AU6653096A - Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccine - Google Patents
Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccineInfo
- Publication number
- AU6653096A AU6653096A AU66530/96A AU6653096A AU6653096A AU 6653096 A AU6653096 A AU 6653096A AU 66530/96 A AU66530/96 A AU 66530/96A AU 6653096 A AU6653096 A AU 6653096A AU 6653096 A AU6653096 A AU 6653096A
- Authority
- AU
- Australia
- Prior art keywords
- protein
- trachomatis
- hybrid protein
- recombinant hybrid
- epitope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)
Description
HYBRID PROTEIN COMPRISING T-HELPER CELL STIMULATING EPITOPES AND B-CELL EPITOPES FROM THE MAJOR OUTER MEMBRANE PROTEIN OF CHLAMYDIA TRACHOMATIS AND ITS USE AS A VACCINE
BACKGROUND OF THE INVENTION
(a) Field of the Invention 5 The present invention relates to a novel pep¬ tide which is of use as vaccine, to a method of prepar¬ ing it and to a pharmaceutical composition containing it. In particular, the invention relates to vaccines against Chlamydia trachomatis . 0 (b) Description of Prior Art
Vaccination as a deliberate attempt to protect humans against disease has a long history, although only in the 20th century has the practice flowered into routine vaccination of large populations. During the 5 last 200 years, since the time of Edward Jenner, vacci¬ nation has controlled nine major diseases, at least in parts of the world, as follows: smallpox, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis, measles, mumps, and rubella. In the case of smallpox, 0 the dream of eradication has been fulfilled, as this disease has disappeared from the world. Vaccinations against influenza, hepatitis B, pneumococci, and Haemophilus influenzae type b have major headway against those infections, but much remains to be done, 5 even in developed countries.
The impact of vaccination on the health of the world's peoples is hard to exaggerate. With the excep¬ tion of safe water, no other modality, not even antibi¬ otics, has had such a major effect on mortality reduc- 0 tion and population growth.
Live vaccines have been used for some time. Protection from small pox by variolation was practiced in Europe for several hundred years prior to 1850. This practice was superseded by the use of naturally occur- 5 ring cow pox viruses discovered by Jenner. An effort to attenuate microorganisms was first made by Pasteur, who
worked with Bacillus anthracis and the rabies virus in the late 19th century. The BCG vaccine against tubercu¬ losis was developed in 1921, and the yellow fever vac¬ cine in 1937. The advent of advanced tissue culture techniques in the last 40 years has promoted many of the live attenuated vaccines in current use. Most live vaccines were produced by extensive passage of virulent forms and isolating nonvirulent derivatives. Alterna¬ tively, nonvirulent forms that occur naturally are iso- lated and can be used as vaccines. The Sabin virus, prepared as an attenuated vaccine, differs from the wild-type neurovirulent form of poliovirus by 57 point mutations. These mutations are distributed throughout the genome and explain the success of this vaccine. It is possible to molecularly engineer microor¬ ganisms using site-directed mutagenesis or by shuffling their various regions from one serotype to another. However, there is always the difficulty of ensuring that these molecular manipulations do not interfere with microorganism assembly. It is also possible to enhance attenuation by growing a microorganism in the presence of a monoclonal antibody (mAb) to a specific region.
Live attenuated vaccines have many advantages. First, they produce high levels of both humoral and cellular immunity. The problems with attenuated vac¬ cines are the mutation and reversion to a more virulent form during the process of attenuation, which, in some cases, can be minimized by inducing several mutations at distinct regions of the genome of the microorganism. Nevertheless, killed vaccines do have certain advantages that contrast with the theoretical short¬ comings of live vaccines. By definition, killed vac¬ cines cannot multiply or disseminate to cause the dis- ease they are intended to prevent or transmit the dis-
ease to another person. Generally, they are better tolerated, especially for the majority of killed vac¬ cines that undergo purification to remove other macro¬ molecules. Killed vaccines usually function by stimu- lating humoral immune responses as well as by priming for immunological memory.
The earliest approach to making killed vac¬ cines, which is akin to the classical strategies for making live vaccines discussed previously, relied on the inactivation of whole bacteria or whole viruses with the objective of eliciting the formation of anti¬ bodies to many antigens, some of which would neutralize the pathogen. However, in some cases ( Chlamydia tra¬ chomatis for example) important adverse effects render the use of the whole pathogen impossible and other strategies have to be consider.
The use of protein-based or peptide-based vac¬ cines represents one such strategy.
Several structures are found on the surface of microorganisms; among them, some are immunogenic, that is they are recognized by the host immune system and may induce protective immune response. It could be speculated that these structures, when they are pro¬ teins, can be used as a vaccine, particularly the region corresponding to the immunoreactive part of the protein. First, the amino acid sequence of this pro¬ tein must be determined by molecular biology techniques and the antigenic determinants identified from the amino acid sequence of a known antigen. Since some antigenic sites contain charged and polar amino acids that are readily determined by a hydrophilicity plot of each known amino acid of the sequence, these hydrophil¬ icity values can therefore be used to predict the hydrophilicity of a particular region along the peptide chain and identify the antigenic sites. However, the
three-dimensional structure from the knowledge of the only primary sequence cannot be predicted. Predictions are complicated by the ways amino acids influence one another that are far removed from the linear sequence. This can be appreciated by the techniques of Chou and Fasman, by x-ray crystallography, by nuclear magnetic resonance or by computer-aided analysis.
Another approach to define antigenic sites could be to produce a neutralizing monoclonal antibody and to identify the sequential peptides bound by the antibody. It is also possible to use predictive model of the structure, for example, to define the solvent accessible areas and simply use the primary structure to predict antigenic regions. While synthetic peptides constitute a very attractive approach for vaccination, due to their secu¬ rity and unlimited supply aspect, they are also subject to the peptide synthesis limits. In fact, it is gener¬ ally admitted that the fMOC and tBOC chemistry have a synthesis limit of 40 to 70 amino acid residues, depending on the instruments. It is therefore inter¬ esting to have the possibility to use a system that permit the production of longer polypeptides or pro¬ teins. Molecular biology techniques offer this possi¬ bility. Moreover, a variety of hybrid constructions can also be achieved, having for result that the desired protein can be designed precisely . Using dif¬ ferent expression systems, it is possible to chose the protein form that possesses the conformation that is the most in accordance with the native protein, for example the respect of glycosylation sites and disul¬ fide bonds.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a pep¬ tide which comprises (i) a region which is able to induce the production of neutralizing antibodies to an epitope of a pathogen in a host organism having an immune system which includes B-cells and T-helper cells, and (ii) a region which is able to stimulate a T-cell response to said pathogen in said organism.
Examples of suitable host organisms include mammals such as humans.
Suitably region (i) comprises a B-cell epitope of said pathogen and region (ii) comprises a T-helper cell epitope of said pathogen. As used herein, the expression "B cell epitope" refers to the sequence of an epitope in a pathogen which stimulates a B cell antibody production response or to a variant of this sequence which produces antibodies which cross-react with said epitope. Similarly, the expressions "T-cell epitope" or "T-helper cell epitope" refers to an epi- tope in a pathogen which can stimulate a T-cell response in a host organism, or to a variant of said sequence which produces a similar response.
