JP2002523049A - Chlamydia antigens and corresponding DNA fragments and uses thereof - Google Patents

Abstract

  (57) [Summary] In the summary of the present disclosure, the present invention provides a method of immunizing a host (including human) for nucleic acid (including DNA) against a disease caused by infection by a strain of Chlamydia (specifically, C. pneumoniae). Provided in the nucleotide sequence and host encoding any of the polypeptides of CPN 100111, CPN 100224, CPN 100230, CPN 100231, CPN 1000023, CPN 100235, CPN 100394, and CPN 100395 of a strain of Chlamydia pneumoniae. A vector is used that contains a promoter that directs the expression of any of these polypeptides. Modifications are possible within the scope of the present invention

Description

DETAILED DESCRIPTION OF THE INVENTION

(Related US Application) This patent application is filed from United States Provisional Application U.S.A. S.
S. N. No. 60 / 097,187, No. 60 / 097,188, No. 60/0
Nos. 97,189, 60 / 097,190, 60 / 097,195,
Nos. 60 / 097,196 and 60 / 097,197, and 19
U.S.A. filed on Aug. 27, 1998 S. S. N. Claim priority to 60 / 097,191.

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

[0003] Chlamydia are prokaryotes. They show morphological and structural similarities to Gram-negative bacteria, including the outer trilayer (including lipopolysaccharides and some membrane proteins). Chlamydia is distinguished from other bacteria by its morphology and unique developmental cycle. These are obligate intracellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replication stage of the life cycle occurs in membrane-bound inclusions that sequester bacteria from the cytoplasm of infected host cells.

[0004] Chlamydia has long been considered a virus because it is small and only amplifies in susceptible cells. However, chlamydia has many features in common with other bacteria: (1) it contains both DNA and RNA, (2) it divides by dichotomy, and (3) its cell envelope differs from other Gram-negative bacteria. Similar, (
4) contains ribosomes similar to those of other bacteria, and (5) is sensitive to various antibiotics. Chlamydia can be observed under light microscopy, and its genome is about one-third the size of the Escherichia coli genome.

[0005] Many different strains of Chlamydia have been isolated from birds, humans, and other mammals, and these strains can be distinguished based on host range, virulence, pathogenicity, and antigenic composition . Although there is strong homology of DNA within each species, surprisingly there is little homology between species, suggesting many years of evolutionary segregation.

C. Trachomatis has a high degree of host specificity and is almost completely restricted to humans; this causes widely varying degrees of eye and urogenital infections. In contrast, C.I. Psittaci strains are rare in humans, but are found in a wide range of birds and are also found in wild, domestic and research mammals, where many organs Amplify in cells.

C. pneumoniae is a common human pathogen, originally from C.
Although described as a TWAR strain of psittaci, it was later recognized as a new species. C. pneumoniae is another Chlamydia species (C. tr.
achomatis, C.I. pecorum and C.I. psittaci)
It is distinguished antigenically, genetically, and morphologically. This is C.I. trachoma
tis or C.I. It shows no more than 10% DNA sequence homology with any of the psittaci, and so far appears to consist only of a single strain TWAR.

C. pneumoniae is a common cause of community-acquired pneumonia;
ococcus pneumoniae and Mycoplasma pneu
Slightly less frequent than S. moniae. Grayston et al. Inf
ect. Dis. 168: 1231 (1995); Campos et al., Inves.
t. Ophthalmol. Vis. Sci. 36: 1477 (1995), each of which is incorporated herein by reference. It can also cause upper respiratory symptoms and diseases, including bronchitis and sinusitis. For example, Grayst
on et al. Infect. Dis. 168: 1231 (1995); Camp.
os et al., Invest. Ophthalmol. Vis. Sci. 36: 147
7 (1995); Grayston et al. Infect. Dis. 161: 6
18 (1990); Marrie, Clin. Infect. Dis. 18: 5
01 (1993). The majority (> 60%) of the adult population is C.I.
pneumoniae (Wang et al., Chlamydial)
Infections, Cambridge University Pre
ss, Cambridge, p. 329 (1986)), which indicates an unrecognized or asymptomatic past infection.

C. Pneumoniae infection usually manifests as an acute respiratory disorder (ie, cough, sore throat, hoarseness, and fever; abnormal chest sounds at auscultation). For most patients, cough persists for 2-6 weeks and recovery is slow. About 10 of these cases
In%, upper respiratory tract infection is followed by bronchitis and pneumonia. Further, C.I. pe
During the numoniae epidemic, subsequent coinfection with pneumococci has been described in about half of these pneumonia patients, especially the debilitated and elderly. As described above,
C. There is more evidence that pneumoniae infection is also associated with diseases different from respiratory infections.

[0010] The reservoir for an organism is probably a human. C. In contrast to psittaci infection, no bird or animal reservoir is known. Propagation is not explicitly defined. This can result from direct contact with secretions, formats, or air-borne transmission. The incubation period is long, which can last for months. Based on epidemic analysis, C.I. pneumoniae slowly grows throughout the population (
In some cases (average 30-day intervals). Because infected people are inefficient spreaders of living things. C. Susceptibility to pneumoniae is universal. Reinfection occurs during adulthood, following the primary infection in children. C. Pneumoniae appears to be endemic throughout the world, with overlapping intervals (lasting 2-3 years) of increased incidence (epidemic). C.
Trachomatis infection is C. trachomatis infection. It does not confer cross immunity against Pneumoniae. Infections are 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 uncomplicated, and up to 90% of infections are subacute or unrecognized. Although infections have been considered uncommon among children in industrialized nations up to the age of five, recent studies have shown that many children in this age group, despite being seronegative, were not It shows evidence and reports an estimated prevalence of 17-19% at 2-4 years. Normann et al., Acta Paediatric
a, 87: 23-27 (1998). In developing countries, C.I. pneumoniae serum prevalence (seroprevalen)
ce) rises and C.I. It is suspected that pneumoniae may be an important cause of acute lower respiratory illness and mortality for infants and children in tropical regions of the world.

[0012] From studies of serum prevalence and studies of local epidemics, the first C. pneumonia
e-infection usually occurs between the ages of 5 and 20 years. For example, in the United States, C.I. pne
Childhood pneumonia caused by Umoniae is estimated to be 30,000 cases each year. Infections can be clustered among groups of children or young adults (eg, school children or conscripts).

C. pneumoniae causes 10-25% of community-acquired lower respiratory tract infections (
As reported by Sweden, Italy, Finland, and the United States).
During the epidemic, C.I. Pneumonia infection can account for 50-60% of cases of pneumonia. During these periods, and More onset of co-infection with Pneumoniae has been reported.

Reinfection during adulthood is common; clinical symptoms tend to be milder. Exposure tends to increase with age based on population serum prevalence studies,
This is especially evident among men. Some investigators report persistent asymptomatic C.I. p
neumoniae infection was presumed to be common.

In middle-aged or older adults, C.I. Pneumoniae infection can progress to chronic bronchitis and sinusitis. Studies in the United States have shown that C. in humans younger than 60 years. pneumoniae caused pneumonia 1 case / 1
2,000 / year; however, in the elderly, the incidence of the disease has tripled. C. Pneumoniae infection rarely results in hospitalization except in patients with underlying disease.

Of great importance are atherosclerosis and C. cerevisiae. pneumoniae infection. C. There are several epidemiological studies that correlate previous infections with pneumoniae with heart attacks, coronary artery disease and carotid artery disease. Saikk
u et al., Lancet 2: 983 (1988); Thom et al., JAMA 268.
: 68 (1992); Linnanmaki et al., Circulation 87.
: 1030 (1993); Saikku et al., Annals Int. Med. 1
16: 273 (1992); Melnick et al., Am. J. Med. 95:49
9 (1993). In addition, the organism has been detected in the atheroma and adipose striatum of the coronary arteries and coronary aorta, carotid artery and carotid aorta, peripheral arteries and peripheral aorta. Shor et al., South Africa Me
d. J. 82: 158 (1992); Kuo et al. Infect. Dis. 1
67: 841 (1993); Kuo et al., Arteriosclerosis a.
nd Thrombosis 13: 1500 (1993); Campbell
J. et al. Infect. Dis. 172: 585 (1995); Chiu et al., C.
irculation 96: 2144-2148 (1997).
Viable C.I. pneumoniae has been recovered from the coronary and carotid arteries. Ramirez et al., Annals Int. Med. 125: 979 (1
996); Jackson et al., Abst. K121, 272 pages, 36th ICA
AC, New Orleans (1996). Further, C.I. pneumonia
e has been shown to be able to induce changes in atherosclerosis in a rabbit model. Fong et al., (1997) Journal of Clinic.
See al Microbiology 35:48. Taken together, these results indicate that C.I. Although pneumoniae is likely to cause atherosclerosis in humans, it shows that the epidemiological significance of chlamydia atherosclerosis remains unproven.

