WO2021063917A1 - Vaccine against chlamydia in swine - Google Patents

Abstract

The present invention relates to means and methods to protect pigs against disease caused by bacteria belonging to the genus Chlamydia. In particular, the present invention relates to isolated B- and T-cell epitopes derived from the major outer membrane protein of Chlamydia suis and/or Chlamydia abortus which can be used against an infection with said species.

Description

Vaccine against Chlamydia in swine

Field of the invention

The present invention relates to means and methods to protect pigs against disease caused by bacteria belonging to the genus Chlamydia. In particular, the present invention relates to isolated B- and T-cell epitopes derived from the major outer membrane protein of Chlamydia suis and/or Chlamydia abortus which can be used against an infection with said species.

Background art

Chlamydia suis (C. suis) is an obligate intracellular Gram-negative bacterium, belonging to the order of Chlamydiales. The pig is the only known natural host of C. suis. Chlamydia suis is currently considered to be the most prevalent chlamydial species in pigs but pigs also can become infected by C. pecorum, C. abortus and C. psittaci (reviewed by Schautteet and Vanrompay, 2011 ). C. suis in pigs has been associated with asymptomatic infections but also with a variety of clinical symptoms such as conjunctivitis, rhinitis, pneumonia, enteritis, reproductive disorders such as irregular return to oestrus, early embryonic dead in inseminated sows and inferior semen quality in boars (decrease of sperm cell motility and death of sperm cells) (Schautteet et al. , 2013; Chahota et al. , 2017). Differences in clinical symptoms and pathology caused by C. suis are thought to be due to a high degree of genetic diversity in C. suis. The latter has recently been proven by Joseph et al., (2016), who performed the first comparative genomic analysis of C. suis, by whole genome alignment of 12 C. suis strains.

C. suis infections could be successfully treated with tetracyclines until the appearance of a tetracycline resistant (Tc^R) phenotype, which was first isolated on pig farms in Iowa and Nebraska (Andersen and Rogers, 1998). Soon thereafter, tetracycline resistant C. suis strains appeared in other countries including Belgium, Cyprus, Germany, Israel, Italy, Switzerland and The Netherlands (Wanninger et al. , 2016; Vanrompay et al. , 2016). The emergence of Tc^R C. suis strains raises considerable concern because C. suis shares 79.8% average nucleotide identity with the human pathogen C. trachomatis. C. trachomatis is the leading cause of sexually transmitted diseases (STD) and preventable blindness (trachoma) worldwide. Recently, Dean et al., (2013), found C. suis mRNA in the eyes of Nepalese trachoma patients.

Chlamydia abortus (C. abortus) infection causes abortion and reproductive failure in several animals, including sheep (ovine enzootic abortion (OEA)), goats and pigs. The pathogen is a great threat to human health and brings enormous economic loss to livestock industry. In the early 1980s an attenuated strain of C. abortus was developed as a live vaccine for sheep and is one of the 5 commercially available vaccines in Europe and the USA, the other four being inactivated whole organism-based vaccines (Longbottom and Livingstone, 2006). These commercial live-attenuated and inactivated vaccines offer good protection against OEA and significantly reduce the shedding of infective organisms, a factor important in limiting the spread of infection to other animals. However, concerns remain over the safety of using live-attenuated vaccines. There may also be a risk of the attenuated strain reverting to virulence, thus having the potential to cause disease and abortion in the vaccinated animal. Furthermore, the vaccine cannot be administered during pregnancy or to animals being treated with antibiotics limiting its use. In contrast, the inactivated vaccines can be administered to pregnant ewes, although care must be taken in handling and administering these vaccines as they are adjuvanted with mineral oils, which have the potential to cause tissue necrosis if accidentally self-injected. The only other animal chlamydial vaccines, which are commercially available are for C. felis infection in cats (Longbottom and Livingstone, 2006).

DNA vaccination was most promising, mimicking a live vaccine, creating protective CD4 as well as CD8 responses, however antibody responses were rather low. In addition, DNA vaccines for swine are still too expensive, mainly because plasmid purification costs too much. Moreover, the public is not (yet) ready to consume products (meat) from DNA vaccinated animals. Hence until now, safe and effective Chlamydia vaccines for pigs are still not available.

It was an object of the present invention to design an innovative vaccine that creates both optimal humoral and cellular immune responses based on a combination of epitopes in one vaccine.

Summary of the invention

Disclosed are peptides, compositions, kits, use and methods for inducing an immune response against a Chlamydia infection and/or for preventing, treating or reducing symptoms of a Chlamydia related disease. The invention provides isolated peptides, and use thereof, comprising B- and T-cell epitopes for producing a vaccine against species of the genus Chlamydia. The composition, kits, and methods contain or utilize these epitopes and (poly)peptides as an antigen.

The compositions, kits, and methods may be utilized to induce a cell-mediated response and/or a humoral response against a pathogen, in particular a cell-mediated and a humoral response. In one embodiment, the pathogen is from the genus Chlamydia as provided herein. The invention also pertains to a method for immunizing a subject, in particular a pig or a small ruminant such as a sheet or a goat, against disease caused by infection with Chlamydia, in particular Chlamydia suis and/or Chlamydia abortus.

The invention also pertains to a method for producing a pharmaceutical composition or vaccine according to the invention, the method comprising preparing, synthesizing or isolating a polyepitope construct according to the invention, and optionally adding other antigens and/or a carrier, vehicle and/or adjuvant substance. Further embodiments of the present invention are a vector which comprises a nucleic acid encoding at least one or more of the epitopes or peptides, in particular the polyepitope construct, as described herein, and which is capable of expressing the respective peptides. A host cell comprising the expression vector and a method of producing and purifying the herein described peptides are also part of the invention. The invention further relates to a micro-organism secreting a (poly)peptide for use in mucosal delivery to treat an immune response related disease in a subject.

Figure legends

With specific reference to the figures, it is to be noted that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention.

Figure 1: C. suis/C abortus- polyepitope (CsCa) construct including a Signal (Usp45 protein precursor) and NanoLuciferase (NLuc) sequence (both underlined in the amino acid sequence).

Figure 2: Nucleic acid and amino acid sequence of a resp. C.suis (polyCs), C. abortus (polyCa) and C.suis/C.abortus polyepitope (polyCsCa) construct. Linker sequences are underlined.

Figure 3: Mean serum antibody titres for pigs immunized with L. lactis transformed with pT1 FX-PolyCsCa and for control animals immunized with L. lactis transformed with pT1 FX-empty. Serum antibody titers were determined against recombinant MOMP of C. suis S45 (A) or recombinant MOMP of C. abortus S26/3 (B). Error bars indicate SD. Significant differences (*) were observed at day 21 and at day 7 post immunization for vaginal administration of pT1 FX-polyCsCa compared to their respective controls. Statisticaly significant differences are indicated with * (p<0,05).

Figure 4: T cell proliferation responses. Peripheral blood mononuclear cells (PBMC’s) of pigs immunized with L. lactis transformed with pT1FX-PolyCsCa were re-stimulated in vitro with recombinant MOMP of the C. suis reference strain S45 and the mean stimulation index was compared to the mean stimulation index of PBMC’s of control pigs immunized with L. lactis transformed with pT 1 FX-empty. Error bars indicate SD. Statisticaly significant differences are indicated with * (p<0,05).