In a preferred embodiment, the peptide com¬ prises two B-cell epitopes (i) and one T-helper cell epitope (ii) linked in any order, preferably in a con¬ secutive or colinear manner. The epitopes may be joined together directly or may be separated by linker sequences. The use of two B-cell epitopes will help to provide a broad protective range covering multiple serovars.
In particular, the present invention provides a peptide comprising substantially only regions (i) and (ii) above i.e. peptides which predominantly comprise a region able to induce neutralizing antibodies to an epitope of a pathogen and a region able to stimulate a
T-cell response to the pathogen. Thus in this particu¬ lar aspect there is the drawing together of regions (i) and (ii) as set out above closely together on a rela¬ tively short peptide sequence. Of course it is impor- tant that the epitopes will be correctly presented in terms of conformation for effective recognition by, and stimulation of the immune system.
Therefore the regions (i) and (ii) may be sepa¬ rated by linker sequences of sufficient length in order to ensure optional conformation and presentation of the regions defined.
For instance, the peptide may include or con¬ sist of any of the following arrangements:
B-T-B T-B-B
B-B-T or in any combination that enhances immunogenicity, whereby "B" represents a B cell stimulating epitope and ni ., represents a T helper cell stimulating epitope. Identification of such regions in the case of a particular pathogen will be within the knowledge of the skilled person or may be determined by routine investi¬ gation, for example as illustrated hereinafter.
A peptide of this nature is able to induce a complete and effective immune response and therefore be useful as a vaccine. The presence of B-cell epitopes induces the production of neutralizing antibodies, and T-helper cell epitopes enhance the immunogenicity of haptenic neutralizing B-cell epitopes and provide T- cell memory.
The peptide as described herein may form part of a fusion protein which incorporates other peptides such as immunogenic carriers like the cholera toxin B subunit.
The peptide of the invention may be synthesized by standard solid-phase methods (fMOC of tBOC) in such a way that the final polypeptide will comprise both a neutralizable B-cell or helper T-cell target epitope. Thus the invention further provides a process for pre¬ paring a peptide which comprises synthesizing an amino acid sequence which comprises an antigenic determinant or target epitope in a repetitive form.
Alternatively, it may be prepared using recom- binant DNA technology as is conventional in the art. For this purpose, a nucleic acid which encodes a pep¬ tide as described above is prepared using conventional techniques. This may involve a total synthesis approach or mutation by deletion of DNA extracted from the relevant pathogen, or a combination of these. A particularly suitable method involves isolation of pathogenic DNA, followed by amplification of the required epitopic regions using for example the polymerase chain reaction (PCR). These regions may then be ligated together using conventional techniques and incorporated into an expression vector.
Nucleic acid which encode the above-described peptide form a further aspect of the invention as do expression vector containing it. The expression vector is then used in the transformation of a host cell. Culture of the transformed host cell will result in the production of the desired peptide which is then iso¬ lated.
These methods, together with the transformed host cell form yet further aspects of the invention.
For instance, a peptide which comprise a recom¬ binant hybrid protein may also be expressed with other genes, to provide a recombinant protein fused to immu¬ nogenic carrier such as the cholera toxin B subunit. Conveniently, the hybrid protein will be inserted in
appropriate reading frame with the fused gene and under the regulatory control of the regulatory system of the fused gene.
The peptide of the invention obtained by recom- binant technology may be glycosylated, partially gly¬ cosylated, or unglycosylated, depending on the nature of the expression host. Generally, prokaryotes such as E. coli will provide no glycosylation of the translated products, while yeast and mammalian cell culture will provide partial or substantial glycosylation. In regard to these different expressions possibilities, the hybrid construction may be cloned for example in pHIL D2 and pPIC 9 for its expression in the yeast, and in pCR III for its expression in mammalian cells. When employing a yeast host, codons which appear at high frequency in the structural genes encoding the yeast glycolytic enzymes may be employed, along with appro¬ priate promoters. In some instances, it may be desir¬ able to add or modify nucleotides in the constructions, to increase stability, enhance immunogenicity or facilitate the conjugation of the hybrid protein to a carrier. Also, the construction may include a secre¬ tory leader as processing signal sequence to effect gene product in the yeast or mammalian cells. The peptide of the invention may be suitable for vaccine use, for which use, it will suitably be formulated as a pharmaceutical composition.
The invention further provides a pharmaceutical composition comprising a peptide as described above in combination with a pharmaceutically acceptable excipient such as carrier, liposomes or adjuvant. Suitable carriers include solid and liquid carriers such as water or saline. Alternatively the peptide may be encapsulated using conventional encapsulation methods. Other carriers and components of pharmaceuti-
cal compositions may be included as is recognized in the art.
The compositions are suitably in a form which is suitable for oral or parenteral administration. The vaccine may comprise a peptide as described above itself in the form of a pharmaceutical composi¬ tion.
Alternatively, a nucleic acid sequence which encodes such a peptide as described above may be incor- porated into a suitable vector such as a virus vector, and this vector administered to the host in such a way that the peptide is expressed. These nucleic acid as well as vector incorporating it and pharmaceutical com¬ position comprising said vector form a further aspect of the invention.
Therefore the invention further provides a method for stimulating an immune response which method comprises administering to a patient an effective amount of a peptide as described above. The method can be used for the prophylaxis or therapeutic treatment of a disease.
Furthermore the peptide of the invention will interact with antibodies specific for particular patho¬ genic epitopes. This has implications in the field of diagnostics where the presence of an antibody specific for a particular epitope is to be determined.
The invention further provides a method for detecting the presence of an antibody to an epitope which method comprises contacting a sample suspected of containing said antibody to a peptide as described above, and detecting antibody bound to said peptide.
The precise techniques used in the methods are those which are conventional in the art and include immunoassay techniques such as radioimmunoassay and enzyme-linked serological assay techniques (ELISA).
For example, the peptide may be immobilized upon a sup¬ port before being contacted by the sample and bound antibody detected using a labeled detection assay. Immunoassays of this type are part of the present invention.
The invention further provides a diagnostic kit for use in these methods which kits comprise a peptide in accordance with the present invention.