A number of recent studies have also described C.I. Figure 2 shows an association between pneumoniae infection and asthma. Infection is associated with asthma, asthmatic bronchitis, adult-onset asthma, and acute relapse of asthma in adults, and small-scale studies suggest that sustained antibiotic treatment may result in severe disease in some individuals. It was shown to be effective in greatly reducing the degree. Hahn et al., Ann Allergy Asthm.
a Immunol. 80: 45-49 (1998); Hahn et al., Epide.
miol Infect. 117: 513-517 (1996); Bjorns.
Son et al., Scan J Infect Dis. 28: 63-69 (199
6); Hahn, J .; Fam. Pract. 41: 345-351 (1995)
Allegra et al., Eur. Respir. J. 7: 2165-2168 (1
994); Hahn et al., JAMA 266: 225-230 (1991).

In light of these results, C.I. Protective vaccines against diseases caused by pneumoniae infection are of considerable importance. Human C. pneum
There is still no effective vaccine for oniae infection. Nevertheless, C.I. tr
achomatis and C.I. Studies using psittaci have shown that
Indicates a achievable 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., Vac
cine 13: 715 (1995). Protection from Chlamydia infection is associated with a Th1 immune response, particularly the induction of INFγ-producing CD4 + T cells. Igies
emes et al., Immunology 5: 317 (1993). Adoptive transfer of CD4 + cell lines or clones to nude or SCID mice confers protection from challenge or eliminates chronic disease (Igietseme).
Et al., Regional Immunology 5: 317 (1993); Ma.
gee et al., Regional Immunology 5: 305 (1992).
And depletion of CD4 + T cells in vivo exacerbated disease following challenge (Landers et al., Infection & Immunity 59: 377).
4 (1991); Magee et al., Infection & Immunity 63.
: 516 (1995)). However, the presence of sufficiently high titers of neutralizing antibodies on mucosal surfaces can also exert a protective effect. Cotter et al., Infection and
Immunity 63: 4704 (1995).

C. The degree of antigenic diversity within the Pneumoniae species is not well characterized. C. Trachomatis serovars are defined based on antigenic diversity in major outer membrane proteins (MOMPs), but have been published by C. trachomatis. pn
The E. moniae MOMP gene sequence does not show variability among several diverse isolates of the organism. Campbell et al., Infection and Imm.
unity 58:93 (1990); McCafferty et al., Infect.
ion and Immunity 63: 2387-9 (1995); Knu
dsen et al., Third Meeting of the European.
Society for Chlamydia Research, Vienna
a (1996). Regions of the protein known to be conserved in other Chlamydia MOMPs are described in pneumoniae. Campbell et al., Infection and Immunity.
y 58:93 (1990); McCafferty et al., Infection.
and Immunity 63: 2387-9 (1995). Certain studies have shown that C.P. pneumoniae
But the gene has not been sequenced. Grayston et al. Infect. Dis. 168: 1231 (1995). The partial sequence of outer membrane protein 2 from 9 diverse isolates was also found to be universal.
Ramirez et al., Annals Int. Med. 125: 979 (1996)
). The genes for HSP60 and HSP70 show little variation from other Chlamydia species, as expected. The gene encoding the 76 kDa antigen is C. pneumoniae has been cloned from a single strain. It has no significant similarity to other known chlamydia genes. Marrie, C
lin. Infect. Dis. 18: 501 (1993).

C. Many antigens recognized by immune sera against P. pneumoniae are conserved across all Chlamydia, but 98 kDa, 76 kDa, and 54 kDa proteins are C. pneumoniae. pneumoniae-specific. C
ampos et al., Invest. Ophthalmol. Vis. Sci. 36:
1477 (1995); Marrie, Clin. Infect. Dis. 18
: 501 (1993); Wiedmann-Al-Ahmad et al., Clin. D
iagn. Lab. Immunol. 4: 700-704 (1997). Immunoblotting of isolates with serum from patients showed a variation in the blotting pattern between the isolates, pneumoniae serotype may be present. Grayston et al. Infect. Dis. 168: 1
231 (1995); Ramirez et al., Annals Int. Med. 12
5: 979 (1996). However, the results can be confused by the infection status of the patient. This is because the immunoblot profile of the patient's serum changes over time after infection. Evaluation of the number and relative frequency of any serotype and determination of antigen is not yet possible.

Thus, there remains a need for compositions that are effective for preventing, treating, and diagnosing Chlamydia infection.

SUMMARY OF THE INVENTION In one aspect, the present invention provides Chlamydia-encoded, purified and isolated DNA molecules, which are used in methods for preventing, treating, and diagnosing Chlamydia infection. Can be done. The present invention provides eight additional preferred DNA molecules (each individually and separately, SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13 or 15). In addition, eight other preferred polypeptides (each individually and separately, SEQ ID NOs: 2, 4, 6, 8, 10, 12,
114, or 16) are provided in the present invention. Those skilled in the art will appreciate that the present invention also provides for DNA molecules encoding variants, variants and derivatives of such polypeptides, which result from the addition, deletion or substitution of non-essential amino acids as described herein. Understand that it includes. The present invention also includes RNA molecules corresponding to the DNA molecules of the present invention.

In addition to DNA and RNA molecules, the invention includes its corresponding polypeptide and monospecific antibodies that specifically bind to such a polypeptide.

The present invention has a wide range of applications and includes expression cassettes, expression vectors, and cells transformed or transfected with a polynucleotide of the present invention.
Accordingly, the invention provides: (i) a method of producing a polypeptide of the invention in a recombinant host system, and related expression cassettes, expression vectors, and producing transformed or transfected cells. (Ii) a live vaccine vector (eg, a viral or bacterial live vaccine vector (eg, a poxvirus vector, an alphavirus vector, a Salmonella typhimurium vector, or a Vib) comprising the polynucleotide of the present invention;
rio cholerae vectors)) (such vaccine vectors, in combination with diluents or carriers, and associated pharmaceutical compositions and associated methods of treatment and / or prevention, for example, to prevent and treat Chlamydia infections). (Iii) an RNA or DNA molecule of the invention (either in naked form or formulated with a delivery vehicle), a polypeptide or combination of polypeptides of the invention, or monospecific. Therapeutic and / or prophylactic methods, including the administration of a specific antibody and a related pharmaceutical composition; (
iv) a method for diagnosing the presence of Chlamydia in a biological sample, which may involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention; and (v) an antibody-based affinity chromatograph. A method for purifying a polypeptide of the invention by chromatography.

(Detailed Description of the Invention) In the pneumoniae genome, an open reading frame (ORF) encoding a Chlamydia polypeptide has been identified. These polypeptides include those that are permanently found in the bacterial membrane structure, those that are located near the exterior of the bacterial membrane, those that are permanently found in the encapsulated membrane structure, And polypeptides released into the cytoplasm of infected cells. These polypeptides are available from Chlamy.
dia infections 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.

The isolated polynucleotide of the invention has an amino acid sequence homologous to the Chlamydia amino acid sequence, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16
Encodes a polypeptide having a Chlamydia amino acid sequence selected from the group consisting of the amino acid sequences shown in Table 1.

The term “isolated polynucleotide” is defined as a polynucleotide that has been removed from its naturally occurring environment. For example, in the bacterial genome,
A naturally occurring DNA molecule has not been isolated, but has been isolated, for example, as a result of a cloning event (amplification), from the rest of the bacterial genome. Typically, an isolated DNA molecule does not contain a DNA region (eg, a coding region) that is immediately contiguous at the 5 ′ end or 3 ′ end in the genome where it naturally occurs. 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. I have.

The polynucleotide of the present invention may be in the form of RNA or DNA (eg, cDNA, genomic DNA, or synthetic DNA), or a variant or combination thereof. DNA can be double-stranded or single-stranded, and if single-stranded,
It can be the coding strand or the non-coding (antisense) strand. SEQ ID NOs: 2, 4, 6,
The sequence encoding the polypeptide of the present invention as shown in 8, 10, 12, 14, or 16 comprises (a) any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15 A coding sequence as indicated; (b) a ribonucleotide sequence induced by the transcription of (a); or (c) a different coding sequence; the latter as a result of the redundancy or degeneracy of the genetic code. Encodes the same polypeptide as the DNA molecule whose nucleotide sequence is set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

“Homologous amino acid sequence” refers to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 only by one or more conservative amino acid substitutions.
Alternatively, it means an amino acid sequence that differs by substitution, deletion or addition of one or more non-conservative amino acids located at positions that do not destroy the specific antigenicity of the polypeptide.

Preferably, such a sequence is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14
Or at least 75%, more preferably 80%
%, And most preferably 90% identical.

The homologous amino acid sequences are shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.
And sequences that are identical or substantially identical to the amino acid sequence as shown in "
An “amino acid sequence that is substantially identical” refers to at least 90 amino acids in the reference amino acid sequence.
%, Preferably 95%, more preferably 97%, and most preferably 99% identical, and preferably means any sequence that differs from its reference sequence, if any, by conservative amino acid substitutions. I do.

