates immunization of mice to achieve protection against a pneumoniae intranasal challenge.

When the mouse has the C. It has previously been shown to be susceptible to intranasal infection with different isolates of Pneumoniae. Yang et al., Infect. Immun. 6
1: 2037-2040 (1993). AR-39 strain (Grayston, 19
89) was used in Balb / c mice as a challenge infection model, and the Chlamydia gene product delivered as naked DNA resulted in the sublethal C. aureus. pn
The ability to elicit a protective response to E. muniae lung infection was tested. Protective immunity is defined as promoting clearance of lung infection.

Groups of 7-9 week old male Balb / c mice (5-9 mice per group) were prepared as described in Examples 1 and 2. Intramuscular (im) and intranasal (in) immunizations were carried out with plasmid DNA containing the coding sequence of pneumoniae omp. Saline or plasmid vectors lacking the inserted Chlamydia gene were given to groups of control animals.

For intramuscular immunization, the left quadriceps and right quadriceps alternately with D in 50 μl of PBS.
100 μg of NA were injected three times at 0, 3, and 6 weeks. For intranasal immunization, anesthetized mice received 50 μl of PBS containing 50 μg of DNA on 0,3,
And aspirated 3 times at 6 weeks. At 8 weeks, immunized mice received 100 μl
5 × 10 ⁵ IFU of C.I. in SPG buffer. pneumoniae AR39 strain was inoculated intranasally, and these mice became sublethal C. aureus. The ability to limit the growth of the pneumoniae challenge was tested.

[0146] Lungs were removed from mice 9 days after challenge and immediately injected with SPG buffer (
It was homogenized to 7.5% sucrose, 5 mM glutamate, 12.5 phosphate, pH 7.5). The homogenate was stored frozen at -70 ° C until assay. Dilutions of this homogenate were assayed by inoculation of susceptible cells onto a monolayer in the presence of infectious Chlamydia. The inoculum was placed on the cells for 3000 r.
After centrifugation at pm for 1 hour, the cells were then incubated at 35 ° C. for 3 days in the presence of 1 μg / ml cycloheximide. After the incubation, the monolayer was fixed with formalin and methanol, and then the C. elegans for the presence of Chlamydia inclusions. Immunoperoxidase staining was performed using convalescent sera from rabbits infected with Pneumoniae, and DAB with metal enhancement as peroxidase substrate.

FIG. 4 shows that mice immunized intranasally and intramuscularly with pCAI314 had Chlamydia lung titers of less than 4600 in three of five cases, whereas the range of values for control mice was Illustrate that mock immunization with saline or immunization with the unmodified vector were 1800-23100 IFU / lung (mean 11811) and 16600-26100 IFU / lung (mean 22100), respectively (Table 1). . Lack of protection with the unmodified vector confirms that the DNA itself does not play a role in the observed protective effect. This is the result obtained for one additional plasmid DNA construct pdagA (pdagA
Did not protect and average lung titers were similar to those obtained for saline-immunized control mice). This construct p
dagA is identical to pCAI314, except that the nucleotide sequence encoding omp is identical to the C. dagA encoding protein dagA. pneumoniae except that it has been replaced with a nucleotide sequence.

[0148]

[Table 1]

(Equivalents) From the foregoing detailed description of certain embodiments of the invention, note that a unique Chlamydia
It should be clear that the antigen has been described. Although specific embodiments are disclosed in detail herein, this is done for illustrative purposes only,
It is not intended to limit the scope of the claims appended hereto. In particular, it is intended by the inventor that various substitutions, changes, and modifications to the invention may be made without departing from the spirit and scope of the invention as defined by the appended claims.

[Brief description of the drawings]

 The invention will be better understood from the following description of the drawings.

FIG. 1 shows the nucleotide sequence of the omp gene from Chlamydia pneumoniae (upper sequence) and the deduced amino acid sequence of the omp protein (full length protein—center sequence; truncated protein—lower sequence).

FIG. 2A shows C.I. Fig. 2 shows a restriction enzyme analysis of a gene encoding a pneumoniae omp gene.

FIG. 2B shows C.I. Fig. 2 shows a restriction enzyme analysis of a gene encoding a pneumoniae omp gene.

FIG. 2C shows C.I. Fig. 2 shows a restriction enzyme analysis of a gene encoding a pneumoniae omp gene.

FIG. 2D shows C.I. Fig. 2 shows a restriction enzyme analysis of a gene encoding a pneumoniae omp gene.

FIG. 3 shows the construction and elements of plasmid pCAI314314.

FIG. 4 shows C. elegans by pCAI314 after DNA immunization. 4 shows protection against Pneumoniae infection. Here, individual data points (open diamonds) are shown for each animal, and mean (solid square) and standard deviation are shown for each group.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 11/06 A61P 27/02 4C085 13/00 31/04 4H045 27/02 C07K 14/295 31/04 16 / 12 C07K 14/295 C12N 1/15 16/12 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/53 D C12P 21 / 02 M C12Q 1/68 33/537 G01N 33/53 33/566 33/569 F 33/537 33/577 B 33/566 C12N 15/00 ZNAA 33/569 5/00 A 33/577 A61K 37/02 ( 31) Priority claim number 09 / 360,434 (32) Priority date July 26, 1999 (July 26, 1999) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, A, UG, US, UZ, VN, YU, Z A, ZW (72) inventors Dan, Pamela El. Canada L5A4B8 Ontario, Mississauga, Kanef Sarkle 3700, Apartment 703 F term (reference) 4B024 AA01 AA13 BA31 CA04 CA09 GA14 HA14 4B063 QA01 QA08 QA19 QQ08 QQ43 QR39 QR55 QR62 QR77 QS25 QS34 QX024 CA20 CE12 DA01 DA15 4B065 AA01Y AB01 BA03 CA24 CA44 CA46 4C084 AA02 AA06 AA13 BA03 CA53 DC50 NA14 ZA332 ZA592 ZA812 ZB352 4C085 AA03 BA45 CC05 CC07 DD22 DD33 GG02 GG03 GG04 GG05 GG06 A4A11A20A10A10A