Figure 5: T cell proliferation responses. Peripheral blood mononuclear cells (PBMC’s) of pigs immunized with L. lactis transformed with pT1 FX-PolyCsCa were re-stimulated in vitro with recombinant MOMP of the C. abortus reference strain S26/3 and the mean stimulation index was compared to the mean stimulation index of PBMC’s of control pigs immunized with L. lactis transformed with pT 1 FX-empty. Error bars indicate SD. Statistically significant differences are indicated with * (p<0,05).

Figure 6: Average total IgG (H + L) serum antibody titres for the different groups are shown. * indicates significant differences between the LL-polyCsCa primo + booster and the LL- empty primo + booster group.

Figure 7: Average mucosal IgA antibody responses for all groups. Significant differences between the immunized groups and the respective controls are indicated with *.

Figure 8: Average stimulation index (SI) for all groups at 14, 28 and 47 days of the experiment. d14 (14 days post primo immunization; d28 (7 days post booster immunization); d47 (12 days post immunization).

Figure 9: Mean percentage (+ SD) of different immune cell populations within PBMC's, isolated at day 47 in the blood.

Figure 10: Mean scores (+ SD) for vaginal C. suis shedding from 0 to 13 days post infection (dpi) in the different groups. Significant differences were observed at day 42, 44 and 47 (p<0.05).

Figure 11: Dot plots show mean presence of C. suis in cervix, vagina, corpus uteri and L/R oviducts, with SD and individual values.

Description of the invention

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound. Throughout the description and claims of this specification the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used herein, "about", "approximately," "substantially," and "significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" and "approximately" will mean plus or minus 10% of the particular value or term. The terms described above and others used in the specification are well understood to those in the art. All references, and teachings specifically referred to, cited in the present specification are hereby incorporated by reference in their entirety.

The present invention provides isolated peptides, and uses thereof, comprising B- and T- cell epitopes for producing a vaccine against species of the genus Chlamydia, and more in particular for inducing a (protective) immune response against an infection with a species of the genus Chlamydia in pigs. In one embodiment, the species of the genus Chlamydia are Chlamydia suis and/or Chlamydia abortus. These epitopes can be used for development of a single or combination vaccine against infection by Chlamydia suis and/or Chlamydia abortus. In addition, the vaccine can also be used against specific disease problems in pigs such as pneumonia, conjunctivitis, diarrhea and/or reproductive failure caused by C. suis and/or C. abortus, thus acting as a multipurpose Chlamydia vaccine for swine.

The current approach is to use a highly immunogenic polyepitope construct comprising only immunostimulatory (amino acid) sequences.

In one embodiment, the present invention relates to a composition comprising at least one isolated peptide comprising a B-cell epitope located in a variable amino acid region of the major outer membrane protein (MOMP) of Chlamydia suis and/or Chlamydia abortus and at least one isolated peptide comprising a T-cell epitope located in a conserved region of the MOMP of Chlamydia suis and/or Chlamydia abortus. In particular, the invention relates to a composition comprising one or more peptides, each of said peptides comprising one or more epitopes selected from the list consisting of a B-cell epitope, a CD4+ Th2 cell epitope, a CD4+ Th1 cell epitope and a CTL epitope; wherein said composition comprises at least one B-cell epitope, at least one CD4+ Th2 cell epitope, at least one CD4+ Th1 cell epitope and at least one CTL epitope, and wherein the epitopes are located in the major outer membrane protein (MOMP) of Chlamydia suis and/or Chlamydia abortus and wherein the composition does not comprise the full-length Chlamydia MOMP protein (e.g. of said particular species).

The composition is specifically designed to induce an immune response, in particular a protective immune response, in a subject (such as e.g. a pig) against an infection with a species of the genus Chlamydia. In particular, the combination of the B- and T-cell epitope containing peptides is not equal to the complete or full-length MOMP protein, in particular the Chlamydia suis and/or Chlamydia abortus MOMP protein. More specific, the B-cell epitope is located in the variable region I, II, III or IV for C. suis, and in variable domain I, II and IV for C. abortus. T-cell epitopes (including CD8+, CD4+ Th1 , and CD4+ Th2 cell epitopes) are located in the conserved regions. The MOMP protein of the Chlamydia suis reference strain S45 is having Genbank accession No. AF269274.1 and can be found in the genome RefSeq ID/ENA database under accession No. PRJNA326179 (Seth-Smith et al. , 2017). The MOMP protein of the Chlamydia abortus strain S26/3 is having Genbank accession No. P16567.1 and can be found in the genome RefSeq ID/SRA database under accession No. CR848038 (Joseph et al., 2015).

The 5 conserved regions (CRs) of MOMP are highly conserved between C. abortus strains (Table 6) and between C. suis strains (Table 7) and thus, all T cell epitopes that can be comprised in the vaccine are present in all so far known C. abortus and C. suis strains (originating from different geographical regions) and might induce a cellular immune response against all so far known C. abortus and C. suis strains.

B cell epitopes of C. abortus and of C. suis are located in the variable domains 1 to 4 (Tables 4 and 5) but for C. abortus also in a more conserved region at the beginning of VD4 (AAVLNLTTWNPTLL, SEQ ID NO 75). To tackle the genetic variability between B cell epitopes, the latter should preferably be included in the vaccine. For C. suis, B cell epitopes in VD1 , VD2 and VD4 are more conserved between strains originating from different geographical regions (i.e. US versus Europe) than the ones in VD3. To tackle the genetic variability between B cell epitopes of C. suis, B cell epitopes of VD1 , VD2 and/or VD4 should therefore preferably be included in the vaccine.

With the term “B cell-epitope” is meant a part of an antigen that induces antibody production upon recognition by the host’s immune system.

A “T-cell epitope” is a part of an antigen that induces a CD4⁺Th1 (T helper, HTL), CD4⁺ Th2 (T helper, HTL) or CD8⁺ cell (cytotoxic, CTL) response upon recognition by the host’s immune system. T helper (Th) cells are considered major players in the response against infectious organisms. To convey their full function, Th cells secrete a variety of cytokines, which define their distinct actions in immunity. Th cells can be subdivided into three different types based on their cytokine signature, Th1 , Th2 and Th17 cells. “Th1 cells” secrete IFN-gamma (pro-inflammatory cytokine), which is the main macrophage activating cytokine and TNF-b, which also activates macrophages, inhibits B cells and is directly cytotoxic for some cells. Th1 cells allow the production of lgG_2a antibodies in mice and of IgM, IgA, IgGi, lgG₂and IgGs antibodies in humans. “Th2 cells” secrete IL-4, IL-5, IL-6, IL-9 and IL-13 all of which activate B cells, and IL10 (important anti-inflammatory cytokine), which inhibits macrophage activation. Th2 cells induce IgGi and IgE antibodies in mice and IgM, lgG₄ and IgE in humans. Th17 cells secrete the pro-inflammatory cytokine IL-17 (Murphy et al. , 2008; Annunziato and Romagnani, 2009).