[0033] Conservative amino acid substitutions typically involve substitutions between amino acids of the same class.
These classes include, for example: (a) amino acids with uncharged polar side chains (eg, asparagine, glutamine, serine, threonine and tyrosine); (b) amino acids with basic side chains (eg, , Lysine, arginine and histidine); (c) amino acids with acidic side chains (eg, aspartic acid and glutamic acid); 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 A).
analysis Software Package of the Gene
tics Computer Group, University of Wi
scossin Biotechnology Center, 1710 Un
(Average Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain a maximum degree of homology (ie, identity). For this purpose, it may be necessary to introduce gaps in the sequence. Once the optimal alignment has been set, the degree of homology (ie, identity) is determined by recording all positions where the amino acids in both sequences are identical to the total number of positions. You.

Alternatively, homology can be determined by alignment tools (eg, Needleman et al.,
J. Mol. Biol. 48: 443 (1970), and Align Program (DNAstar, Inc.).
(A commercial software package produced by) (the teachings of which are incorporated herein by reference)) can be determined by aligning the candidate and reference sequences. After an initial alignment has been created, 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 has been created and refined, a percent homology score is calculated. The individual amino acids in each sequence are
They are continuously 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 ATLA.
S OF PROTEIN SEQUENCE AND Structure
345-352 (1978 and addenda), incorporated herein by reference.
It is a PAM25O matrix described in (1). The similarity score is initially calculated as the sum of the aligned paired amino acid similarity scores. 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 the raw score by the geometric mean of the scores of the candidate compound and the reference sequence. The geometric mean is the square root of the product of these scores. The normalized raw score is the percent homology.

Preferably, the homologous sequence is SEQ ID NO: 1, 3, 5, 7, 9, 10, 11, 13,
And 15 coding sequences are at least 45%, more preferably 60%, and most preferably 85% identical to the 15 coding sequences.

One of the sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16
Polypeptides having sequences homologous to each other include naturally-occurring allelic variants and polypeptides having sequences as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16. Variants include variants and variants similar in antigenicity or any other non-naturally occurring variants.

An allelic variant does not substantially alter the biological function of the polypeptide.
An alternative form of the polypeptide characterized by having the above amino acid substitutions, deletions or additions. By "biological function" is meant the function of a polypeptide in a cell where the polypeptide is naturally present, even if the function is not necessary for the growth or survival of the cell. For example, the biological function of porin is to allow the entry of compounds present in the extracellular medium into cells. Biological functions differ from antigenic functions. A polypeptide may have more than one biological function.

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

[0041] Support for the use of allelic variants of a polypeptide antigen is described, for example, by Chlamy.
generated by studies of the dial MOMP antigen. Although the amino acid sequence of MOMP varies from strain to strain, cross-strain antibody binding and neutralization of infectivity still occur, which is a consequence of the amino acid variation when MOMP is used as an immunogen. Indicates resistance.

A polynucleotide (eg, a DNA molecule) encoding an allelic variant can include:
Polymerase chain reaction (P) of bacterial genomic DNA extracted by conventional methods
By CR) amplification, it can be easily recovered. This involves the use of synthetic oligonucleotide primers that fit upstream and downstream of the 5 'and 3' ends of the coding domain. Suitable primers are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 1
3 and 15 can be designed according to the nucleotide sequence information provided. Typically, a primer may consist of 10 to 40 nucleotides, preferably 15 to 25 nucleotides. It is also possible to select primers containing C and G nucleotides in sufficient proportions to ensure efficient hybridization (eg, at least 40%, preferably 50% C and G nucleotides) of the total nucleotides. It can also be advantageous.

Useful non-naturally occurring homologs can be designed utilizing known methods for identifying regions of the antigen that are resistant to amino acid sequence changes and / or deletions. For example, sequences of antigens 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 has SEQ ID NOs: 2, 4, 6, 8, 10 to the extent that the fragment retains the desired substantial antigenicity (specific antigenicity) of its parent polypeptide. , 12, 14 or 16 can be derived from a polypeptide having a sequence homologous to any of the sequences shown in FIG. Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial portion of the parent polypeptide while retaining the desired specific antigenicity. Generally, a polypeptide derivative should be at least about 12 amino acids in length to maintain its antigenicity. Advantageously, this is at least 20 amino acids in length, preferably at least 50 amino acids in length,
More preferably at least 75 amino acids in length, and most preferably at least 1
It can be as long as 00 amino acids.

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. Hughes et al., Infect. Immun. 60: 3497 (1992).

Polypeptide fragments and polypeptides with large internal deletions are otherwise masked in the parent polypeptide and may be, for example, epitopes that may be important for inducing a protective T cell-dependent immune response Can be used to reveal Deletions can also remove highly variable immunodominant regions between strains.

[0048] The use of fragments and variants of protein immunogens as vaccines and immunogens is a recognized routine in the field of immunology. This is because only a small (eg, 8-10 amino acid) region of the protein may be required to elicit an immune response to the protein. This has been done for many vaccines against pathogens other than Chlamydia. For example, a short synthetic peptide corresponding to the surface-exposed antigen of a pathogen (eg,
Peptide containing 11 amino acids of mouse mammary adenocarcinoma virus (Dion et al., Viro
logy 179: 474-477 (1990));
Peptides containing 16 amino acids (Snijders et al., J. Gen. Virol.
72: 557-565 (1991)); and two overlapping peptides of canine parvovirus, each containing 15 amino acids (Langeveld et al., Vaccin.
e 12: 1473-1480 (1994))) have been shown to be effective vaccine antigens against each of these pathogens.

[0049] Polypeptide fragments and polynucleotides encoding polypeptides with large internal deletions can be produced, for example, by PCR (inverse P
(Including CR), by restriction enzyme treatment of the cloned DNA molecule, or by Kunkel et al. (Proc. Natl. Acad. Sci. USA 82:44).
8 (1985)) by standard methods (eg, Ausubel et al., C.I.
URENT PROTOCOLS IN MOLECULAR BIOLOG
Y, John Wiley & Sons Inc. (See (1994)) (biological material is available from Stratagen).

[0050] Polypeptide derivatives also include a polypeptide or polypeptide derivative of the invention fused to any other polypeptide, for example, at the N-terminus or C-terminus.
It can be produced as a fusion polypeptide. To construct a DNA encoding the amino acid sequence corresponding to the hybrid fusion protein, CPN100111, CPN1
00224, CPN100230, CPN100231, CPN100232,
The first DNA encoding the amino acid sequence corresponding to the portion of the nucleotide sequence (SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15) of CPN100233, CPN100394, CPN100395 is described in, for example, US Pat. 844,0
No. 95, incorporated by reference herein, is ligated to the second DNA. The product can then be easily obtained by translation of the gene fusion. Vectors for expressing the fusion polypeptide are commercially available (eg, the New England Biolabs pMal-c2 or pMal-p2 systems, where the fusion peptide is a maltose binding protein), and 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, such as subunit B of either cholera toxin or E. coli non-thermostable toxin). And a polypeptide or polypeptide derivative of the present invention fused to Several possibilities can be used to achieve fusion. First, the polypeptides of the present invention can be fused to the N-terminus, or preferably the C-terminus, of a polypeptide having adjuvant activity. Second, polypeptide fragments of the present 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, which may mature into a polypeptide of the invention. "Heterologous signal peptide" means a signal peptide that is not found in naturally occurring precursors of a polypeptide of the invention.

A polynucleotide of the present invention having a homologous coding sequence comprises SEQ ID NOs: 2, 4,
It hybridizes to a polynucleotide having a sequence as set forth in 6, 8, 10, 12, 14, and 16, preferably under stringent conditions. Hybridization procedures are described, for example, in Ausubel et al., CURRENT PRO.
TOCOLS IN MOLECULAR BIOLOGY, John Wil
eye & Sons Inc. (1994); Silhavey et al., EXPERIME.
NTS WITH GENE FUSIONS, Cold Spring Ha
rbor Lanoratory Press (1984); Davis et al., A.
MANUAL FOR GENETIC ENGINEERING: ADVA
NCED BACTERIAL GENETICS, Cold Spring
Harbor Laboratory Press (1980), each of which is incorporated herein by reference. An important parameter that can be considered for optimizing hybridization conditions is the critical value (two complementary D
The melting temperature at which the NA chains separate from each other) is reflected in the equation that allows the calculation. Case
y and Davidson, Nucl. Acid Res. 4: 1539 (19
77). This equation is as follows: Tm = 81.5 + 0.5 × (% G + C) +1.6 log (cation concentration) −0
. 6x (% formamide).

Under appropriate stringency conditions, the hybridization temperature (Th) can be about 20-40 ° C., 20-25 ° C., or preferably 30 ° C. above the calculated Tm.
~ 40 ° C lower. 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 include (i) within 4-16 hours at 42 ° C. in 6 × SSC containing 50% formamide for both prehybridization and hybridization incubation, or (ii) 6 ×
SSC aqueous solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)
)) At 65 ° C. within 4 to 16 hours.