A particular application of the invention relates to the development of vaccines for Chlamydia trachomatis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. IA to IC illustrate the hybrid construc- tion coding for VDI B-T-VDIV K;
Fig. 2 illustrates the expression of the VDI B- T-VDIV K hybrid construction in pET 3b in an experiment in which E. coli DE3 were stimulated for expression of the recombinant protein with IPTG; Fig. 3 shows the results of antibody reactivity on E. coli TBl expressing the VDI B-T-VDIV K hybrid construction;
Fig. 4 shows the reactivity profile of six strains of mice immunized with the recombinant hybrid protein;
Figs. 5A to 5F show the results of direct-bind¬ ing radioimmunoassay measurements of specific vaginal IgA on synthetic peptides 4 VDI B and 4 VDIV K as well as on elementary bodies (EB) of serotype B and K on six inbred strains of mice;
Figs. 6A to 6F show the results of direct-bind¬ ing radioimmunoassay measurements of specific serum IgA on synthetic peptides 4 VDI B and 4 VDIV K as well as on elementary bodies (EB) of serotype B and K on six inbred strains of mice;
Figs. 7A to 7F show the results of direct-bind¬ ing enzyme-linked immunosorbent assay measurements of specific serum IgG on synthetic peptides 4 VDI B and 4 VDIV K as well as on elementary bodies (EB) of serotype B and K on six inbred strains of mice.; and
Figs. 8A to 8F show the results of direct-bind¬ ing enzyme-linked immunosorbent assay measurements of specific vaginal IgG on synthetic peptides 4 VDI B and 4 VDIV K as well as on elementary bodies (EB) of sero- type B and K on six inbred strains of mice.
DETAILED DESCRIPTION OF THE INVENTION
Chlamydia trachomatis, an obligate intracellu¬ lar bacterium, is a leading cause of sexually transmit- ted diseases in industrialized countries and is also a major cause of preventable blindness in underdeveloped countries. It is estimated that over 300 millions of people are infected worldwide by this microorganism. Moreover, 55 millions of cases appear each year. Based on pathogenicity, antigenicity and nucleic acid composition, human pathogen strains of C. trachomatis have been subdivided into 15 serovars. The Ll, L2, and L3 serovars are responsible for the lymphogranuloma venereum, a relatively rare infection. The A, B, Ba, and C serovars provoke the trachoma, which has a strong incidence in underdeveloped countries, while the serovars D through K are responsible for the genital tract infections. The late diagnostic, due to the lack of clinical symptoms, often lead to severe complications such as ectopic pregnancy and even sterility.
The first studies on human and other primates have suggested that a vaccination using inactivated chlamydiae frequently resulted in a more severe form of trachoma following reinfection. The characterization
of the antigen implicated in the pathogenic response revealed that this antigen (the 57-kDa protein) was common to the three Chlamydia species and that it was associated with elementary bodies as well as reticulate bodies. The 57-kDa protein, provokes an ocular delayed hypersensitivity reaction in animals and is implicated as mediator of the pathogenic response associated with severe chlamydial infections. The histopathology of the inflammatory response stimulated by the 57-kDa is comparable to that found in severe trachoma cases and in post-salpingitis infertility.
Although the 57-kDa has been implicated in the chlamydial infections pathogenesis, its precise role in the stimulation of pathogenic responses of human dis- eases has not been yet determined. However, studies have demonstrated a correlation between the capability to develop an immune response against this protein and severe infections. A correlation has been observed between the antibody response to that protein and acute salpingitis and ectopic pregnancy. An association between the disease and an anti-57kDa antibody response has also been observed in other studies implicating patients suffering of tubal infertility.
These observations clearly indicate that vacci- nation against chlamydiae cannot be achieved by using the whole bacteria. Novel strategies using only a part of the bacteria, such as synthetic peptides or recombi¬ nant proteins, have to be employed.
Surface-exposed outer membrane constituents of chlamydial elementary bodies (EBs) are the likely tar¬ gets of the immune responses that prevent reinfection. The predominant constituents of the outer membrane are the cysteine-rich major outer membrane protein (MOMP), the cysteine-rich 60-kDa and 12-kDa proteins, and lipopolysaccharides (LPS). Only the MOMP and LPS are
immunoaccessible on the EB surface, as defined by anti¬ body reactivity. Though surface exposed, LPS do not stimulate the production of neutralizing antibodies, whereas antibodies to the MOMP are neutralizing. The MOMP, an antigenically complex protein of
40 kDa that accounts for 60%, by weight, of the chlamy¬ dial outer membrane proteins, exhibits both unique and common antigenic moieties, which account for the serovariation among isolates of Chlamydia trachomatis . MOMP-encoding genes of several Chlamydia trachomatis serovars and Chlamydia psi ttaci strains have been cloned, sequenced and compared (Baehr, Proc. Natl . Acad. Sci . USA j55_: 4000, 1988; Stephens, J. Bacteriol . 169: 3879, 1987). These studies revealed that the MOMP has highly conserved portions but contains four regions of substantial sequence variability, referred to as variable sequence domains (VD) I, II, III, and IV. The nucleotide and amino acid sequences of these four variable domains are the subject of the United States Patent application No. 7/324,664 filed on March 17, 1989 in the name of the U.S. Department of Health and Human Services (Washington, D.C). These four regions contain specific sequences, referred to as neutralizable epitopes, which are the targets for neutralizing antibodies. They have been characterized for several C. trachomatis serovars and their ability to induce the production of a neutralizing immune response has been shown (Baehr, Proc. Natl . Acad. Sci . USA 5: 4000, 1988; Qu, Infect . Immun. J L: 1365, 1993; Villeneuve, Infect . Immun. £2:: 3547, 1994; Villeneuve, Microbiol . 140: 2481, 1994).
Using overlapping synthetic peptides corre¬ sponding to the entire MOMP sequence in T-cell prolif¬ eration assays and as in vivo priming immunogens for the production of an anamnestic IgG antibody response,
Su et al. demonstrated that amino acid residues 106-130 of the MOMP possessed functional T-helper activity. This region of the MOMP is largely sequence invariant among the different C. trachomatis MOMP suggesting that the T-helper cell epitope contained within its sequence is antigenically conserved across serovars (Su, J. Exp. Med. 175: 227, 1992). This conserved T-helper stimulating epitope, synthesized colinearly with B-cell neutralizing antibody stimulating epitope, has been used to produce an immune response to C. trachomatis in a vertebrate (International Patent Application No. WO 94/06827, published on March 31, 1994 in the name of The Government of the United States of America). The peptides of WO 94/06827 were only shown to be immuno- genic enough to cause the production of serum antibodies, but were not shown to elicit the production of mucosal IgA antibodies, which would be a great advantage for an effective vaccine against Chlamedia.
In addition, one region encompassing the non- surface exposed VDIII has been reported to elicit T- cell help in vivo for the production of antibodies to protective B-cell epitopes (Allen, J. Immunol . 147: 674, 1991) and a T-cell epitope recognized by the majority of naive human volunteers, adjacent to VDIII has also been described (Stagg, Immunology 2i: 1/ 1993) .
Therefore, there is a need for a peptide or protein vaccine capable of generating a protective im¬ mune response against a variety of C. trachomatis serovars and peptides of the invention fulfill this function.
Hence in a preferred embodiment of the peptide of the invention, the said pathogen is C. trachomatis or a serovar thereof.
Suitably the B-cell and T-cell epitopes which form the basis of the peptide constitute part of the major outer membrane protein of C. trachomatis .
Thus in a particularly preferred embodiment, the present invention provides a peptide which is capa¬ ble of inducing an immunological response to C. trachomatis comprising at least one T-helper stimulat¬ ing epitope from the major outer membrane protein of C. trachomatis and at least two B-cell neutralizing anti- body stimulating . epitope from the major outer membrane protein of C. trachomatis .