A peptide comprising a CTL epitope (CD8+) usually consists of about 13 or less amino acid residues in length, 12 or less amino acids in length, or 11 or less amino acids in length, preferably from 8 to 13 amino acids in length, most preferably from 8 to 11 amino acids in length (i.e. 8, 9, 10, or 11). A peptide comprising a HTL epitope (CD4+) consists of about 50 or less amino acid residues in length, and usually from 6 to 30 residues, more usually from 12 to 25, and preferably consists of 12 to 20 (i.e. 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids in length. Peptides comprising B cell epitopes do not have a defined length and can vary from 5 to 30 amino acids in length, preferably from 5 to 20 amino acids, more preferably from 5 to 15 amino acids in length, i.e. 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids. In a specific embodiment, a peptide comprising a T cell epitope has a length of 6 to 50 (including e.g. 8-50, 10-50 and 15-50) amino acids and a peptide comprising a B cell epitope has a length of 5 to 30 (including e.g. 5-25, 8-30, 8-25, 5-20 and 8-20) amino acids. Hence, it is to be understood that peptides with a defined length and comprising the epitopes specified herein are part of the present invention and can be used in the compositions and methods as described herein. In a specific embodiment, the peptides are immunogenic peptides, i.e. peptides capable of eliciting an immune response in an organism, including a cellular and/or humoral response, for example as determined by the methods described herein. More particular, the present invention comprises an innovative vaccine that creates both humoral and cellular immune responses by combining B cell and CD4 Th2 cell epitopes (humoral) and CD4 Th1 and CD8⁺ cytotoxic T cell epitopes (cellular) in one composition or construct.

The term “isolated” is used to indicate that a cell, peptide or nucleic acid is separated from its native environment. Isolated peptides and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may bound in nature.

With the term ‘induction of an protective immune response’ is meant a (humoral and/or cellular) immune response that reduces or eliminates one or more of the symptoms of disease, i.e. clinical signs, lesions, bacterial excretion and bacterial replication in tissues in the infected subject compared to a healthy control. Preferably said reduction in symptoms is statistically significant when compared to a control. Particular outcome parameters are provided in the Examples disclosed herein.

In a particular embodiment, the present invention provides peptides, and nucleic acids encoding them, suitable for vaccine design.

For C. abortus, peptides comprising a B-cell epitope correspond to SEQ ID NO 73 to SEQ ID NO 75 and SEQ ID NO 200.

For C. suis, peptides comprising a B-cell epitope correspond to SEQ ID NO 46 to SEQ ID NO 72 and SEQ ID NO 207 to SEQ ID NO 211 . For C. abortus, suitable peptides comprising a T-cell epitope correspond to SEQ ID NO 124 to SEQ ID NO 141.

As can be derived from the data in Table 8, said peptides are characterized as follows: -Peptides comprising a CTL (CD8+) epitope correspond to SEQ ID NO 124, 126,127, 128, 133, 134, 135, 136, 138, 139, 140, and 141 ;

-Peptides comprising a CD4+ Th1 cell epitope correspond to SEQ ID NO 124-126, 128, 130, 131 , 133-135, 137, 138 and 139; and

-Peptides comprising a CD4+ Th2 cell epitope correspond to SEQ ID NO 129, 132, 136, 139 and 141.

For C. suis, suitable peptides comprising a T-cell epitope correspond to SEQ ID NO 142 to SEQ ID NO 196. As can be derived from the data in Table 9, said peptides are characterized as follows:

-Peptides comprising a CTL (CD8+) epitope correspond to SEQ ID NO 142, 143, 144, 145, 147, 148, 149, 152, 153, 154, 155, 156, 159, 160, 163, 165, 166-173, 177, 178, 180, 183, 185, 186, 187, 193, 194 and 195;

-Peptides comprising a CD4+ Th1 cell epitope correspond to SEQ ID NO 142-144, 146- 149, 153-157, 159-162, 167, 169, 178, 181 , 186 and 187; and

-Peptides comprising a CD4+ Th2 cell epitope correspond to SEQ ID NO 145, 150, 151 , 158, 164, 166, 168, 170, 173-176, 179, 182, 184, 185, 188-192, 195 and 196.

With the peptides identified herein, at least 3 types of vaccines can be designed, such as a vaccine specific against C. suis, a vaccine specific against C. abortus and a “combination vaccine” against both C. suis and C. abortus.

The composition of the present invention preferably comprises:

1. specific combinations of the herein provided peptides of one species (i.e. C. suis or C. abortus) thereby inducing a humoral as well as a cellular immune response; or

2. specific combinations of the herein provided peptides of both species (i.e. C. suis and C. abortus ; herein referred to as a combination vaccine). Such a composition typically comprises one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) peptide(s) comprising at least one B cell epitope, at least one CD4 Th2 cell epitope, at least one CD4 Th1 cell epitope and at least one CD8⁺ cytotoxic T cell epitope.

More specifically and for C. abortus, the present invention relates to the composition as indicated above, wherein said isolated B cell epitope consists of the amino acid sequence PTGTAAANYKTPT (SEQ ID NO 73), PTGTAAANYKTP (SEQ ID NO 200), GSSIAADQLP (SEQ ID NO 74) or AAVLNLTTWNPTLL (SEQ ID NO 75), or variants thereof, which are located in the variable domains of the MOMP of Chlamydia abortus strain S26/3; and, wherein said isolated T-cell epitope consists of the amino acid sequence selected from the group consisting of: SEQ ID NO 124 to SEQ ID NO 141 , and variants thereof, which are located in the conserved domains of the MOMP of Chlamydia abortus strain S26/3. In a particular embodiment, the isolated B-cell epitope consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 200, SEQ ID NO 74 and SEQ ID NO 75, and a variant thereof; and/or, the isolated T-cell epitope consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 124 and SEQ ID NO ISO- 138, and a variant thereof.

More specifically and for C. suis, the present invention relates to the composition as indicated above, wherein said isolated B cell epitope consists of the amino acid sequence NTGNTTSPTQL (SEQ ID NO 70) or NTGNTTSPT (SEQ ID NO 211) in VD1 , TTAAQATA (SEQ ID NO 71) in VD2, or KVEDKGSA (SEQ ID NO 72) in VD4, or variants thereof, which are located in the variable domains of the MOMP of Chlamydia suis strain S45 (SEQ in VD1 , VD2 and VD4), H5 (SEQ in VD2 and VD4) and R19 (SEQ in VD2 and VD4) and, wherein said isolated T-cell epitope consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 142 to SEQ ID NO 196, and a variant thereof, which are located in the conserved regions of the MOMP of Chlamydia suis strain H7 or R24. In a particular embodiment, the isolated B-cell epitope consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 70, SEQ ID NO 71 and SEQ ID NO 72, and a variant thereof; and/or, the isolated T-cell epitopes consist of the amino acid sequence selected from the group consisting of: SEQ ID NO 142-145, 149-151 , 154- 159, 162-164, 168-170, 181-185 and 187-196, and a variant thereof.

Optionally, flanking amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids) or flanking regions, e.g. as present in the natural MOMP protein, of the specified B- and T- cell epitopes can be part of the peptide sequences to be included in the composition.