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

Hybridization conditions for oligonucleotides shorter than 20-30 bases do not exactly follow the rules described above. In such a case, the formula for calculating Tm is as follows: Tm = 4 × (G + C) +2 (A + T). For example, a 50% G + C 18 nucleotide fragment has a Tm of about 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, a DNA molecule is (
A vaccine, such as a poxvirus, further used in (i) a process for producing the encoded polypeptide in a recombinant host system, (ii) in a method and composition for preventing and / or treating Chlamydia infection. In the construction of the vector, (iii) as a vaccine agent (and RNA molecule) in naked form or formulated in a delivery vehicle, and (iv) the polynucleotide of the invention can be overexpressed or, if appropriate, An attenuated C that can express the polynucleotide in a modified, mutated (eg, non-toxic) form
hlamydia strains can be used in construction. For vaccine compositions and uses of the proteins and peptides and coding nucleotides of the invention for protection against diseases caused by Chlamydia, naked DNA encoding the protein or peptide, and intranasal or intramuscular administration of these nucleotides Is not preferred. For these proteins, administering the encoding nucleic acid by another route (eg, intradermal); and / or
Alternatively, it is preferred to formulate the encoding nucleic acid to improve (or assist) the immune response. 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.

Thus, according to a second aspect of the present invention there is provided: (i) the present invention arranged under the control of elements required for expression, especially under the control of a suitable promoter. (Ii) an expression vector containing the expression cassette of the present invention; (iii) a prokaryotic cell or eukaryotic transformed or transfected with the expression cassette and / or the expression vector of the present invention. Biological cells, and (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, wherein the process is transformed or transfected with an expression cassette and / or expression vector of the invention. A prokaryotic or eukaryotic cell that allows expression of a DNA molecule of the present invention. And recovering the encoded polypeptide or polypeptide derivative from the cell culture.

[0060] Recombinant expression systems can be selected from prokaryotic and eukaryotic hosts. Eukaryotic hosts include yeast cells (eg, Saccharomyces cerevis).
iae or Pichia pastoris, mammalian cells (eg, CO
S1 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 are derived from many different sources (eg, Amer
ican Type Culture Collection (ATCC; Ro)
ckville, Maryland)) available to those skilled in the art.

The choice of the expression system depends on the desired characteristics for the expressed polypeptide.
For example, it may be useful to produce a polypeptide of the invention in a particular lipidated or 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 is functional in the selected host system and can be constitutive or inducible;
Ribosome binding site; initiation codon (ATG) if necessary; signal peptide (
For example, a region coding for a lipidated signal peptide); a DNA molecule of the present invention; a stop codon; The signal peptide coding region is adjacent to the polynucleotide of the invention and is located in a suitable reading frame. The signal peptide coding region 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 constituted by the DNA molecule of the present invention, alone or together with a signal peptide, is placed under the control of a promoter, resulting in transcription and translation in the host system. Promoters, signal peptide coding regions, are widely known and available to those of skill in the art, and include, for example, the promoter (and derivative) of Salmonella typhimurium, which is inducible by arabinose (promoter araB), and gram. Functional in negative bacteria (eg, E. coli) (US Pat. No. 5,028,530; and Cagnon
(Cagnon et al., Protein Engineering 4: 843 (
1991))); the promoter of the gene for bacteriophage T7, which encodes an RNA polymerase, which is functional in many E. coli strains expressing T7 polymerase (US Pat. 952
No. 496)); OspA lipidated signal peptide; and RlpB
Lipidation signal peptide (Takase et al., J. Bact. 169: 5692 (
1987)).

The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system. Expression vectors (eg,
Plasmid vectors or viral vectors) are described in Pouwels et al.
ing Vectors: Laboratory Manual, 85, addendum,
1997). These can be purchased from various commercial sources.

Methods for transforming / transfecting host cells with expression vectors are described in Ausubel et al., Current Protocols in Mole.
cultural Biology, John Wiley & Sons Inc. (1
994), depending on the host system chosen.

Upon expression, a recombinant polypeptide (or polypeptide derivative) of the invention is produced in the intracellular compartment and remains there or secreted / excreted into the extracellular medium or into the periplasmic space. Or embedded in the cell membrane. The polypeptide may then be recovered in substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification, or any other method that can be readily adapted by one of skill in the art, such as by fusion of the gene to a small affinity binding domain. obtain. Antibody-based affinity purification methods can also be used to purify polypeptides of the invention extracted from Chlamydia strains. Useful antibodies for purifying the polypeptide of the present invention by immunoaffinity can be obtained as follows.

The polynucleotides of the present invention may also be used in the field of vaccines (eg, DNA
(To achieve 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). The therapeutic or prophylactic efficacy of a polynucleotide of the present invention can be assessed as described below.

Thus, in a third aspect of the invention there is provided: (i) a vaccine vector (eg, a vaccine vector comprising a DNA molecule of the invention which is placed under the control of elements required for expression) (Poxvirus); (ii) a composition comprising a vaccine vector of the invention together with a diluent or carrier; in particular, (iii) a pharmaceutical composition comprising a therapeutic or prophylactic amount of a vaccine vector of the invention; iv) Immune response against Chlamydia in mammals (eg, humans; alternatively, this method can be used in veterinary applications to treat or prevent Chlamydia infection in animals (eg, cats or birds)) A method for inducing a mammal with an immunogenically effective amount of a vaccine vector of the present invention. Administering, to elicit an immune response (eg, a prophylactic or therapeutic immune response against Chlamydia); and, in particular, (v)
Chlamydia (eg, C. trachomatis, C. psitta)
ci, C.I. pneumonia, C.I. pecorum) A method for preventing and / or treating an infection, comprising administering to a person in need thereof a prophylactically or therapeutically effective amount of a vaccine vector of the present invention. .
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. In addition, it may express a cytokine (eg, interleukin-2 (IL-2) or interleukin-12 (IL-12)) that enhances the immune response (adjuvant effect). 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 include a further Chlamydia antigen or subunit, fragment, homolog, variant, or derivative thereof; or a vaccine vector capable of expressing a cytokine (eg, IL-2 or IL-12).

In a method of administering a vaccine to treat or prevent infection in a mammalian cell, the vaccine vector of the invention may be administered by any conventional route of use in the field of vaccines, especially mucosal (eg, ocular, nasal) Intra-orally, stomach, lung, intestinal, rectal, vaginal, or urinary tract, or parenterally (eg, subcutaneously, intradermally, intramuscularly, intravenously,
Or intraperitoneal) route. The preferred route depends on the choice of vaccine vector. Administration can be accomplished in a single dose or repeatedly at intervals. The appropriate dosage depends on various parameters understood by those skilled in the art, such as the vaccine vector itself, the route of administration, or the condition (body weight, age, etc.) of the mammal to be vaccinated.

Live vaccine vectors available in the art include viral vectors (eg, adenovirus, alphavirus, and poxvirus), and bacterial vectors (eg, Shigella, Salmonella, Vibr)
io cholerae, Lactobacillus, Bacille bi
lie de Calmette-Guerin (BCG) and Strep
tococcus).

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, vaccinia virus and canarypox virus, which are described in U.S. Patent Nos. 4,722,848 and 5,364,773, respectively (e.g., For a description of vaccinia virus vectors, see Tartaglia et al.,
Virology 188: 217 (1992); and for references on canarypox virus, see Taylor et al., Vaccine 13: 539 (199).
See also 5)). Poxvirus vectors capable of expressing the polynucleotides of the present invention can be obtained by homologous recombination, as described in Kieny et al., Nature 312: 163 (1984), so that the polynucleotides of the present invention can be used in mammals. Inserted into the viral genome under conditions appropriate for expression in animal cells. Generally, the dose of the vaccine viral vector will range from about 1 × 10 ⁴ to about 1 × 10 ¹¹ per kilogram for therapeutic or prophylactic use, advantageously
About ^{1 × 10 7 ~1 × 10 10} , may be preferably about 1 × 10 ⁷ ~ about 1 × 10 ⁹ plaque forming units. Preferably, the viral vector is administered parenterally; for example, three administrations at four week intervals. One skilled in the art will recognize that it is preferable to avoid adding a chemical adjuvant to the composition comprising the viral vector of the present invention, thereby minimizing the immune response to the viral vector itself.

Non-toxin producing Vibrio cholerae mutants useful as live oral vaccines are described in Mekalanos et al., Nature 306: 551 (1983) and US Pat. No. 4,882,278 (the coding sequence for each of the two ctxA alleles). WO 92/11354 (strain in which the irgA locus is inactivated by mutation; this mutation is And WO 94/1533 (deletion mutant lacking functional ctxA and attRS1 DNA sequences). These strains are described in WO 94/1948.
2, can be engineered to express a heterologous antigen. An effective vaccine dose of a Vibrio cholerae strain capable of expressing the polypeptide or polypeptide derivative encoded by the DNA molecule of the present invention will be in a volume appropriate for the route of administration selected, for example, from about 1 × 10 ⁵ to about It may contain 1 × 10 ⁹ , preferably from about 1 × 10 ⁶ to about 1 × 10 ⁸ viable bacteria. Preferred routes of administration include all mucosal routes; most preferably, these vectors
It is administered intranasally or orally.

Attenuated Salmonella typhimurium strains, engineered or unengineered for recombinant expression of heterologous antigens, and their use as oral vaccines are described in Nakayama et al., Bio / Tech.
No. 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 are described in 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)
).