Suitably, this peptide comprises a recombinant hybrid protein.
In a preferred embodiment of this invention, the T-helper cell stimulating epitope and the B-cell neutralizing antibody stimulating epitopes are colin- ear. Preferably the T-helper stimulating epitopes are located within the sequence
ALNIWDRFDVFCTLGATTGYLKGNS (SEQ ID NO:l); or TINKPKGYVGKEFPLDLTAGTDAAT (SEQ ID NO:2).
Preferably the B-cell neutralizing antibody stimulating epitopes are located within the sequence
LDVTTLNPTI (SEQ ID NO:3 ) (referred to as VDIV K) ; or TTTGNAVAPS (SEQ ID NO:4)(referred to as VDI B).
Preferably the T-helper stimulating epitope is located in the middle of the construction, bordered to its left with the sequence VDI B and to its right with the sequence VDIV K. In another preferred embodiment of this inven¬ tion, the T-helper stimulating epitope is located on the protein N-terminus side of the B-cell neutralizing antibody stimulating epitopes, or on their C-terminus side, or in a combination that enhances immunogenicity.
Suitably, a linker sequence is provided so as to separate the sequences ALNIWDRFDVFCTLGATTGYLKGNS
(SEQ ID NO:l) or TINKPKGYVGKEFPLDLTAGTDAAT (SEQ ID
NO:2) from the sequences LDVTTLNPTI (SEQ ID NO:3) or TTTGNAVAPS (SEQ ID NO:4).
It is also contemplated that the peptide addi¬ tionally comprises other known or yet unknown B-cell neutralizing eρitope(s) of the outer membrane protein or other known or yet unknown T-helper stimulating epi- tope(s) from the major outer membrane protein of C. trachomatis or from another protein.
Further, this invention relates to a peptide comprising at least one T-helper cell stimulating epi¬ tope and at least two B-cell neutralizing antibody stimulating epitopes, or isolated nucleic acid molecule coding for the said peptide, for use in preparation of a vaccine for C. trachomatis, wherein said recombinant protein of nucleic acid molecule is formulated in a pharmaceutically acceptable excipient for use as a vac- cine.
This invention also relates to an immunoassay for detecting the presence of antibody to C. trachomatis in a sample, comprising a recombinant hy¬ brid protein and means for detecting antibody bound to said protein, wherein the amino acid sequence of said protein comprises at least one of the T-cell stimulat¬ ing epitope and at least two of the B-cell neutralizing antibody epitopes described herein.
Peptides of the present invention may be useful as substitutes for the naturally-occurring major outer membrane proteins of C. trachomatis.
The invention also relates to a peptide com¬ prising a B-cell neutralizing antibody stimulating epi¬ tope which comprises either (a) an epitope located within the sequence VDIV K (SEQ ID NO:3) or the
sequence VDI B (SEQ ID NO:4); or (b) an epitope immu¬ nologically cross-reactive with an epitope as in (a) above.
Such a peptide may be prepared and used as described above in relation to the peptides comprising regions (i) and (ii).
A panel of monoclonal antibodies (mAbs) were produced, following immunizations with Chlamydia trachomatis whole EBs or purified MOMP. Reactivity of anti-MOMP mAbs with the 15 serovars of Chlamydia trachomatis was further evaluated. Serovar-specific, subspecies-specific and species-specific mAbs were so purified and characterized. The in vi tro neutralizing activity of an antibody is defined as the ability of this mAb to inhibit the infection of host cells by the bacteria. The neutralizing activity of mAbs of differ¬ ent specificities against Chlamydia trachomatis serovar K, which is one of the serovar responsible of the geni¬ tal tract infections, was investigated further. Results indicated that five mAbs were neutralizing, one which is serovar-specific, two subspecies-specific and two species-specific (react with the 15 serovars of Chlamydia trachomatis) .
In order to define the neutralizable epitopes of the major outer membrane protein of Chlamydia trachomatis serovar K, fifteen peptides of ten amino acid residues, overlapping by five residues, corre¬ sponding to the four variable domains (VDI to VDIV: residues 64-85, 139-160, 224-237 and 287-319) of the MOMP were synthesized. The fine specificity of seven mAbs produced (five neutralizing and two non- neutralizing mAbs) and three polyclonal antibodies, was defined by direct binding on synthetic peptides. Two neutralizable epitopes were identified, one located in VDI (69SDVEGLQNSP78) and one located in VDIV
(292LDVTTLNPTI302) . Neutralizable epitope located in the VDIV is a species-specific epitope of 9 uncharged residues that are conserved among all 15 serovars. This epitope has been reported by many others for its ability to induce neutralizing antibodies in some Chlamydia trachomatis serotypes. However, such results have never been reported for serovar K.
Neutralizable epitope located in the VDI is located in the same amino acid region as a neutraliz- able epitope for some C and C-related serovars such as serovars A (Baehr, Proc . Natl . Acad. Sci . USA .85.: 4000, 1988; Su, J. Exp. Med. 172: 203, 1990; Su, J. Exp. Med. 175: 227, 1992); C, I, J, and L3 (Qu, Infect . Immun. 61: 1365, 1993). In order to extend these findings to B-complex serovar VDI, the corresponding peptide of the VDI of serovar B, 69TTTGNAVAPS78 was synthesized. Synthetic peptides defining neutralizable epitopes (VDI-K, VDI-B and VDIV-K) were able to inhibit the binding of respective mAbs to MOMP and to whole EBs and were also able to inhibit the neutralizing activity of mAbs. Moreover, mice immunized with synthetic peptides defining neutralizable epitopes, developed a humoral response against these peptides and the corresponding native EBs, and mouse sera possess neutralizing activity. These data confirm the functional value of these synthetic peptides and their applicability in vaccine development.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE 1 Construction of the hybrid recombinant protein
Genomic DNA was extracted from purified EBs of C. trachomatis serovars B and K using proteinase K
(lOOμg/ml) in 0.5% SDS, 1 hr at 37°C. Following pre¬ cipitation of cell debris with CTAB/NaCl solution, nucleic acids were phenol/chloroform/isoamyl alcohol extracted, and isopropanol precipitated. The DNA was rinsed in 70% ethanol, dried, and then suspended in tris-EDTA (TE) buffer.