More specifically and for C. abortus and C. suis, the invention relates to the composition and use as indicated herein, wherein the peptide comprising a B-cell epitope is selected from the group consisting of: SEQ ID NO 46 to SEQ ID NO 75, SEQ ID NO 200 and SEQ ID NO 207 to SEQ ID NO 210, and a variant thereof; and wherein the peptide comprising a T-cell epitope comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 124 to SEQ ID NO 196, and a variant thereof.

In particular, the peptide comprising a B-cell epitope is selected from the group consisting of: SEQ ID NO 200, SEQ ID NO 70 to SEQ ID NO 75, and a variant thereof, and the peptide comprising a T-cell epitope comprises or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO 124, SEQ ID NO 130-138, SEQ ID NO 142-145, 149-151 , 154-159, 162-164, 168-170, 181-185 and 187-196, and a variant thereof.

In one embodiment, the composition comprises 2, 3 or 4, preferably 3, copies of one or more (2, 3, 4, ...) or all of the herein provided B-cell epitopes. In particular, for C. abortus the composition comprises 2, 3 or 4 copies of a peptide comprising the B-cell epitope represented by SEQ ID NO: 200, 74 and/or 75. In a further embodiment and for C. abortus, the composition comprises 2, 3 or 4 copies of a peptide comprising the B- cell epitope represented by SEQ ID NO: 70, 71 and/or 72.

In a particular embodiment, the composition comprises:

- a B-cell epitope consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO 70 to SEQ ID NO 75, and SEQ ID NO 200, or a variant thereof having at least 80% identity;

- a peptide comprising a CD4+ Th2 cell epitope selected from the group consisting of: SEQ ID NO 132, 136, 141 , 145, 150, 151 , 158, 166, 168, 170, 173, 174, 179, 182, 184, 185, 188, 189, 190, 191 , 192, 195 and 196, or a variant thereof having at least 80% identity;

- a peptide comprising a CTL (CD8+) epitope selected from the group consisting of: SEQ ID NO 124, 126, 127, 128, 133, 134, 135, 136, 138, 140, 141 , 142, 143, 144,

145, 147, 148, 149, 152, 153, 154, 155, 156, 159, 163, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 180, 183, 185, 186, 187, 193, 194 and 195, or a variant thereof having at least 80% identity; and

- a peptide comprising a CD4+ Th1 cell epitope selected from the group consisting of: SEQ ID NO 124, 126, 128, 130, 131 , 133, 134, 135, 137, 138, 142, 143, 144,

146, 147, 148, 149, 153, 154, 155, 156, 157, 159, 167, 169, 181 , 186 and 187, or a variant thereof having at least 80% identity.

In one embodiment, the composition can comprise 2, 3, 4, or more copies of one or more (2, 3, 4, ...) or all of the herein provided T-cell epitopes.

In another embodiment, the peptide comprising the B-cell epitope is selected from the group consisting of: PTGTAAANYKTP (SEQ ID NO 200), GSSIAADQLP (SEQ ID NO 74), AAVLNLTTWNPTLL (SEQ ID NO 75), NTGNTTSPTQL (SEQ ID NO 70), TTAAQATA (SEQ ID NO 71), and KVEDKGSA (SEQ ID NO 72), or a variant thereof; and/or the peptide comprising a T-cell epitope is selected from the group consisting of: MKKLLKSALLFAATGSALSLQ (SEQ ID NO 197);

NGYFKASSAAFNLVG (SEQ ID NO 130);

QGIVEFYTDTTFSWSVGARGALWECGCATLGAEFQYAQSNPKIEMLNVVSSPAQ (SEQ ID NO 198);

SATIKYHEWQVGLALSYRLNMLVPYISVNWSRATFDADAIRIAQPKLA (SEQ ID NO 199);

SVLVFAALGSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPC (SEQ ID NO 201); IWDRFDVFCTLGATNGYLKGNSAAFNLVGL (SEQ ID NO 202); VSLSQSVIELYTDTAFAWSVGARAALWE (SEQ ID NO 203); TKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKLATAVFDVT (SEQ ID NO 205); and MQIVSMQINKMKSRKSCGLAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF (SEQ ID NO 206); or a variant thereof.

Optionally, the peptide comprising a T-cell epitope is ELNVLCNAAEFTINKPQGYVG (SEQ ID NO 204) or a variant thereof, and can be part of a composition as provided herein.

The compositions and methods of the present invention also encompass variants of the above specified peptides comprising the epitopes. “Variants” of the B and T-cell epitopes on the corresponding peptide sequences of the different strains or species are also part of the invention, i.e. those peptide sequences at corresponding amino acid positions when aligned to a reference sequence. Moreover, a "variant" as used herein, is a peptide that differs from the native antigen only in 1 , 2, 3, 4, 5, or more (in particular 1 or 2, more in particular 1) conservative substitutions and/or modifications, such that the ability of the peptide to induce an immune response is retained. Peptide variants preferably exhibit at least about 70%, preferably at least about 80% or 85%, more preferably at least about 90% and most preferably at least about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the identified peptides/epitopes disclosed herein. Alternatively, such variants may be identified by modifying one of the above peptide sequences and evaluating the immunogenic properties of the modified peptide using, for example, the representative procedures described herein. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the nature of the peptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be peptides as described herein modified by, for example, the deletion or addition of 1 , 2, 3, 4, 5, or more amino acids that have minimal influence on the immunogenic properties, secondary structure and hydropathic nature of the peptide. The present invention relates, even more specifically, to the composition or use as indicated above wherein said species of the genus Chlamydia is Chlamydia abortus and/or Chlamydia suis. As such, the present invention relates to (methods, compositions, kits, peptides and/or epitopes for use in) the prevention, reduction and/or treatment of specific disease problems/symptoms such as rhinitis, pharyngitis, bronchitis, pneumonia, enteritis, conjunctivitis, diarrhea, abortion and/or reproductive failure due to infection with Chlamydia abortus and/or suis in a subject, or to the prevention or reduction of morbidity or mortality due to said infections. Subjects are humans or animals, but preferably are pigs or swine (including piglets, sows and boars) and in the context of C. abortus also ruminants, in particular small ruminants such as sheep and goat.

A preferred means of administration of the peptides of the present invention is mucosal delivery or at a mucosal site, wherein said mucosal delivery is chosen from the group consisting of rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, vaginal delivery and oral delivery. More particular, administration is by vaginal delivery. Other means of administration are also possible and include all other systemic and mucosal administration routes well known to the skilled person (e.g. intramuscular, intradermal, etc.). “Mucosa” (mucus membrane) as used herein refers to an epithelial membrane containing mucosal cells that secret mucus, a gel-like fluid containing mainly water (-95%), mucins (0.5-5%), inorganic salts (-1%), proteins (0.5-1%), lipids, and mucopolysaccharides, and can be any mucosa such as oral mucosa, rectal mucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa, pulmonary mucosa and nasal mucosa. In one embodiment, mucosal delivery encompasses the delivery into or onto to the mucosa by injection or any injection (needle)-free method.