Depending on the bacterial vector, the polynucleotides of the present invention may be inserted into the bacterial genome or may be included in a plasmid and remain free.

An adjuvant may 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.

In 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) a therapeutic or A pharmaceutical composition comprising a prophylactically effective amount; (iii) a method for inducing an immune response against Chlamydia in a mammal, the method comprising: providing a mammal with an immunogenically effective amount of a polynucleotide of the invention. By inducing an immune response (eg, a protective immune response against Chlamydia); and, in particular, (iv) Chlamydia (eg, C. trachomatis, C. psittaci, C. pneumon).
iae, or C.I. pecorum) A method for preventing and / or treating an infection, the method comprising administering to a subject in need thereof a prophylactic or therapeutic amount of a polynucleotide of the present invention. Further, a fourth aspect of the present invention encompasses the use of a polynucleotide of the present invention in the preparation of a medicament for preventing and / or treating Chlamydia infection. A fourth aspect of the present invention relates to a DNA molecule, preferably placed under conditions for expression in a mammalian cell (eg, unable to replicate in a mammalian cell and substantially integrated into a mammalian genome). (In plasmids that cannot be used).

The polynucleotides (DNA or RNA) of the present invention can also be administered to a mammal for vaccine purposes, eg, 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 be replicated in mammalian cells and that cannot be integrated into the mammalian genome. Typically, the DNA molecule will be under the control of a promoter appropriate for expression in mammalian cells. The promoter can function ubiquitously or tissue-specific. Examples of non-tissue specific promoters include early cytomegalovirus (CMV)
) Promoter (described in U.S. Patent No. 4,168,062), and the Rous sarcoma virus promoter (Norton & Coffin, Molec. Ce).
11 Biol. 5: 281 (1985)). Desmin promoter (Li et al., Gene 78: 243 (1989), Li & Paul)
in, J .; Biol. Chem. 266: 6562 (1991), and Li &
Paulin, J. et al. Biol. Chem. 268: 10403 (1993)) are tissue-specific and drive 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. If it encodes a precursor type, the precursor type can be homogeneous or heterogeneous. In the latter case, a eukaryotic leader sequence such as a tissue type plasminogen factor (tPA) leader sequence may be used.

[0081] The compositions of the invention may comprise one or several polynucleotides of the invention. It may also include at least one additional polynucleotide encoding another Chlamydia antigen or fragment, derivative, variant, or analog thereof. Cytokines (eg, interleukin-2 (IL-2)
Alternatively, a polynucleotide encoding interleukin-12 (IL-12)) can also be added to the composition, resulting in an enhanced immune response. These additional polynucleotides are under appropriate control for expression. Advantageously, the DNA molecules of the invention and / or further DNA molecules contained in the same composition may be contained in the same plasmid.

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

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

[0084] Alternatively, the polynucleotide may be associated with an agent that aids cellular uptake. It includes, inter alia, (i) bupivacaine (eg, WO 94/16737)
Supplemented with chemical agents that modify the permeability of cells, such as (i.
i) encapsulated in liposomes or (iii) cationic lipids,
Alternatively, it can be accompanied by silica fine particles, gold fine particles, or tungsten fine particles.

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

[0086] Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids are known as Lipofectin ^™ (DOTMA (N
-[1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride)), DOTAP (1,2-bis (oleyloxy) -3- (trimethylammonio) ) Propane), DDAB (dimethyldioctadecyl ammonium bromide), DOGS (dioctadecylamidologlysyl spermine)
ermine)) and cholesterol derivatives (eg, DC-Chol (3β
-(N- (N ', N'-dimethylaminomethane) -carbamoyl) cholesterol)). Descriptions of these cationic lipids are given in EP 187,702, WO
90/11092, U.S. Pat. No. 5,283,185, WO 91/1550
1, WO 95/26356, and US Pat. No. 5,527,928. The cationic lipid for gene delivery is preferably, for example, WO 90
/ 11092, as used in conjunction with neutral lipids such as DOPE (dioleyl phosphatidylethanolamine).

[0087] Other transfection facilitating compounds may be added to the formulation containing the cationic liposomes. Many of them are described, for example, in WO 93/18759, WO 9
3/19768, WO 94/25608, and WO 95/2397. They include, inter alia, spermine derivatives useful for facilitating the transport of DNA across the nuclear membrane (see, eg, WO 93/18759), and membrane permeable compounds (eg, GALA, gramicidin S, and cations). Sex bile salts) (see, for example, WO 93/19768).

Gold or tungsten microparticles are also described in WO 91/359, WO 93
/ 17706, and Tang et al. (Nature 356: 152 (1992)
), Can be used for gene delivery. In this case,
Microparticle-coated polynucleotides have been described in needleless injection devices ("gene guns") (see, e.g., U.S. Patent Nos. 4,945,050, 5,015,580, and WO 94/24263). Can be injected via the intradermal or intraepithelial route.

The amount of DNA used in a vaccine recipient depends, for example, on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product,
It depends on the intended condition of the mammal for administration (eg, body weight, age, and general health of the mammal), the mode of administration, and the type of formulation. Generally, from about 1 μg to
About 1 mg, preferably about 10 μg to about 800 μg, and more preferably about 2 μg
A therapeutically or prophylactically effective dose of 5 μg to about 250 μg can be administered to adult humans. This administration can be accomplished in a single dose or repeatedly at intervals.

The route of administration may be any of the conventional routes used in the field of vaccines. As general guidance, polynucleotides of the present invention may be administered to mucosal surfaces (eg, ocular, nasal, lung, oral, intestinal, rectal, vaginal, and urinary tract surfaces); or parenteral routes (eg, intravenous, (By subcutaneous, intraperitoneal, intradermal, intraepithelial, or intramuscular route). The choice of the route of administration depends, for example, on the formulation chosen. The polynucleotide formulated with bupivacaine is advantageously administered to muscle. If neutral or anionic liposomes or cationic lipids (eg, DOTMA or DC-Chol) are used, the formulation may be intravenous, intranasal (aerosol applied), intramuscular, intradermal. , And via the subcutaneous route. Naked forms of the polynucleotides may be advantageously administered via the intramuscular, intradermal, or subcutaneous routes.

[0091] Although not absolutely necessary, such compositions may also include an adjuvant. If so, a systemic adjuvant that does not require co-administration to exhibit an adjuvant effect (eg, QS21, which is described in US Pat.
057,546) are preferred.

The sequence information provided in the present application allows for the design of particular nucleotide probes and primers that may be useful in diagnosis. Accordingly, in a fifth aspect of the invention, the sequences found in the sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15 or derived from these sequences by degeneracy of the genetic code A nucleotide probe or primer having the sequence to be provided is provided.

As used in this application, the term “probe” refers to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15 under stringent conditions as defined above. A DNA molecule (preferably single-stranded) or RNA molecule (or a modification or combination thereof) that hybridizes to a nucleic acid molecule having a sequence homologous to the indicated sequence, or to a complementary or antisense sequence. Typically,
Probes are significantly shorter than the full-length sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15; for example, they are about 5 to about 100, preferably about 10
〜80 nucleotides. In particular, the probes have SEQ ID NOs: 1, 3, 5, 7
, 9, 11, 13 and 15 have a sequence that is at least 75%, preferably at least 85%, more preferably 95% homologous, or a sequence that is complementary to such a sequence. 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: 1).
497 (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'-hydroxyl group of a ribonucleotide can be modified, for example, by including an alkyl group.

The probes of the present invention can be used in diagnostic tests as capture or detection probes. Such capture probes can be conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption. The detection probe is a radioisotope; an enzyme (eg, an enzyme capable of hydrolyzing peroxidase, alkaline phosphatase, and a chromogenic, fluorogenic, or luminescent substrate); a compound that is chromogenic, fluorogenic, or luminescent; Nucleotide base analogs; and biotin.

The probes of the present invention can be used, for example, in dot blots (Maniatis et al., Mol.
ecological Cloning: A Laboratory Manual (1
982) Cold Spring Harbor Laboratory Pr
ess, Cold Spring Harbor, New York, Southern Blot (Southern, J. Mol. Biol. 98: 503 (1975).
)), Northern blots (same as Southern blots except that RNA is used as the target), or sandwich technology (Dunn et al., Cell 12:
23 (1977)) can be used in any conventional hybridization technique. The latter technique involves the use of specific capture probes and / or specific detection probes, with nucleotide sequences at least partially different from each other.

A primer is typically a probe of about 10 to about 40 nucleotides used to initiate enzymatic polymerization of DNA in an amplification process (eg, PCR), in an extension process, or in a reverse transcription method. . In diagnostic methods, including PCR, primers can be labeled.

Accordingly, the present invention also provides (i) a reagent comprising a probe of the present invention for detecting and / or identifying the presence of Chlamydia in a biological material; (ii)
A method for detecting and / or identifying the presence of Chlamydia in a biological material, wherein (a) the sample is recovered from or derived from the biological material, DNA or RNA is extracted from this material and denatured; and (c) exposed to a probe of the invention (eg, a capture probe, a detection probe, or both) under stringent hybridization conditions, and A method for detecting and / or identifying the presence of Chlamydia in a biological material, wherein (a) recovering a sample from the biological material Or derived from biological material, (b) DNA is extracted therefrom, and (c) the DNA of the present invention One even without preferably includes using two primers, the extracted DNA is primed, and is amplified by polymerase chain reaction, and (d) the amplified DNA fragment is produced, a method, a.