Claims (37)

[Claims] 1. An isolated polynucleotide comprising: (a) a polynucleotide having a sequence comprising the nucleotide sequence SEQ ID NO: 1;
(B) a polynucleotide encoding a polypeptide having a sequence at least 75% homologous to SEQ ID NO: 2, and a functional fragment thereof; and (c) a sequence comprising the nucleotide sequence SEQ ID NO: 1. An isolated polynucleotide selected from the group consisting of a polynucleotide capable of hybridizing under stringent conditions to a polynucleotide having the same, and a functional fragment thereof. 2. The polynucleotide of claim 1, wherein said polynucleotide is linked to a second nucleotide sequence encoding a fusion polypeptide. 3. The nucleotide according to claim 2, wherein said fusion polypeptide is a heterologous signal peptide. 4. The nucleotide of claim 2, wherein said polynucleotide encodes a functional fragment of a polypeptide having SEQ ID NO: 2. 5. An isolated polypeptide having a sequence that is at least 75% homologous to SEQ ID NO: 4, and a functional fragment thereof. 6. The polypeptide of claim 5, wherein said polypeptide has the sequence of SEQ ID NO: 2 or a functional fragment thereof. 7. A polypeptide comprising the polypeptide of claim 5 linked to a fusion polypeptide. 8. The method according to claim 7, wherein the fusion polypeptide is a signal peptide.
2. The polypeptide according to item 1. 9. The polypeptide of claim 7, wherein said fusion polypeptide comprises a heterologous polypeptide having adjuvant activity. 10. An expression cassette comprising the polynucleotide of claim 1 operably linked to a promoter. An expression vector comprising the expression cassette according to claim 10. 12. A host cell comprising the expression cassette according to claim 10. 13. The host cell according to claim 12, wherein said host cell is a prokaryotic cell. 14. The host cell according to claim 12, wherein said host cell is a eukaryotic cell. 15. A method for producing a recombinant CPN100314 polypeptide, comprising: (a) culturing the host cell of claim 12 under conditions that permit expression of the polypeptide. And (b) recovering the recombinant polypeptide. 16. A vaccine vector comprising the expression cassette according to claim 10. 17. The vaccine vector according to claim 16, wherein said host mammal is a human. 18. The vaccine vector according to claim 16, wherein the vaccine vector is in a pharmaceutically acceptable excipient. 19. A pharmaceutical composition comprising an immunologically effective amount of the vaccine vector according to claim 16. 20. A method for inducing an immune response in a mammal, comprising administering an immunologically effective amount of the vaccine vector of claim 16 to said mammal. Wherein the administration induces an immune response. 21. A pharmaceutical composition comprising an immunologically effective amount of the polypeptide of claim 5 and a pharmaceutically acceptable diluent. 22. The pharmaceutical composition according to claim 21, further comprising an adjuvant. 23. The pharmaceutical composition according to claim 21, further comprising one or more known Chlamydia antigens. 24. A method for inducing an immune response in a mammal, comprising administering to the mammal an immunologically effective amount of the pharmaceutical composition of claim 21. Wherein the administration induces an immune response. 25. A polynucleotide probe reagent capable of detecting the presence of Chlamydia in a biological material, wherein the reagent hybridizes under stringent conditions to the polynucleotide of claim 1. A polynucleotide probe reagent comprising: 26. The polynucleotide probe reagent according to claim 25, wherein the reagent is a DNA primer. 27. A hybridization method for detecting the presence of Chlamydia in a sample, the method comprising: (a) obtaining a polynucleotide from the sample; (b) obtaining the polynucleotide; 22. hybridizing the polynucleotide probe reagent according to claim 21 under conditions that allow hybridization of the probe and the sample; and (c) combining the polynucleotide of the detection reagent with the polynucleotide in the sample. Detecting the hybridization of. 28. An amplification method for detecting the presence of Chlamydia in a sample, the method comprising: (a) obtaining a polynucleotide from the sample; and (b) claiming the obtained polynucleotide. 26. A method comprising: amplifying using one or more polynucleotide probe reagents according to item 25; and (c) detecting the amplified polypeptide. 29. A method for detecting the presence of Chlamydia in a sample, comprising: (a) contacting the sample with a detection reagent that binds to a CPN100314 polypeptide to form a complex. And (b) detecting the formed complex. 30. The method of claim 29, wherein said detection reagent is an antibody. 31. The method according to claim 30, wherein said antibody is a monoclonal antibody. 32. The method according to claim 30, wherein said antibody is a polyclonal antibody. 33. An affinity chromatography method for substantially purifying a CPN100314 polypeptide, comprising: (a) a sample containing the CPN100314 polypeptide;
(4) contacting a detection reagent that binds to the polypeptide to form a complex; (b) isolating the formed complex; (c) dissociating the formed complex; And (d) isolating the dissociated CPN100314 polypeptide. 34. The method of claim 33, wherein said detection reagent is an antibody. 35. The method of claim 34, wherein said antibody is a monoclonal antibody. 36. The method according to claim 34, wherein said antibody is a polyclonal antibody. 37. An antibody that immunospecifically binds to the polypeptide of claim 5, or a fragment or derivative of the antibody comprising a binding domain thereof.