The composition of the present invention can further comprise a pharmaceutically acceptable carrier, buffer, diluent and/or excipient conventional in the art, including mixtures thereof. Non limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In the composition, sometimes also referred to as poly- or multi-epitope vaccine, the peptides can be present as a mixture of individual peptides and/or (part of) the peptides can be linked to each other and/or can be part of vector/carrier construct. In a specific embodiment, the composition comprises a polyepitope construct. The term polyepitope construct or vaccine as used herein denotes a composition that does not occur as such in nature. Hence, the "polyepitope vaccine" of the present invention does not encompass a wild-type full- length protein but includes two or more isolated epitopes of the present invention, not necessarily in the same sequential order or number (repetitions might be used) as in nature. The polyepitope vaccine of the present invention preferably comprises 2 or more, 5 or more, 10 or more, 13 or more, 15 or more, 20 or more, or 25 or more epitopes of the present invention. More specific, the polyepitope vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12. 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more epitopes as provided herein. The epitopes of the polyepitope vaccine can be prepared as synthetic peptides or recombinant peptides. These synthetic peptides or recombinant peptides can be used either individually or directly or indirectly linked to one another. Optionally, two or more of the epitopes (either B-cell and/or T-cell epitopes) can be linked in a construct, referred to herein as a polyepitope construct, and are either contiguous or are separated by a linker or one or more spacer amino acids. "Link" or "join" refers to any method known in the art for functionally connecting epitopes. Examples of spacer amino acids are Gly (G) and Ser (S), including combinations thereof. More particular, the polyepitope vaccine of the present invention is a synthetic or recombinant string of two or more peptides harboring (part of) the epitopes as described herein. Methods for preparing a polypeptide, which may comprise a polyepitope (polyepitope vaccine/construct), are known in the art and are described in for example the book Molecular Cloning; a laboratory manual by Joseph Sambrook and David William Russell 2001

In a particular embodiment, the composition or polyepitope vaccine of the present invention comprises at least two, and in particular all, of the following peptide sequences: MKKLLKSALLFAATGSALSLQ (SEQ ID NO 197); NGYFKASSAAFNLVG (SEQ ID NO 130);

QGIVEFYTDTTFSWSVGARGALWECGCATLGAEFQYAQSNPKIEMLNVVSSPAQ (SEQ ID NO 198);

SATIKYHEWQVGLALSYRLNMLVPYISVNWSRATFDADAIRIAQPKLA (SEQ ID NO 199)

PTGTAAANYKTP (SEQ ID NO 200);

GSSIAADQLP (SEQ ID NO 74);

AAVLNLTTWNPTLL (SEQ ID NO 75);

SVLVFAALGSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPC (SEQ ID NO 201); IWDRFDVFCTLGATNGYLKGNSAAFNLVGL (SEQ ID NO 202); VSLSQSVIELYTDTAFAWSVGARAALWE (SEQ ID NO 203); TKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKLATAVFDVT (SEQ ID NO 205);

MQIVSMQINKMKSRKSCGLAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF (SEQ ID NO 206);

NTGNTTSPTQL (SEQ ID NO 70);

TTAAQATA (SEQ ID NO 71) and KVEDKGSA (SEQ ID NO 72); or variants thereof.

Optionally, 2-5, in particular 2-3, copies of the disclosed B-cell epitopes can be included. In an alternative embodiment, one or more of the peptides or T-cell epitopes in the above provided composition can be replaced by a peptide comprising an epitope selected from the group consisting of: SEQ ID NO 126-128, SEQ ID NO 140-141 , SEQ ID NO 146-148, SEQ ID NO 152-153, SEQ ID NO 165-167, SEQ ID NO 171-174, SEQ ID NO 179-180, and SEQ ID NO 186.

In a further embodiment, the composition provided herein can further comprise one or more peptides comprising an epitope selected from the group consisting of: SEQ ID NO 126-128, SEQ ID NO 140-141 , SEQ ID NO 146-148, SEQ ID NO 152-153, SEQ ID NO 165-167, SEQ ID NO 171-174, SEQ ID NO 179-180, and SEQ ID NO 186. In a further specific embodiment, the composition or polyepitope vaccine of the present invention comprises or encodes the (poly)peptide sequence as provided Table 11 , or in Figure 1 or Figure 2 (as represented by resp. SEQ ID NO 212-219).

The present invention further includes an isolated nucleic acid encoding an epitope, peptide or polyepitope construct as described herein and the use of said nucleic acid for preparing a (pharmaceutical) composition or vaccine. Particular nucleic acids encoding the peptides of the invention (see also Table 11 and Figures 1 or 2) are the following.

For C. abortus

Corresponding to T cell CD8 + T cell CD4 Th1 epitope MKKLLKSALLFAATGSALSLQ (SEQ ID NO 197):

AT G AAAAAATT ATT AAAAT C AG C ACTTTT GTTT G CTG C AAC AG GTT C AG CTTT AT CAT TACAA (SEQ ID NO 220)

Corresponding to T cell CD4 Th1 epitope NGYFKASSAAFNLVG (SEQ ID NO

130):

AATG G AT ATTTT AAA GCTTCAAGTGCTG C ATTT A ATTT G GTTG G A (SEQ ID NO 221)

Corresponding to one T cell CD4 Th2 + cluster* of T cell CD4Th1 and T cell CD8 epitope:

QGIVEFYTDTTFSWSVGARGALWECGCATLGAEFQYAQSNPKIEMLNVVSSPAQ (SEQ ID NO 198):

C AAG GT ATT GTT G AATTTT AT AC AG AT AC AACTTTTT CAT GGAGTGTTGGTGCTC GTG GAG CT CTTT G G G AAT GTGGTTGTG C AAC ATT G G G AG C AG AATTT C AAT AT G C AC AAAGT AAT C C AAAAATT G AAAT G CTT AAT GTTGTAT C AAGT C C AGC AC AA (SEQ ID NO 222)

Corresponding to one T cell CD4 Th2 + cluster of T cell CD4Th1 and T cell CD8 epitope: SATIKYHEWQVGLALSYRLNMLVPYISVNWSRATFDADAIRIAQPKLA (SEQ ID NO 199):

TCAGCTACT ATT AAAT AT CAT G AAT G G C AAGTT G GTCTTG C ATT ATCTT AT C GAT T AAAT ATGTTG GTTC CAT AT ATTT C AGTAAATT G GT C AAG AG C AAC ATTT GAT G C T GAT G C AATT C GT ATT G CT C AAC CT AAATT AG C A (SEQ ID NO 223) Corresponding to B cell epitope: PTGTAAANYKTP (SEQ ID NO 200):

C C AAC AG G AAC AG CT G C AG C AAATT AT AAAACT C C A (SEQ ID NO 224); CCTACAGGTACTGCAGCTGCTAATTATAAAACACCA (SEQ ID NO 225); or CCAACTGGTACTGCAGCAGCAAATTATAAAACACCT (SEQ ID NO 226) Corresponding to B cell epitope: GSSIAADQLP (SEQ ID NO 74):

G G AAGTT C AATT G CTG C AG AT C AATT AC C A (SEQ ID NO 227);

G GTTCTT C AATT GOT G C AG AT C AACTT C CT (SEQ ID NO 228); or G G ATC AT C A ATT GCTGCTGAT C A ATT G C C T (SEQ ID NO 229)

Corresponding to B cell epitope: AAVLNLTTWNPTLL (SEQ ID NO 75):