As previously mentioned, polypeptides that can be produced upon expression of a newly identified open reading frame are useful 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 it naturally occurs and / or the polypeptide present in the environment in which it was synthesized. It is defined as a polypeptide that does not contain a moiety. For example, a substantially purified polypeptide does not include a cytoplasmic polypeptide. One skilled in the art will appreciate that the polypeptides of the present invention can be obtained from natural sources (ie, Chla).
mydia strain) or may be produced by recombinant means.

A homologous polypeptide or polypeptide derivative encoded by a polynucleotide of the present invention is converted to a reference polypeptide having the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. Testing for cross-reactivity with the raised antisera can screen for specific antigenicity. Briefly, a monospecific hyperimmune antiserum is purified by itself or as a fusion polypeptide, using a purified reference polypeptide (eg, MBP, GST, or His-
Tag-based expression products, or synthetic peptides that are predicted to be antigenic). Homologous polypeptides or derivatives to be screened for specific antigenicity can be produced as such or as fusion polypeptides. In the latter case, where an antiserum is also raised against the fusion polypeptide, two different fusion systems are used. Specific antigenicity was determined by Western blot (Towbin et al., Proc. Natl. Acad.
Sci. USA 76: 4350 (1979)), dot blots, and EL
It can be determined according to a number of methods, including ISA.

In a Western blot assay, the products to be screened were purified as purified preparations or E. coli. E. coli whole extract, Lae
Subject to SDS-PAGE electrophoresis as described by mmli (Nature 227: 680 (1970)). After transferring to nitrocellulose membrane,
The material is further added to about 1: 5 to about 1: 5000, preferably about 1: 100 to about 1
: Incubate with monospecific hyperimmune antiserum diluted in a range of 500 dilutions. Specific antigenicity is indicated once the band corresponding to the product shows reactivity at any dilution in the above range.

In an ELISA assay, the product to be screened is preferably used as a coating antigen. Although purified products are preferred, whole cell extracts can also be used. Briefly, about 100 μl of the preparation at 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). Fill wells 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 plate is
Wash with BS / Tween buffer. The antiserum was prepared using P containing 0.5% BSA.
Dilute serially in BS / Tween buffer. Add 100 μl of diluent per well. The plate is incubated at 37 ° C. for 90 minutes,
Wash and evaluate according to standard procedures. For example, if specific antibodies were raised in rabbits, goat anti-rabbit peroxidase conjugate is added to the wells. Incubation is performed at 37 ° C. for 90 minutes, and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under the experimental conditions described above, a positive reaction resulted in a greater O.D. than non-immune control sera. 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 about 100 μl / ml of the product is
Dilute serially 2-fold in 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 were filled with 50 mM Tris-HCl
Wash by adding (pH 7.5) and air dry the membrane. The membrane was treated with a blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M N
aCl, 10 g / L skim milk), from about 1:50 to about 1: 5000,
Incubate with antiserum, preferably at a dilution of about 1: 500. The reaction is revealed 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 the appropriate substrate and terminated. The reaction is quantified visually by the appearance of a colored spot (eg, a colorimetric method). Under the experimental conditions described above, once a colored spot is associated with a dilution of at least about 1: 5, preferably at least about 1: 500, a positive reaction is indicated.

The therapeutic or prophylactic efficacy of a polypeptide or derivative of the present invention can be assessed 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) therapeutic or prophylactic treatment of a polypeptide of the present invention. (Iii) administering to a mammal an immunologically effective amount of a polypeptide of the present invention to elicit an immune response, such as an immune response to Chlamydia (e.g.,
A method of inducing a protective immune response against Chlamydia);
iv) by administering to a subject in need thereof a prophylactically or therapeutically effective amount of a polypeptide of the present invention, a Chlamydia (eg, C. tra).
chomatis, C.I. psittaci, C.I. pneumoniae, or C.I. pecorum) A method of preventing and / or treating infection. Furthermore, a seventh aspect of the present invention includes the use of a polypeptide of the present invention in the preparation of a medicament for preventing and / or treating Chlamydia infection.

The immunogenic compositions of the present invention may be used in the field of vaccines by any conventional route, especially mucosal (eg, ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urine) Tract) surface or parenterally (eg, subcutaneous, intradermal, intramuscular, intravenous,
Or via the 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 intranasal or oral route is preferred,
When using lipid formulations or aluminum compounds, the parenteral route is preferred.
In the latter case, the subcutaneous or intramuscular route is most preferred. The choice may also depend on the nature of the vaccine. For example, a polypeptide of the invention fused to CTB or LTB is best administered to mucosal surfaces.

A composition of the present invention may include one or several polypeptides or derivatives of the present invention. The composition of the present invention may also comprise at least one additional Chlamydia
It may comprise an antigen, or a subunit, fragment, homolog, variant or derivative thereof.

For use of the compositions of the present invention, polypeptides or derivatives thereof are 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 the immune response. These compounds are readily available to those skilled in the art; for example, LIPOSOME
S: A PRACTICAL APPROACH (see above).

Adjuvants other than liposomes and the like can also be used and are known in the art. Appropriate selections can be made conventionally by one of ordinary skill in the art (eg, from the list provided below).

Administration can be accomplished in a single dose, or can be repeated at intervals as needed, as can be determined by one skilled in the art. For example, a priming dose may be followed by three booster doses at weekly or monthly intervals. Appropriate doses can be determined by one skilled in the art, as can the recipient (eg, adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence / absence or type of adjuvant, and the desired effect (eg, , Protection and / or treatment). Generally, the vaccine antigens of the present invention have a
It may be administered by a mucosal route in an amount of from g to about 500 mg, preferably from about 1 mg to about 200 mg. For parenteral routes of administration, the dose should generally not exceed about 1 mg (preferably, about 100 μg).

When used as a vaccine, 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 initially primed (eg, via a parenteral route) with a vaccine vector of the invention, such as a poxvirus, and then with a polypeptide encoded by the vaccine vector. Two boosts (eg, via the mucosal route) can be provided. In another example, liposomes associated with a polypeptide or derivative of the present invention can also be used for priming, wherein a booster is administered with a soluble polypeptide of the present invention in combination with a mucosal adjuvant (eg, LT) or It is performed mucosally using derivatives.

The polypeptide derivatives of the present invention are also useful as diagnostic reagents for detecting the presence of an anti-Chlamydia antibody (eg, in a blood sample). Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length, and may be labeled or unlabeled depending 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 containing a fused tail that facilitates purification. The fusion product can be used to immunize a small mammal (eg, a mouse or rabbit) to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies). Accordingly, an eighth aspect of the present invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the present invention.

By “monospecific antibody” is meant an antibody capable of reacting with a unique, naturally occurring Chlamydia polypeptide. The antibodies of the present invention can be polyclonal or monoclonal antibodies. Monospecific antibodies are recombinant (eg, chimeric (eg, composed of a variable region of murine origin associated with a human constant region)).
, Humanized (human immunoglobulin constant scaffold with hypervariable regions of animal (eg, mouse) origin), and / or single-chain. Both polyclonal and monospecific antibodies are also immunoglobulin fragments (eg, F (ab
) '2 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 comprise a mixture of isotypes.

Antibodies of the invention raised against a polypeptide or polypeptide derivative of the invention can be produced and standard immunological assays (eg, Western blot analysis, dot blot analysis, or ELISA (eg, , Colligan et al.
CURRENT PROTOCOLS IN IMMUNOLOGY (1994
) John Wiley & Sons, Inc. , New York, NY)). This antibody can be used in diagnostic methods to detect the presence of Chlamydia antigen in a sample (eg, a biological sample). This antibody may also be used in affinity chromatography methods for purifying the polypeptide or polypeptide derivative of the present invention. As discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods.

Thus, a ninth aspect of the invention provides: (i) a Chlam in a biological sample comprising an antibody, polypeptide or polypeptide derivative of the invention.
reagents for detecting the presence of ydia; and (ii) contacting a biological sample with an antibody, polypeptide, or polypeptide derivative of the invention, thereby forming an immune complex; and A diagnostic method for detecting the presence of Chlamydia in a biological sample by detecting the complex and indicating the presence of Chlamydia in the sample or in the organism from which the sample is derived.

[0118] One of skill in the art will recognize that whether an antibody, polypeptide, or polypeptide derivative is used, an immune complex will be formed between the components of the sample and the antibody, polypeptide, or polypeptide derivative; And it is understood that any unbound material can be removed prior to detection of the complex. As can be readily appreciated, polypeptide reagents are useful for detecting the presence of anti-Chlamydia antibodies in a sample (eg, a blood sample), while the antibodies of the invention are useful for detecting the presence of a sample (eg, a gastric extract). ,
Or a biopsy) can be used to screen for the presence of a Chlamydia polypeptide.