The MOMP genes fragments were obtained by polymerase reaction amplification from genomic DNA. As is illustrated in Fig. IA, sense primer used for the MOMP B fragment amplification was 5' G-AAA-GAA-TTC-AAA- ATG-GGT-G 3' (SEQ ID NO:5), comprising an Eco RI diges¬ tion site, and antisense primer was 3' CCT-TTG-AGA-CGA- AGA-AAG-TTA-GAC-GTC-ATC 5' (SEQ ID NO:6), comprising a Pst I digestion site, in order to amplify the region coding for the B-cell neutralizing antibody stimulating epitope (TTTGNAVAPS) (SEQ ID NO:4) and the T-helper stimulating epitope (ALNIWDRFDVFCTLGATTGYLKGNS) (SEQ ID N0:1). As is illustrated in Fig. IB, sense primer used for the MOMP K fragment amplification was 5 ' G-ATC-CGT- ATC-CTG-CAG-CCT-AAA-TTG 3" (SEQ ID NO:7), comprising a Pst I digestion site, and antisense primer was 3' CCT- GAC-CGA-CTA-TGT-TAC-ATT-CTT-AAG-AGG 5' (SEQ ID NO:8), comprising an Eco RI digestion site, in order to am¬ plify the region coding for the B-cell neutralizing antibody stimulating epitope (LDVTTLNPTI)(SEQ ID NO:3). The amplification was carried out in a 100 μl volume containing 100 ng of genomic DNA. A DNA Thermal Cycler ™ (Perkin-Elmer Model 9600) was used with 30 sec of melting at 94°C, 30 sec of annealing at 50°C, and 30 sec of polymerization at 72°C for 35 cycles. Amplifi¬ cation products of the correct size were isolated from a 1.2% agarose gel.
The amplified ompi DNA fragments were phos¬ phorylated and blunted. The plasmid vector pBluescript II KS- (BS) was digested with Eco RV and Sma I, in
order to remove the Eco RI and Pst I digestion sites of the polylinker and further alkaline phosphatase treated. The VDI B-T fragment and the VDIV K fragment were excised from a 1.2% agarose gel. Each one of the fragments were ligated in BS, and transformed in E. coli DH5α, leading to the formation of clones desig¬ nated BS-VDI B-T and BS-VDIV K. Clones of both type were further digested with Pst I and Xba I, in order to 1) release the VDIV K fragment (see Fig. IA) and 2) open the BS-VDI B-T vector (see Fig. IB). Isolated VDIV K fragment from Fig. IB was then ligated in BS-VDI B-T from Fig. IA, resulting in BS-VDI B-T-VDIV K of Fig. IC, with both gene fragments in the appropriate reading frame as shown by DNA sequencing. The hybrid construction, flanked either end by an Eco RI digestion site (Fig. IC), was released after an Eco RI digestion, isolated from the gel, blunted and further ligated in blunted-pET 3b for transformation in E. coli BL21 (DE3). EXAMPLE 2
Recombinant hybrid protein expression The expression of the recombinant hybrid pro¬ tein by DE3/pET 3b-VDI B-T-VDIV K cells was evaluated by SDS-PAGE and Western blot analysis. Culture of DE3/pET 3b-VDI B-T-VDIV K was grown overnight in LB broth plus 100 μg of ampicillin per ml. Overnight cul¬ tures were diluted 1/100 in fresh LB and grown to mid- log phase
, and recombinant hybrid protein expression was induced by the addition of 0.3 mM IPTG (isopropyl-β-D-thiogalactopyranoside) . Culture super¬ natant, whole cells, cell membrane and cytoplasm were isolated and the activity of characterized monoclonal and polyclonal antibodies was then assessed on these fractions by direct-binding ELISA and by immunoblot analysis.
SDS-PAGE analysis shows the apparition of a protein of an expected molecular weight of approxi¬ mately 16 kDa in cytoplasm (Fig. 2). Monoclonal anti¬ body directed against the described neutralizable epi- tope of the VDIV K, along with polyclonal antibody spe¬ cific of the neutralizable epitope of the VDI B, were used for the characterization of the recombinant hybrid protein expressed by E. coli . Recognition of the expressed epitopes by their respective antibody was shown by direct-binding ELISA (Fig. 3), as well as by immunoblot analysis, indicating that neutralizable epi¬ topes were expressed in an appropriate conformation. Furthermore, knowing that antibodies used here reacted with chlamydial EBs in their native form (Villeneuve, Infect . Immun. J52.: 3547, 1994; Villeneuve, Microbiol . 140: 2481, 1994), the recognition of the recombinant hybrid protein by these antibodies confirms the rele¬ vancy of its use as a vaccine.
The recombinant hydrid proteins, as described herein or in accordance to the possible modifications described previously, may also be expressed with other genes, to provide a recombinant protein fused to immu¬ nogenic carrier such as the cholera toxin B subunit. Conveniently, hydrid protein will be inserted in appro- priate reading frame with the fused gene and under the regulatory control of the regulatory system of the fused gene.
The recombinant hydrid proteins may be glycosy¬ lated, partially glycosylated or unglycosylated, depending on the nature of the expression host. Gener¬ ally, prokaryotes such as E. coli will provide no gly¬ cosylation of the translated products, while yeast and mammalian cell culture will provide partial or substan¬ tial glycosylation. In regard to these different expression possibilities, we have cloned the hybrid
construction in pHIL D2 and pPIC 9 for its expression in the yeast, and in pCR III for its expression in mam¬ malian cells. When employing a yeast host, codons which appear at high frequency in the structural genes encoding the yeast glycolytic enzymes may be employed, along with appropriate promoters. In some instances, it may be desirable to add or modify nucleotides in the constructions, to increase stability, enhance immuno¬ genicity or facilitate the conjugation of the hybrid protein to a carrier. Also, the constructions may include a secretory leader and processing signal sequence to effect gene product in the yeast or mammal¬ ian cells.
EXAMPLE 3
Immunogenicity of the recombinant hydrid protein
BALB/cJ (H-2d), A/J (H-2a), C57BL/10SnJ (H-2b), CBA/J (H-2k), DBA/1J (H-2q) and SJL/J (H-2s) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Females at 8-12 weeks of age were used for experimenta¬ tions- Groups of five mice were immunized by intrape¬ ritoneal injection of 50 μg of VDI B-T-VDIV K emulsi¬ fied in Complete Freund's Adjuvant (CFA) on day 0, and boosted once three weeks later with the same dose of recombinant protein in Incomplete Freund's Adjuvant. Mice were bled three weeks after the secondary immuni¬ zation.
A (G-17), B (TW-S), Ba (AP-2), C (TW-3), D (UW- 3), E (UW-5), F (UW-55), G (UW-57), H (UW-4), I (UW- 12), J (UW-36), K (UW-31), Ll (440), L2 (434), and L3 (404) elementary bodies (EBs) were purified from infected cells by centrifugation on a Renografin den¬ sity gradient. The presence of C. trachomatis specific IgG was evaluated by enzyme linked immunoabsorbent assay (ELISA). This experiment was done in order to assess the functionality of the T-cell stimulating
epitope. In fact, the presence of IgG indicates that memory T-helper cells have been primed and recalled. The IgG reactivity on the 15 C. trachomatis serovars was therefore tested. Briefly, microtiter plates were coated overnight at 4°C with 100 μl of formalin-killed EBs (5μg/ml) in phosphate-buffered saline (PBS, pH 7.4). After saturation with bovine serum albumine (3%) in PBS, sera were added at a dilution of 1/2000 for lh30 at 37°C. After appropriate washes, mouse immunoglobulin G was detected by using a biotin-labeled anti-mouse IgG (Jackson Immunoresearch Laboratories), lh30 at 37°C. The reaction was developed in a peroxidase substrate solution [2, 2 '-azino-di (3- ethylbenzthiazoline sulfonate) and H2O2] following the addition of a streptadivine-peroxidase conjugate. The A 05 was then measured. Pooled pre-immune sera were used as the negative controls.