G CTG CT GTTTT G AAT CTTACTACTTG G AAT C CT ACTTT ATT G (SEQ ID NO 230); G CTG C AGT ATT AAATTT GAC AAC AT G G AAT C CT AC ATT GTTA (SEQ ID NO 231); or GCAGCAGTATTGAATTTAACAACTTGGAATCCAACATTACTT (SEQ ID NO 232)

For C. suis

Corresponding to One T cell CD4 Th2 + cluster of T cell CD4Th1 and T cell CD8 epitope: SVLVFAALGSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPC (SEQ ID NO 201 ):

AGCGTGCTGGTGTTTGCGGCGCTGGGCAGCGCGAGCAGCCTGCAGGCGCTG CCGGTGGGCAACCCGGCGGAACCATCATTAATGATTGATGGAATTTTATGGGA AGGTTTTGGTGGTGATCCATGTGATCCTTGT (SEQ ID NO 233)

Corresponding to T cell CD4 Th2 + T cell CD8 epitope: IWDRFDVFCTLGATNGYLKGNSAAFNLVGL (SEQ ID NO 202): ATTTGGGATCGTTTTGATGTATTTTGTACTTTAGGAGCAACAAATGGTTATTTGA AAGGAAATTCTGCAGCTTTTAATCTTGTAGGTTTA (SEQ ID NO 234) Corresponding to Cluster of T cell CD4 Th2 epitope: VSLSQSVIELYTDTAFAWSVGARAALWE (SEQ ID NO 203):

GTTT C ATT AAGT C AAT C AGTT GTAGAACTTT AT AC AG ATAC AG CTTTT G CAT G GT CTGTAGGTGCTCGTGCAGCATTATGGGAA (SEQ ID NO 235) Corresponding to CD4 Th1 + CD8 epitope: TKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKLATAVFDVT (SEQ ID NO 205):

AC AAAAGAT G C AAGTATT GATT AT CAT G AAT G G C AAG CTT C ATT G G C ATT AT CAT AT C GTTT AAAT AT GTTTACT C CTTAT ATT GGTGT AAAAT GGTCACGTG C AAGTTT TGATGCTGATACTATTAGAATTGCACAACCTAAACTTGCAACTGCAGTTTTTGAT GTTACA (SEQ ID NO 236)

Corresponding to Cluster of T cell CD4 Th2, T cell CD4Th1 and T cell CD8 epitope:

MQIVSMQINKMKSRKSCGLAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF (SEQ ID NO 206):

AT G C AAATT GTAT C AAT G C AAATT AAT AAAAT G AAAAGT C GTAAAAGTT GTG GAT TAGCAGTTGGTACAACAATTGTTGATGCTGATAAATATGCTGTAACAGTTGAAAC AC GACTT ATT GAT GAAC GAGCTGCACAT GTT AAT GCTCAATTTCGTTTT (SEQ ID NO 237)

Corresponding to B cell epitope: NTGNTTSPTQL (SEQ ID NO 70): AATACAGGTAATACTACATCACCTACACAATTG (SEQ ID NO 238); AATACTGGTAATACAACATCACCTACTCAATTG (SEQ ID NO 239); or AATACAGGAAATACAACATCACCAACTCAATTG (SEQ ID NO 240)

Corresponding to B cell epitope: TTAAQATA (SEQ ID NO 71 ): ACAACTGCAGCTCAAGCAACAGCT (SEQ ID NO 241); ACTACAGCTGCTCAAGCAACAGCA (SEQ ID NO 242); or ACAACAGCTGCACAAGCTACAGCA (SEQ ID NO 243)

Corresponding to B cell epitope: (SEQ ID NO) KVEDKGSA (SEQ ID NO 72): AAAGTT G AAGAT AAAG GTTCTG C A (SEQ ID NO 244);

AAAGTT G AAG AT AAAG G AT C AG C A (SEQ ID NO 245); or AAAGTAGAAGATAAAGGTAGTGCA (SEQ ID NO 246)

In one embodiment, a specific example of a nucleic acid (e.g. as used for a vaccine) is the sequence as shown in SEQ ID NO 213, 215, 217 and 219 (Figure 1 and 2). The linker sequences are optional and/or can be any linker as described herein. In one embodiment the linkers are the following: i) a non-cleavable, flexible GPGPG (SEQ ID NO 247) between and after the T cell epitopes and ii) short diglycine (GG) repeats between/after the B cell epitopes.

In a further embodiment, the invention encompasses an expression system comprising a genetic construct comprising at least one nucleotide sequence encoding one or more of the peptide(s) (including combinations as provided herein), preferably operably linked to a promoter capable of directing expression of the sequence in the hosting micro-organism. Suitably the peptide(s) to be expressed can be encoded by a nucleic acid sequence that is adapted to the preferred codon usage of the host. The construct may further contain (all) other suitable element(s), including enhancers, transcription initiation sequences, signal sequences, reporter genes, transcription termination sequences, etc., operable in the selected host, as is known to the person skilled in the art. The nucleic acid construct or constructs may further comprise a secretory signal sequence.

The construct is preferably in a form suitable for transformation of the host and/or in a form that can be stably maintained in the host, such as a vector or mini-chromosome. Suitable vectors comprising nucleic acid for introduction into micro-organisms, e.g. bacteria, can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid’, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al. , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acids, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures relating to said protocols of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

In one embodiment, the present invention relates to a C. suis and/or C. abortus polyepitope transformed micro-organism, for example transformed Lactococcus bacteria, in particular Lactococcus lactis (L. lactis). Transformation of micro-organisms is achievable by application of known genetic engineering techniques such as those described in, e.g. Sambrook and Russell (2001) “Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

In general, heterologous gene expression is achieved by cloning of the heterologous gene such as the polyepitope construct into a plasmid, which replicates within the recipient. The term “plasmid” is used to refer to a molecule capable of autonomous replication that is suitable for transformation of a recipient bacterial strain and contains DNA sequences that direct and/or control the expression of the inserted heterologous DNA sequence. Various types of plasmids may be used such as low and high copy number plasmids, narrow and broad-host range plasmids, expression plasmids, and cosmids. As an example, pT1 FX- polyCsCa, containing the C. suis and C. abortus polyepitope (CsCa) was used to transform L. lactis.

Transformation methods of micro-organisms are known to the person skilled in the art, such as for instance chemical transformation and electroporation.

The micro-organism according to the invention can be any micro-organism, including bacteria, yeasts or fungi, preferably suitable for mucosal delivery. More specific, said micro-organism is a non-pathogenic micro-organism. Examples of bacteria are Salmonella typhi, BCG ( Bacille Calmette Guerin), Shigella and Listeria. In a specific embodiment, said micro-organism is a lactic acid bacterium, such as Lactococcus lactis (L. lactis). Delivery of heterologous proteins (i.e. non lactic acid-bacterial proteins) by lactic acid bacteria into the mucosa, including both oral and vaginal delivery, has been described, which makes these lactic acid bacteria suitable for delivery of the present (poly)peptide(s). L. lactis is a non-pathogenic, non-invasive, non-colonizing gram-positive bacterium. Examples of L. lactis strains are L. lactis subsp. cremoris MG1363, but other L. lactis strains might be used including the next generation L. lactis strains used for gene expression in an antibiotic resistance marker free system. L. lactis systems devoid of an antibiotic resistance marker have been described for instance by Glenting et al.,(2002), Steidler et al. , (2003), Berlec et al. , (2018), and de Castro et al. , (2018). Examples of viruses are poxvirus, Alphaviruses (Semliki Forest Virus, Sindbis Virus, Venezuelan Equine Encephalitis Virus (VEE), Herpes simplex Virus (HSV), Kunjin virus, Vesicular Stomatitis Virus (VSV) replication-deficient strains of Adenovirus (human or simian), polyoma vectors (such as SV40 vectors, bovine polyoma), CMV vectors, papilloma virus vectors, influenza virus, measles virus, and vectors derived from Epstein Barr virus. A wide variety of other delivery vehicles useful for therapeutic administration or immunization, e.g. lentiviral vectors, retroviral vectors, and the like, will be apparent to those skilled in the art. Examples of yeasts are a Hansenula cell or Saccharomyces cerevisiae cell.