For use in diagnostic applications, the reagents (ie, the antibodies, polypeptides, or polypeptide derivatives of the invention) may be in a free state, even when in solid form (eg, tubes, beads, or the like). Any other conventional support used in the field)
May be immobilized. Immobilization can 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,
When using a polypeptide reagent, an antibody that binds to the reagent can serve as an anti-reagent if the antibody that binds to that reagent binds to an epitope that is not involved in the recognition of the antibody in a biological sample. Indirect means may also utilize a ligand-receptor system (eg, molecules such as vitamins can be implanted on a polypeptide reagent and the corresponding receptor can be immobilized on a solid phase). This system is exemplified by the biotin-streptavidin system. Alternatively, indirect means
For example, a peptide tail can be used chemically or by genetic manipulation to add to the reagent, and the implanted or fused product immobilized 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. This process is
It involves performing an antibody-based affinity chromatography using a biological sample, wherein the antibody is a monospecific antibody of the invention.

For use in the purification process of the invention, the antibodies may be polyclonal or monospecific, and are preferably 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 of antibody implantation, are described, for example, in ANTIBODIES: A LABORATORY.
MANUAL, D. Lane, E .; Harlow (1988).

Briefly, a biological sample (eg, a C. pneumoniae extract, preferably in a buffer solution) is converted into a chromatographic material (preferably a buffer used to dilute the biological sample). ), So that the polypeptide or polypeptide derivative of the invention (ie, antigen) is
Adsorb on the material. The chromatography material (eg, a gel or resin bound to the antibody of the invention) may be in batch form or in a column. Wash off unbound components and then elute the antigen with a suitable elution buffer (eg, glycine buffer or a buffer containing a chaotropic agent (eg, guanidine HCl) or a high salt concentration (eg, 3M MgCl ₂ )). . The eluted fraction is removed and the presence of the antigen is detected (eg, by measuring the absorbance at 280 nm).

The antibodies of the 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) therapeutic use of a monospecific antibody of the invention. Or a pharmaceutical composition comprising a prophylactically effective amount; and (iii)
)) Administering to a person in need thereof a prophylactically or therapeutically effective amount of a monospecific antibody of the invention, thereby allowing Chlamydia (eg, C. trac) to be administered.
homatis, C.I. psittaci, C.I. pneumoniae, or C
. pecorum) A method of treating or preventing an infection. Further, the eleventh aspect of the present invention
Aspects include 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 preferably may be of the IgA isotype (mainly).
In passive immunization, the antibodies may be administered to the mucosal surface of a mammal, such as the gastric mucosa (eg, orally or intragastrically), advantageously in the presence of a bicarbonate buffer.
Alternatively, systemic administration (which does not require bicarbonate buffer) can be performed. The monospecific antibodies of the invention can be used as a single active ingredient or as different Chlamydi
a can be administered as a mixture with at least one monospecific antibody specific for the polypeptide. The amount of antibody used and the particular therapy can be readily determined by one skilled in the art. For example, daily administration of about 100-1,000 mg of antibody for one week, or three doses per day of about 100-1,000 mg of antibody for 1-3 days, is an effective therapy for most purposes. Can be

[0125] Therapeutic or prophylactic efficacy may be assessed, for example, by C.I. Pneumoniae mouse models can be used to assess using standard methods in the art (eg, by inducing a mucosal immune response, or by measuring the induction of protective and / or therapeutic immunity). One skilled in the art will appreciate the C.I. It recognizes that the strain pneumoniae can be replaced by another strain of Chlamydia. For example, C.I. pneumoniae-derived DN
The potency of the A molecule and polypeptide is preferably that of C.A. pneumoniae strain is evaluated in a mouse model. Protection can be determined by comparing the extent of Chlamydia infection to the control group. Protection is indicated if the infection is reduced by comparing to the control group. Such evaluation can be made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, and antibodies of the 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 hydroxide phosphate. The antigen can be precipitated with the aluminum compound or adsorbed to the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), can be used for parenteral administration.

Adjuvants for mucosal administration include bacterial toxins such as cholera toxin (C
T), E. coli heat labile toxin (LT), Clostridium dif
ficile toxin A and pertussis toxin (PT), or a combination, subunit, toxoid, or variant thereof. For example, a purified preparation of natural cholera toxin subunit B (CTB) can be used. If fragments, homologs, derivatives and fusions of any of these toxins retain adjuvant activity, they are also appropriate. Preferably, variants with reduced toxicity are used. Suitable variants are described, for example, in WO 95/17211.
(Arg-7-Lys CT mutant), WO96 / 6627 (Arg-192-
Gly LT variants), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT variants). Additional LT variants that can be used in the methods and compositions of the present invention include, for example, Se
r-63-Lys, Ala-69-Gly, Glu-110-Asp, and G
lu-112-Asp mutant. For example, E. coli, Salmo
nella minnesota, Salmonella typhimuri
um, or bacterial monophosphoryl lipid A of Shigella flexneri
Other adjuvants such as (MPLA); saponins, or polylactide glycolide (PLGA) microspheres, may also be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral administration include polyphosphazene (WO95 / 2415), DC-chol (3b- (N- (N ', N
'-Dimethylaminomethane) -carbamoyl) cholesterol (DC-chol
(3b- (N- (N ', N'-dimethylaminomethane))
-Carbamoyl) cholesterol) (U.S. Pat. No. 5,283,1)
No. 85 and WO 96/14831) and QS-21 (WO 88/9336)
Is mentioned.

[0130] Any of the pharmaceutical compositions of the present invention, including the polynucleotides, polypeptides, polypeptide derivatives or antibodies of the present invention, may be manufactured in conventional manner. In particular, the composition can be formulated with a pharmaceutically acceptable diluent or carrier, for example, water or saline solution, such as phosphate buffered saline. Generally, the diluent or carrier may be selected based on the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical requirements for their use in pharmaceutical formulations, can be found in Remington
's Pharmaceutical Sciences (standard reference text in the field) and in USP / NF.

The invention also includes a method of treating a Chlamydia infection by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant in combination with an antibiotic, antacid, sucralfate or a combination thereof. Examples of such compounds that can be administered with vaccine antigens and adjuvants include, for example, antibiotics, including macrolides, tetracyclines and derivatives thereof (particular examples of antibiotics that can be used include azithromycin or doxycycline). Or immunomodulators such as cytokines or steroids. Additionally, compounds that include more than one of the above-listed components bound together may be used. The present invention also provides compositions for practicing these methods (ie, a Chlamydia antigen of the present invention, an adjuvant, and more than one of the above-listed compounds in a pharmaceutically acceptable carrier or diluent). Comprising a composition).

The amount of the above-listed compounds used in the methods and compositions of the present invention can be readily 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 can be obtained with reference to the following specific examples. These examples are set forth for illustrative purposes only and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are:
It may be considered as an incidental matter and may be suggested as convenient or may be convenient. Although specific terms have been used herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Equivalents From the foregoing detailed description of the specific embodiments of this invention, note that the unique Chlamyd
It should be clear that the ia antigen has been described. Although particular embodiments have been disclosed in detail herein, this disclosure is made by way of example only for illustrative purposes, and is not intended to be limiting with respect to the appended claims. . In particular, it is contemplated by the inventors that various substitutions, changes, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined in the claims. .

[Sequence list]

[Brief description of the drawings]

 The invention can be better understood from the following description with reference to the following drawings.

FIG. 1 shows the nucleotide sequence (upper sequence) and the deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100111 polypeptide from Chlamydia pneumoniae.

FIG. 2A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2G shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2H shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2I shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2J shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 2K shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100111.

FIG. 3 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100224 polypeptide from Chlamydia pneumoniae.

FIG. 4A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4G shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4H shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4I shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4J shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4K shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4L shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4M shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 4N shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100224.

FIG. 5 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100230 polypeptide from Chlamydia pneumoniae.

FIG. 6A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100230.

FIG. 6B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100230.

FIG. 6C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100230.

FIG. 6D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100230.

FIG. 6E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100230.

FIG. 7 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100231 polypeptide from Chlamydia pneumoniae.

FIG. 8A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8G shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8H shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8I shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8J shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 8K shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100231.

FIG. 9 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100232 polypeptide from Chlamydia pneumoniae.

FIG. 10A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 10B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 10C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 10D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 10E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 10F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100232.

FIG. 11 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100233 polypeptide from Chlamydia pneumoniae.

FIG. 12A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100233.

FIG. 12B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100233.

FIG. 12C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100233.

FIG. 13 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100394 polypeptide from Chlamydia pneumoniae.

FIG. 14A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14G shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14H shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14I shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 14J shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100394.

FIG. 15 shows the nucleotide sequence (upper sequence) and deduced amino acid sequence of the full-length (middle sequence) and processed (lower sequence) CPN100395 polypeptide from Chlamydia pneumoniae.