As shown in Fig. 4, sera from the six strains of mice that were immunized with the recombinant hybrid protein reacted with several serotypes. Although the A/J response was generally weaker, results of this study are consistent with the hypothesis that the T helper cell epitope contained in the SEQ ID N0:1 is recognized by multiple MHC class II haplotypes and that these T-cell determinants provide cognate help for antibody production. Also the serotypes corresponding to the sequences enclosed in the hybrid protein, or serotypes closely related (B, Ba and K, L3) were better recognized. Therefore, it is contemplated that the preferred recombinant hybrid protein vaccine of this invention can additionally incorporate B-cell neutral¬ izing epitope in order to evoke a broadly cross reac¬ tive antibody response.
EXAMPLE 4 In vitro neutralization of chlamydial infectivity
Neutralization assays were done without cen¬ trifugation. Briefly, purified EBs were diluted in 0.25M sucrose - 10 mM sodium phosphate - 5mM L-gluamic acid (SPG) (pH 7.2) for a final concentration of 4 X 10^ inclusion forming units (IFUs) per ml. Two fold dilutions of pooled mouse sera (diluted in SPG-0.5% decomplemented fetal calf serum (FCS)) were mixed with an equal volume of chlamydiae. The mixture was incubated at 37°C for 60 minutes, and 400 μl of the appropriate C. trachomatis-serum mixture dilutions were inoculated in triplicate onto confluent HaK cell monolayers. After 2h of incubation, the inocula were removed and the monolayers were washed and fed with 500 μl of Eagle's minimal essential medium supplemented with 10% FCS containing 0.5μg of cycloheximide per ml and incubated at 37°C for 72h. The monolayers were fixed with methanol, and chlamydial inclusions were identified by fluorescent-antibody staining with a genus specific mAb. A 50% or greater reduction from the number of control inclusion-forming units in infectivity was considered positive for neutralization. Immune sera from mice immunized with the recom¬ binant hybrid protein were assayed for their in vitro neutralizing activity as described above. As shown in Table I, serum from mouse strains tested displayed a significant neutralizing activity against serovar K. Although activity against serovar B and others remains to be tested, these results indicate that antibodies generated in animals, in response to the recombinant hybrid protein, were functional neutralizing antibodies and that this protein constitutes a very attractive vaccine structure.
Table 1 In vitro neutralization of Chlamydia trachomatis serovar K by immune sera from mice immunized with the recombinant hydrid protein
* Neutralizing titers are expressed as the reciprocal of pooled serum dilutions that neutralized 50% of chlamydial infectivity. Preimmune or normal sera had neutralizing titers of less than 20.
EXAMPLE 5 Induction of a Chlamydia-specific mucosal response
The local response has a critical role to play to prevent invasion of the host by organisms, such as Chlamydia trachomatis, penetrating through the mucosa. The importance of secretory IgA antibodies has been clearly shown but cellular immunity is probably also involved particularly in the view that Chlamydia trachomatis is an intracellular organism. In fact, the production of IgA reflect activation of both B and T cells. The development of such a protective immune response in the mucosa is the target of the Chlamydia vaccine program. However, this objective should be achieved with the concern that the vaccine has to be designed for human use. Therefore, adjuvants, carriers or vectors acceptable by the human regulatory agencies were used in the development of this vaccine.
The objectives of this experiment were to induce, following intra-nasal immunization with repeti¬ tive synthetic peptides or the recombinant hybrid pro¬ tein, alone or in combination with the cholera toxin B subunit (CTB) as "adjuvant", a specific mucosal IgA
antibody response. Also, to analyze the MHC restric¬ tion of the induced immune response by using strains of mice differing at the H-2 locus. Finally, to assess the functionality of this antibody response and its ability to neutralize infectivity in vitro, which is an essential criteria for the development of an effective vaccine.
Briefly, BALB/cJ (H-2d), A/J (H-2a), C57BL/10 SnJ (H-2b), CBA/J (H-2k), DBA/1J (H-2q) and SJL/J (H- 2s) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Females at 8-12 weeks of age were used for experimentations and did not receive any hormonal treatment. Groups of 8 mice were immunized intra¬ nasally with 20 μg of cholera toxin B subunit (CTB), 25 μg 4 VDI B + 25 μg 4 VDIV K with or without 20 μg of CTB, or 50 μg of rVDI B-T1-VDIV K with or without 20 μg of CTB. Antigens were delivered to mice by introducing 10 μl of PBS in each nostril using a micropipette. Intra-nasal immunizations were carried out on days 0, 14, 28 and 62. Bleeding and vaginal washes (2 X 30μl) were performed on days 1, 13, 27, 41, 55, 69 and 75. Sera were decomplemented by heating 30 minutes at 56°C. Vaginal washes were centrifuged and supernatants, as well as sera, were stored at -70°C until tested. Specific serum and vaginal IgA were monitored by solid-phase radioimmunoassay (RIA). For specific IgA, microtiter plates were coated overnight at 4°C with lOOμl of EBs (5μg/ml) of serotype B or K in PBS (pH 7.4), or with synthetic peptides (50μg/ml) 4 VDI B, 4 VDIVK (corresponding to four-times repeats of target epitopes) or irrelevant control peptide (from the sequence of the allergen Lolium perenne) in 50 mM car¬ bonate buffer (pH 9.6). After saturation with BSA (3%) in PBS, appropriate dilutions of pooled sera or vaginal washes were added for lh30 at 37°C. After washes,
mouse immunoglobulin A were detected by using a 125j_ anti-mouse IgA (Pierce Biochemical Co., USA), lh at room temperature. Radioactivity was measured on a 1261 Multigamma™ counter (LKB Wallace). Non specific bind- ing of vaginal washes and sera on an irrelevant peptide was less than 1 and 10% respectively.
Total vaginal IgA was also monitored by solid- phase RIA. Briefly, microtiter plates were coated overnight at 4°C with 100 μl of a sheep anti-mouse IgA (The Binding Site, Birmingham, England) (lOμg/ml) in 50 mM carbonate buffer (pH 9.6). After saturation with BSA (3%) in PBS, appropriate dilutions of pooled sera or vaginal washes were added for lh30 at 37°C. Puri¬ fied mouse IgA was used for the standard curve and was added to the anti-IgA antibody in the same conditions. After washes, mouse immunoglobulin A were detected as described above.
Results were reported in nanograms of Chlamy- dia-specific IgA per total IgA (micrograms per millili- ter) to minimize differences between groups due to variability in total immunoglobulin concentrations resulting from the vaginal washes.