In an alternative embodiment, the composition according to the present invention can comprise an antigen delivery system, which optimizes the presentation of the peptide(s)/antigen. In a specific embodiment, the antigen delivery system is a recombinant protein, for instance an adhesin such as the adenylate cyclase adhesin (CyaA) from Bordetella pertussis (the causative agent of whooping cough) (Ladant et al. , 1999; Fayolle et al., 2004; W0200173108; EP1576967).

A promoter employed in accordance with the present invention can be inducible or constitutive, but is preferably expressed constitutively in the vector micro-organism used (bacterium, virus or yeast) or by a eukaryotic expression vector for DNA vaccination. In case of a micro-organism as vector, the promoter directs expression at a level at which the host cell remains viable, i.e. retains some metabolic activity, even if growth is not maintained. The promoter may be homologous to the micro-organism employed, i.e. one found in that micro-organism in nature, or heterologous. For example, a Lactococcal promoter may be used in a Lactococcus. A promoter for use in Lactococcus lactis (or other Lactococci) can be the constitutive P1 promotor used in plasmid pT1 FX (BCCM Belgian coordinated collection of micro-organisms, LMBP 10260), or for example the inducible promotor described in the commercial NICE expression system for L lactis being PnisA or PnisF for nisin controlled gene expression (MoBiTec GmbH, Germany; Mierau and Kleerebezem, 2005) or any other promotor functional in Lactococcus spp. for instance pORI23, pll_253, pll_252, pWV01 , the groESL promotor (SICE system), the pczcD promotor (ZIREX system) or the pXylT promotor (XI ES system).

In case of a eukaryotic expression vector for DNA vaccination the promotor might be any eukaryotic expression promotor for instance SV40, CMV, UBC, EF1A, PGK, CAGG.

In one embodiment, the invention provides a L lactis strain (GRAS) comprising a plasmid, being pT1 FX including the nucleic acids as provided herein and the P1 promotor.

Further Suitable L lactis strains and plasmids are provided in Table 1 of Azizpour et al. 2017.

The present invention also encompasses the preparation of above mentioned polyepitope construct from a recombinant or transformed micro-organism, in particular a bacterium, more particular Lactococcus. In a further embodiment, the invention provides a plasmid comprising a nucleic acid sequence encoding one or more of the epitopes or peptides, in particular the polyepitope, as disclosed herein. The invention also encompasses a recombinant antigen-delivery system, or a micro-organism, such as a Lactococcus bacterial cell, comprising a nucleotide sequence coding for one or more of the epitopes or peptides, in particular the polyepitope, as disclosed herein, as well as the use thereof to treat, prevent and/or alleviate symptoms associated with C. suis or C. abortus infection in pigs, or in the case of C. abortus also in small ruminants.

The composition of the present invention can further comprise an adjuvant, in particular a mucosal adjuvant. Suitable adjuvants are 1) receptor specific (mucosal) adjuvants such as for instance adjuvants binding to pathogen recognition receptors (PRRs) and ganglioside receptor binding toxins, 2) antigen presenting cell targeting (mucosal) adjuvants such as for instance the ones described by Gerdts et al., (2006). Further examples of adjuvants include, but are not limited to, tensoactive compounds (such as Quil A), mineral salts (such as aluminium hydroxide), micro-organism derived adjuvants (such as muramyl dipeptide), oil-in-water and water-in-oil emulsions (such as Freund’s incomplete adjuvant), particulate antigen delivery systems (such as liposomes, polymeric atmospheres, nanobeads, ISCOMATRIX, lipid/polymer e.g. ENABL® (No. 7010101 , VaxLiant), polysaccharides (such as micro-particulate inulin), nucleic acid based adjuvants (such as CpG motivs), cytokines (such as interleukins and interferons), activators of Toll-like receptors and eurocine L3 en N3 adjuvantia. In a specific embodiment, the adjuvant is an ISCOM™ (ISCOTEC AB, Uppsala, Sweden) adjuvant.

The epitopes, peptides or composition of the present invention can be used as a medicament, and more specific can be used against an infection with a species of the genus Chlamydia, preferably wherein said species is Chlamydia abortus and/or Chlamydia suis. In one embodiment, the composition is a vaccine. With the term ‘vaccine’ is meant a biological preparation that elicits a protective immune response in a subject to which the vaccine has been administered. Preferably, the immune response confers some beneficial, protective effect to the subject against a subsequent challenge with the infectious agent. More preferably, the immune response prevents the onset of or ameliorates at least one symptom of a disease associated with the infectious agent, or reduces the severity of at least one symptom of a disease associated with the infectious agent upon subsequent challenge.

Individual and isolated peptides comprising B- and/or T-cell epitopes and comprising the amino acid sequences as described herein (e.g. in tables 4-11 ) are also part of the present invention, as well as the nucleic acids encoding them. As demonstrated, said peptides are particular useful for the development of a vaccine against a species of the genus Chlamydia, and more specific Chlamydia abortus and/or Chlamydia suis. Hence, any combination of two or more of these peptides for producing a polyepitope vaccine and for use in developing a composition or vaccine is part of the present invention. In a specific embodiment, the polyepitope vaccine comprises or consists of a combination of two or more peptides comprising a T cell epitope as described herein. In a further embodiment, the polyepitope vaccine comprises or consists of a combination of two or more peptides comprising a B-cell epitope of the present invention.

In a further aspect of the current invention, administration of the composition as described herein, and in particular a vector comprising the peptides or nucleic acids of the invention, can overcome the inactivating (i.e. neutralizing) effects of maternal antibodies. It is well- known in the art that maternally-transmitted antibodies interfere with the efficacy of early vaccination programs in young subjects. Accordingly, the present invention provides a composition and the use thereof for effectively vaccinating a subject infected with a species of the genus Chlamydia that has maternal antibodies against said species. Additionally, the methods disclosed herein may be carried out to vaccinate a young animal soon after birth (e.g. within 6 weeks). In particular, in pigs, the composition of the present invention is administered to boars and sows, preferably at weaning, and optionally repeated after 3 to 4 weeks, and also in gilts and boars before the first insemination or semen production for artificial insemination, respectively.

However, the composition of the present invention can also be used to treat specific disease problems in adult pigs such as pneumonia, conjunctivitis and diarrhea caused by C. suis and/or reproductive failure caused by C. abortus (in pigs, goats and sheep).