FIG. 16A shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

FIG. 16B shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

FIG. 16C shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

FIG. 16D shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

FIG. 16E shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

FIG. 16F shows C.I. 1 shows a restriction enzyme analysis of the nucleotide sequence encoding pneumoniae CPN100395.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/295 C07K 16/12 4C085 16/12 C12N 1/15 4C087 C12N 1/15 1/19 4H045 1 / 19 1/21 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 C12Q 1/68 A 21/08 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 33/566 33/50 33/569 F 33/566 33/577 B 33/569 C12N 15/00 ZNAA 33/577 5/00 A (31) Priority claim number 60 / 097,189 (32) Priority date 1998 (20 August 1998) (33 Aug. 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 097,190 (32) Priority date August 20, 1998 (1998. 20) (33) Priority claim country United States (US) (31) Priority claim number 60/097 195 (32) Priority date August 20, 1998 (August 20, 1998) (33) Priority country United States (US) (31) Priority claim number 60 / 097,196 (32) Priority date 1998 August 20, 1998 (August 20, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 097,197 (32) Priority date August 20, 1998 (1998. 8.20) (33) Priority claim country United States (US) (31) Priority claim number 60 / 097,191 (32) Priority date August 27, 1998 (August 27, 1998) (33) Priority Claiming country United States (US) (31) Priority claim number 09 / 376,770 (32) Priority date August 17, 1999 (August 17, 1999) (33) Priority claiming country United States (US) ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, M , 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, CR, CU, CZ, DE, DK, DM, 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, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 AA25 AA28 AA34 AA35 AA40 CB17 CB21 DA12 DA13 DA14 DA36 FB01 FB02 FB03 FB04 4B024 AA01 AA13 BA31 CA02 CA07 CA09 CA20 DA01 DA02 DA05 DA11 FA18 GA11 HA13 HA14 HA15 HA17 4B063 QA01 QA13 QA18 QA19 QQ06 QQ43 QQ53 QQ79 QR32. CA11 CA19 CA20 CC01 CC24 DA01 DA15 4B065 AA01X AA01Y AA58X AA72X AA87X AB01 AC14 BA02 CA24 CA45 CA46 4C085 AA03 BA45 DD22 DD23 DD62 EE01 EE06 FF24 4C087 AA01 AA03 BB64 BB65 CA12 NA14 AB AA ABA A31 AB A31 AA BAA ABA A31 AA BAA FA74

Claims (53)

[Claims] 1. An isolated polynucleotide selected from the group consisting of: (a) a nucleotide selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15 A polynucleotide having a sequence, and a functional fragment thereof; (b) a sequence that is at least 75% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 And a functional fragment thereof; and (c) a polynucleotide having a sequence further comprising the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15. Either a polynucleotide capable of hybridizing under stringent conditions, or a functional fragment thereof. 2. The method of claim 2, wherein the polynucleotide is SEQ ID NO: 2, 4, 6, 8, 10,
2. The nucleotide of claim 1, which encodes a functional fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of 12, 14, and 16. 3. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 1, and wherein the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 2. The polynucleotide according to claim 1, which encodes a polypeptide having: 4. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 3 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 4 The polynucleotide according to claim 1, which encodes a polypeptide having: 5. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 5 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 6 The polynucleotide according to claim 1, which encodes a polypeptide having: 6. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 7 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 8 The polynucleotide according to claim 1, which encodes a polypeptide having: 7. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 9 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 10 The polynucleotide according to claim 1, which encodes a polypeptide having: 8. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 11 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 12 The polynucleotide according to claim 1, which encodes a polypeptide having: 9. A sequence wherein the polynucleotides of (a) and (c) have the nucleotide sequence of SEQ ID NO: 13 and the polynucleotide of (b) is at least 75% homologous to the amino acid sequence of SEQ ID NO: 14 The polynucleotide according to claim 1, which encodes a polypeptide having: 10. The polynucleotide of (a) and (c) is SEQ ID NO: 15
And the polynucleotide of (b) has the nucleotide sequence of SEQ ID NO: 16
The polynucleotide of claim 1 which encodes a polypeptide having a sequence that is at least 75% homologous to the amino acid sequence of 11. SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16
An isolated polypeptide having an amino acid sequence that is at least 75% homologous to an amino acid sequence selected from the group consisting of: 12. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof. 13. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof. 14. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 6 or a functional fragment thereof. 15. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 8 or a functional fragment thereof. 16. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 10 or a functional fragment thereof. 17. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 12 or a functional fragment thereof. 18. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 14 or a functional fragment thereof. 19. The polypeptide according to claim 11, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 16 or a functional fragment thereof. 20. The polynucleotide of claim 1, wherein said polynucleotide is linked to a second nucleotide sequence encoding a fusion polypeptide. 21. The fusion polypeptide is a heterologous signal peptide.
A nucleotide according to claim 20. 22. A polypeptide comprising the polypeptide of claim 21 linked to a fusion polypeptide. 23. The polypeptide of claim 22, wherein said fusion polypeptide is a signal peptide. 24. The polypeptide of claim 23, wherein said fusion polypeptide comprises a heterologous polypeptide having adjuvant activity. 25. An expression cassette comprising the polynucleotide of claim 1 operably linked to a promoter. 26. An expression vector comprising the expression cassette according to claim 25. 27. A host cell comprising the expression cassette according to claim 25. 28. The host cell of claim 27, wherein said host cell is a prokaryotic cell. 29. The host cell of claim 27, wherein said host cell is a eukaryotic cell. 30. A method for producing a recombinant polypeptide, comprising the following steps: (a) culturing the host cell according to claim 27 under conditions allowing expression of said polypeptide. And (b) recovering the recombinant polypeptide. 31. The recombinant polypeptide may be CPN 100111, CP
N 100224, CPN 100230, CPN 100231, CPN 1
00232, CPN 100235, CPN 100394, and CPN 1
31. The method of claim 30, wherein the method is selected from the group consisting of: 00395. 32. A vaccine vector comprising the expression cassette according to claim 25. 33. The vaccine vector according to claim 32, wherein said host mammal is a human. 34. The vaccine vector of claim 32, wherein said vaccine vector is in a pharmaceutically acceptable excipient. 35. A pharmaceutical composition comprising an immunologically effective amount of the vaccine vector of claim 32. 36. A method for inducing an immune response in a mammal, comprising the steps of: administering to said mammal an immunologically effective amount of the vaccine vector of claim 32. Wherein the administration induces an immune response. 37. A pharmaceutical composition comprising an immunologically effective amount of the polypeptide of claim 11, and a pharmaceutically acceptable diluent. 38. The pharmaceutical composition according to claim 37, further comprising an adjuvant. 39. The pharmaceutical composition according to claim 37, further comprising one or more known Chlamydia antigens. 40. A method for inducing an immune response in a mammal, comprising the steps of: administering to said mammal an immunologically effective amount of the pharmaceutical composition of claim 37. And wherein said administering induces an immune response. 41. A polynucleotide probe reagent capable of detecting the presence of Chlamydia in a biological material, wherein the reagent is a stringent condition.
A polynucleotide probe reagent comprising a polynucleotide that hybridizes to the polynucleotide according to 1. 42. The polynucleotide probe reagent according to claim 41, wherein the reagent is a DNA primer. 43. A hybridization method for detecting the presence of Chlamydia in a sample, comprising the following steps: (a) obtaining a polynucleotide from the sample; (b) 43. Hybridizing the polynucleotide probe reagent of claim 41 with the probe and the sample under conditions that permit hybridization; and (c) combining the detection reagent with a polynucleotide in the sample. Detecting hybridization. 44. An amplification method for detecting the presence of Chlamydia in a sample, the method comprising: (a) obtaining a polynucleotide from the sample; (b) obtaining the polynucleotide from the sample 42. A method comprising: amplifying using one or more polynucleotide probe reagents according to item 41; and (c) detecting the amplified polypeptide. 45. A method for detecting the presence of Chlamydia in a sample, comprising: (a) contacting the sample with a detection reagent that binds a polypeptide to form a complex; In the process, the polypeptide is CPN 100111, CPN 100224, CPN 100230, CPN 100231, CPN 10
0232, CPN 100235, CPN 100394, and CPN 10
0395; and (b) detecting the formed complex. 46. The method of claim 45, wherein said detection reagent is an antibody. 47. The method of claim 46, wherein said antibody is a monoclonal antibody. 48. The method according to claim 46, wherein said antibody is a polyclonal antibody. 49. An affinity chromatography method for substantially purifying a Chlamydia antigen, comprising the steps of: (a) contacting a sample containing the Chlamydia antigen with a detection reagent that binds to a polypeptide; Forming a complex, wherein the polypeptide comprises C
PN 100111, CPN 100224, CPN 100230, CPN
100231, CPN 100232, CPN 100235, CPN 100
394, and CPN 100395; (b) isolating the formed complex; (c) dissociating the formed complex; and (d) dissociating. Isolating the isolated Chlamydia antigen. 50. The method of claim 49, wherein said detection reagent is an antibody. 51. The method of claim 50, wherein said antibody is a monoclonal antibody. 52. The method of claim 50, wherein said antibody is a polyclonal antibody. 53. An antibody that immunospecifically binds to the polypeptide of claim 11, or a fragment or derivative of the antibody comprising a binding domain thereof.