Specific serum and vaginal IgG responses were further monitored by enzyme-linked immunosorbent assay. Briefly, microtiter plates were coated overnight at 4°C with 100 μl of EBs (5μg/ml) of serotype B or K in PBS (pH 7.4), or with synthetic peptides (50μg/ml) 4 VDI B, 4 VDIV K or irrelevant control peptide in 50 mM carbon¬ ate buffer (pH 9.6). After saturation with BSA (3%) in PBS, appropriate dilutions of pooled sera or vaginal washes were added for lh30 at 37°C. After appropriate washes, mouse immunoglobulin G were detected by using a peroxidase-conjugated goat anti-mouse IgG. The reac¬ tion was developed in a peroxidase substrate solution t2, 2 '-azino-di(3-ethylbenzthiazoline sulfonate) and
H2O2]. The A405 was then measured. Non specific bind¬ ing of vaginal washes and sera on an irrelevant peptide gave O.D. of less than 0.15 and 0.5 respectively.
Complement-independent neutralization assays were carried out without centrifugation. Briefly, purified EBs were diluted in 25 μl of 0.25 M sucrose/10 mM sodium phosphate/5mM L-glutamic acid (SPG, pH 7.2) to give a final concentration of 6 X IO4 inclusion-forming-units (IFUs) per ml. The bacteria were then mixed with an equal volume of serially diluted pooled vaginal washes (diluted in SPG plus 0.5% fetal calf serum). The mixture was incubated at 37°C for 60 min an further diluted to give a final volume of 400 μl, before to be inoculated onto confluent HaK cell monolayers grown in 24-well plates (Costar). After 2h incubation, the inocula were removed and the monolayers washed with Hank's balanced salts solution. Monolayers were fed with 500 μl of Eagle's minimal essential medium supplemented with 10% fetal calf serum containing 0.5 μg cycloheximide/ml and incubated at 37°C, 5% CO2 for 72h. The monolayers were fixed with methanol, and chlamydial inclusions were identified by indirect fluorescent antibody staining using a genus- specific mAb to chlamydial LPS. The total number of inclusions per well were counted. Samples were tested in duplicate.
Groups immunized with the CTB alone or with the repetitive synthetic peptides, alone or in combination with the CTB, did not produce significant amounts of specific vaginal or serum IgA. However, as shown in Fig. 5, BALB/c mice immunized with the recombinant hybrid proteins developed significant amounts of spe¬ cific vaginal IgA antibodies. These antibodies effi¬ ciently recognized the VDIV K epitope in its synthetic form, and to a lesser extent the native epitope on the
EB of Chlamydia trachomatis serotype K. The IgA also weakly reacted with the synthetic peptide 4 VDI B, although the latter response was less significant. The response occurred following the fourth immunization on day 62. Surprisingly, mice which received the recombi¬ nant hybrid protein along with the CTB, did not develop Chlamydia-specific IgAs. To date however, the efficacy of CTB as a mucosal adjuvant remains equivocal. Indeed, the CTB has been reported to enhance the immune response to some mucosally administered antigens, while failing to have such an effect to other antigens unless holotoxin was added. The immunogenicity of antigens was reported to be better when they were conjugated to CTB instead of simply mixed together with it. The Chlamydia-specific vaginal IgA response was also studied in other strains of mice, varying at the H-2 haplotype of MHC class II. Although slight vari¬ ations can be observed among the different strains of mice, a significant specific IgA antibody response was seen at mucosal site in all strains. Results showed that some strains of mice developed a better response to the VDIV epitope (BALB/c and C57BL/10 SnJ), while for other strains, no significant differences can be seen between the reactivity for the VDI B and the VDIV K epitopes (A/J, DBA/1J, and SJL/J). It is important to note that levels of mucosal IgA antibodies against the native form of the epitopes (elementary bodies), as well as their synthetic forms, were comparable except for BALB/c where higher anti-peptide responses were observed and CBA/J where a strong anti-VDI B response can be seen.
In some strains of mice (BALB/c, CBA/J and DBA/J), an increase in the IgA antibody production fol¬ lowing the last immunization on day 62 was observed.
On another hand, the specific serum IgA response (Fig. 6), although detectable, was around ten fold weaker than the vaginal response, suggesting that
IgA in the vaginal fluids does not originate from serum transudation through the vaginal epithelium.
As mentioned for the BALB/c, the IgA antibody response observed for mice immunized with the recombi¬ nant protein along with the CTB was lower or comparable to the one observed for mice immunized with rVDI B-Tl- VDIV K alone. Higher levels of mucosal IgA were not seen in any of the mouse strains analyzed.
As shown on Fig. 7, mice immunized intra¬ nasally with rVDI B-Tl-VDIV K also generated a signifi¬ cant serum IgG response. The levels of specific IgG increase over time and seem to remain stable, in con¬ trast to the vaginal IgA. This indicates that in addi¬ tion to the induction of a mucosal IgA response, immu¬ nization with the recombinant hybrid protein led to the production of specific serum IgG. It is interesting to note that the peak in IgG and IgA response often coin¬ cided further confirming the memory type of these responses. This is well illustrated in the case of BALB/c, where a strong IgA and IgG anti-4 VDIV K response developed after the last immunization on day 62.
Groups immunized with the CTB alone or with the repetitive synthetic peptides, alone or in combination with the CTB, did not develop a significant specific serum IgG response. However, mice immunized with the CTB in addition to rVDI B-Tl-VDIV K developed IgG anti¬ bodies. Finally, no specific IgG were detectable in the vaginal washes (Fig. 8).
Preliminary results suggest that the specific IgA antibodies found in vaginal washes were functional. Indeed, C57BL/10 SnJ mice immunized with the rVDI B-Tl-
VDIV K protein developed an in vi tro neutralizing activity against C. trachomatis serovar K. A 39% reduction of chlamydial infectivity was observed when using vaginal washes harvested at day 55 following immunization. Such an inhibition was not observed with vaginal washes obtained from non immunized mice. These data are promising taking into account the dilution factor in the vaginal washes. Indeed, the Inventor reported that 3 μg/ml of anti-C. trachomatis mAb (DP10) were required to obtain a 50% reduction of chlamydial infectivity; these concentrations are higher than those measured in the vaginal washes.
These results suggest that a Chlamydia-specific vaginal IgA response was achieved upon nasal immuniza- tion with the recombinant hybrid protein and without the use of any adjuvant. Moreover, the induced vaginal IgA response was functional since it significantly reduced the in vitro chlamydial infectivity. Finally, results showed that the helper T-cell epitope of the recombinant hybrid protein was recognized in the con¬ text of multiple MHC haplotypes, since the responses were observed in all strains of mice. A Chlamydia-spe¬ cific serum IgG response was also seen.
While the invention has been described in con- nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
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(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 951623.9
(B) FILING DATE: 09-AUG-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 1 5 10 15
Thr Thr Gly Tyr Leu Lys Gly Asn Ser 20 25
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu Phe Pro Leu Asp 1 5 10 15
Leu Thr Ala Gly Thr Asp Ala Ala Thr 20 25
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Leu Asp Val Thr Thr Leu Asn Pro Thr Ile 1 5 10
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Thr Thr Thr Gly Asn Ala Val Ala Pro Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Sense DNA Primer"
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAAAGAATTC AAAATGGGTG
20
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Anti-Sense DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTACTGCAGA TTGAAAGAAG CAGAGTTTCC 30
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Sense DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATCCGTATC CTGCAGCCTA AATTG 25
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Anti-Sense DNA Primer"
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGAGAATTCT TACATTGTAT CAGCCAGTCC 30
Claims (17)
1. A recombinant hybrid protein comprising one or several T-helper cell stimulating epitope from the major outer membrane protein of C. trachomatis and two or more B-cell neutralizing antibody stimulating epitopes from the major outer membrane protein of C. trachomatis.
2. The recombinant hybrid protein of claim 1, wherein said T-helper cell stimulating epitope comprises the amino acid sequence SEQ ID NO:l or SEQ ID NO:2.
3. The recombinant hybrid protein of claim 1, wherein said B-cell neutralizing antibody stimulating epitope comprises the amino acid sequence SEQ ID NO:3 or SEQ ID NO:4.
4. The recombinant hybrid protein of claim 2 or 3, wherein said T-helper stimulating epitope is either flanked by said B-cell neutralizing antibody stimulating epitopes, or on their N-terminus side, or on their C-terminus side, or in a combination that enhances immunogenicity.
5. The recombinant hybrid protein of claim 4, further comprising a linker sequence inserted between the sequences SEQ ID N0:1 or SEQ ID NO:2 from the sequences SEQ ID NO:3 or SEQ ID NO:4.
6. The recombinant hybrid protein of Claim 4, wherein said protein further comprises at least one other T- helper stimulating epitope from a protein other than the outer membrane protein of C. trachomatis .
7. A nucleic acid molecule coding for the expression of the recombinant hybrid protein of claim 1.
8. Use of a recombinant hybrid protein according to claim 1 for the preparation of a vaccine for C. trachomatis .
9. Use of a nucleic acid molecule according to claim 7 for the preparation of a vaccine for C. trachomatis.
10. Use of the recombinant hybrid protein according to claim 1 in an immunoassay for detecting the presence of antibodies to C. trachomatis in a sample.
11. Composition comprising a recombinant hybrid protein according to claim 1, 2, 3, 4, 5 or 6 in association with a pharmaceutically acceptable excipient.
12. Use of a composition according to claim 11 as a vaccine for C. trachomatis.
13. A method for preparing a recombinant hybrid protein according to claim 1, 2, 3, 4, 5 or 6, which comprises the step of synthesizing an amino acid sequence, wherein said amino acid comprises an antigenic determinant or target epitope in a repetitive form.
14. Method for stimulating an immune response, said method comprising the step of administering to a patient an effective amount of a recombinant hybrid protein according to claim 1, 2, 3, 4, 5 or 6.
15. The method of claim 14, for the prophylaxis or treatment of a disease.
16. Method for detecting the presence of an antibody to an epitope, said method comprising the steps of i) contacting a sample suspected of containing said antibody to a recombinant hybrid protein according to claim 1, 2, 3, 4, 5 or 6, and ii) detecting said antibody bound to said peptide.
17. A diagnostic kit for use in a method according to claim 16, said kit comprising a recombinant hybrid protein comprising one or several T-helper cell stimulating epitope from the major outer membrane protein of C. trachomatis and two or more B-cell neutralizing antibody stimulating epitope from the major outer membrane protein of C. trachomatis .
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9516293 | 1995-08-09 | ||
| GBGB9516293.9A GB9516293D0 (en) | 1995-08-09 | 1995-08-09 | Novel peptides and their use as vaccines |
| PCT/CA1996/000538 WO1997006263A1 (en) | 1995-08-09 | 1996-08-08 | Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU6653096A true AU6653096A (en) | 1997-03-05 |
Family
ID=10778973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU66530/96A Abandoned AU6653096A (en) | 1995-08-09 | 1996-08-08 | Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccine |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0846176A1 (en) |
| AU (1) | AU6653096A (en) |
| CA (1) | CA2228522A1 (en) |
| GB (1) | GB9516293D0 (en) |
| MX (1) | MX9801089A (en) |
| NZ (1) | NZ313950A (en) |
| WO (1) | WO1997006263A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003731A2 (en) * | 1998-07-17 | 2000-01-27 | Spectrum Medical Sciences Ltd. Oy | Vaccine to prevent and treat chlamydial infections |
| CA2366462C (en) * | 1998-12-04 | 2011-08-09 | University Of Manitoba | Two-step immunization procedure against chlamydia infection |
| US6432916B1 (en) | 1998-12-08 | 2002-08-13 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| US6565856B1 (en) * | 1998-12-08 | 2003-05-20 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| US6555115B1 (en) | 1998-12-08 | 2003-04-29 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| US20020061848A1 (en) | 2000-07-20 | 2002-05-23 | Ajay Bhatia | Compounds and methods for treatment and diagnosis of chlamydial infection |
| US6447779B1 (en) | 1998-12-08 | 2002-09-10 | Corixa Corporation | Compounds for the diagnosis of Chlamydial infection |
| US6448234B1 (en) | 1998-12-08 | 2002-09-10 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| US6919187B2 (en) | 2000-04-21 | 2005-07-19 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| EP2192128A3 (en) | 2000-04-21 | 2010-09-22 | Corixa Corporation | Compounds and methods for treatment and diagnosis of chlamydial infection |
| EP1221317A1 (en) | 2000-12-28 | 2002-07-10 | SHAN-Beteiligungsgesellschaft m.b.H. | Vaccines containing hybrid polypeptides consisting of at least two different allergenic proteins |
| WO2011147975A1 (en) | 2010-05-28 | 2011-12-01 | Spixia Biotechnology Ab | Chimeric momp antigen, method and use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2144882A1 (en) * | 1992-09-18 | 1994-03-31 | Harlan D. Caldwell | Synthetic peptide vaccine for chlamydia trachomatis |
| CA2162664A1 (en) * | 1993-05-13 | 1994-11-24 | Andrew David Murdin | Hybrid picornaviruses expressing chlamydial epitopes |
-
1995
- 1995-08-09 GB GBGB9516293.9A patent/GB9516293D0/en active Pending
-
1996
- 1996-08-08 AU AU66530/96A patent/AU6653096A/en not_active Abandoned
- 1996-08-08 CA CA002228522A patent/CA2228522A1/en not_active Abandoned
- 1996-08-08 EP EP96926290A patent/EP0846176A1/en not_active Withdrawn
- 1996-08-08 NZ NZ313950A patent/NZ313950A/en unknown
- 1996-08-08 WO PCT/CA1996/000538 patent/WO1997006263A1/en not_active Application Discontinuation
-
1998
- 1998-02-09 MX MX9801089A patent/MX9801089A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997006263A1 (en) | 1997-02-20 |
| MX9801089A (en) | 1998-11-29 |
| CA2228522A1 (en) | 1997-02-20 |
| EP0846176A1 (en) | 1998-06-10 |
| GB9516293D0 (en) | 1995-10-11 |
| NZ313950A (en) | 1999-09-29 |
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| Date | Code | Title | Description |
|---|---|---|---|
| MK5 | Application lapsed section 142(2)(e) - patent request and compl. specification not accepted |