In an even further embodiment, the invention includes a prime-boost immunization or vaccination against a species of the genus Chlamydia. The priming can be done with the composition, peptides or nucleic acids as described herein. Equally, boosting can be done with the composition, peptides or nucleic acids as described herein.

The invention thus also relates to a method of immunizing a subject against a species of the genus Chlamydia, more specific C. suis and/or C. abortus, comprising administering to the subject the composition as described herein in a prime-boost regimen. In its broadest sense, the term of "prime-boost" refers to at least two successive administrations of a composition or of two different vaccine types or immunogenic compositions having at least one epitope or immunogen in common. The priming administration is the administration of a first vaccine or composition (type) and may comprise one, two, three or more administrations. The boost administration is the administration of the vaccine or composition at a later time point or of a second vaccine or composition type and may comprise one, two, three or more administrations, and, for instance, may comprise or consist essentially of weekly, monthly or annual administrations. The "boost" may be administered from about 2 weeks to about 6 months after the "priming", such as from about 2 to about 8 weeks after the priming, and advantageously from about 2 to about 6 weeks after the priming, and more advantageously, about 2, 3 or 4 weeks after the priming. In a specific embodiment, the prime and boost compositions are the same, and in particular include or encode the same peptide composition as described herein.

The present invention is illustrated by the following Examples, which should not be understood to limit the scope of the invention to the specific embodiments therein.

Examples

1. B- and T-cell epitope mapping of C. suis and C. abortus MOMP during in vivo experiments in pigs.

IN VITRO PRODUCTION OF RECOMBINANT MOMP OF C. SUIS AND C. ABORTUS

The outer membrane protein A ( ompA ) of 8 different Major Outer Membrane Proteins (MOMP) of the Chlamydia (C.) suis strains R19, R22, R24, R27, R130, S45, H5, and H7 and of the C. abortus reference strain S26/3 were cloned using the eukaryotic expression vector pcDNA4::MOMP-V5-His. Plasmids were multiplied in transformed Escherichia coli and purified by Qiagen endofree plasmid GIGA-kit (Qiagen GmbH, Hilden, Germany). Purity and concentration were determined using standard techniques and plasmids were stored at -20°C until use. Recombinant MOMP proteins were produced in pcDNA4::MOMP-V5-His transfected COS-7 cells as previously described by Vanrompay et al. (1998) as the MOMP of Chlamydia is glycosylated. Briefly, COS-7 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat- inactivated fetal bovine serum (Invitrogen, Merelbeke, Belgium) and 1% gentamicin (Gibco, ThermoFisher scientific, Merelbeke, Belgium). Transfections with plasmid DNA were performed by the DEAE dextran method as described by Tregaskes and Young (Ί997) and rMOMP-his production in transfected COS-7 cells was evaluated by immunofluorescence staining using an anti-V5-FITC antibody (Invitrogen, Thermo Fisher, Merelbeke, Belgium). Forty-eight hours post transfection, cell culture medium was removed, 1x wash- buffer (First 200ml of 10x wash- buffer was prepared: 500mM NaH₂P0₄.2H₂0, 3mM NaCL, pH 7.5; for the preparation of 1x wash buffer: 100 ml of 10x wash buffer + 5-10 mM imidazole, 0.1 % Triton X-100, 0.1 % Tween, 900 ml bidest, pH 7.5) was added and tissue culture flasks were placed at -80°C. After 2 freeze- thaw cycles, the wash buffer was collected and centrifuged for 5 min at 450 x g. Supernatants was filtered and his-tag labeled rMOMP proteins were purified by affinity chromatography using the Akta purifier (Akta purifier 100, GE Healthcare Life Sciences, Diegem, Belgium) with a HisPrepFF 16/10 column (GE Healthcare Life Sciences). Purified proteins were transferred into a dialysis membrane Spectra/por® MW 12-14 (Spectrum®, Breda, The Netherlands) and placed in a PBS solution at 4°C on a magnetic stirrer, to eliminate imidazole. SDS-PAGE and Western-Blotting were used to analyze the purified rMOMP- His proteins. Protein concentration was determined using the Pierce™ BCA protein kit (Thermo Scientific, Merelbeke, Belgium).

IN VIVO EXPERIMENT FOR RECOMBINANT MOMP B CELL EPITOPE MAPPING

Ten 9-weeks-old Chlamydiaceae- negative female SPF pigs were randomly assigned to three groups of three pigs and housed in separate isolation units. The Chlamydia negative status of the animals was verified by two in house developed antibody ELISA’s, the first being a MOMP-based antibody ELISA (De Clercq et al. , 2014) and the second one being a PmpC-based antibody ELISA (De Puysseleyr et al., 2018). In addition, a pan- Chlamydiaceae PCR detecting the Chlamydiaceae 16S rRNA and 23S rRNA (Everett et al., 1999) was used as well as a C. suis- specific real-time PCR (De Puysseleyr et al., 2014a) and a C. abortus- specific PCR (Livingstone et al., 2009).

The animals were immunised intramuscularly at the age of 9 weeks using 250 pg adjuvanted protein per pig for the primo vaccination and the same dose of adjuvanted protein for the booster vaccination at day 21. One pig served as control and the animal received PBS + adjuvant (Table 1). Table 1. Pigs immunized with respective recombinant MOMP proteins

Recombinant Gene ID (European Nucleotide Archive Animal MOMP (ENA)) pig 1 R19 ENA study acc. No. PRJNA326179* pig 2 R22 ENA study acc. No. PRJNA326179 pig 3 R24 ENA study acc. No. PRJNA326179 pig 4 R27 ENA study acc. No. PRJNA326179 pig 5 R130 Non-published result pig 6 S45 ENA study acc. No. PRJNA326179 pig 7 H5 ENA study acc. No. PRJNA326179 pig 8 H7 ENA study acc. No. PRJNA326179 pig 9 S26/3 SRA database acc No. CR848038.1** pig 10 PBS /

* Seth-Smith et al., 2017; ** Joseph et al., 2016

B-cell epitope identification was performed using overlapping synthetic peptides of 8 amino acids with 7 amino acids overlap of the variable domains (VD) I to IV of MOMP- sequences of C. abortus (Table 3) and C. suis (Table 2). A total amount of 100 nmol of these peptides were coupled to each pin via an extra C-terminal cysteine residue (Pepscan systems, The Netherlands). B-cell epitope mapping was performed using a pin- peptide ELISA with sera of: (i) pigs immunised with rMOMP of C. suis or C. abortus ; (ii) C. suis or C. abortus infected SPF pigs from previous experiments (De Clercq et al. , 2014; De Puysseleyret al. 2015) and (iii) sera of non-immunised, non-infected SPF animals (De Clercq et al., 2014). In addition, field sera of infected pigs collected in the slaughterhouse (De Puysseleyr et al. 2014b) or collected on pig farms (De Puysseleyr et al. 2015) were used.

Table 2. MOMP sequences for C. suis B cell epitope mapping based on VD1 to 4

Table 3. Strains and MOMP VD sequences used for C. abortus B cell epitope mapping

P.S. MOM p sequence of all herein provided C. abortus strains is identical except for VD3 of 4873p (1 different AA) IDENTIFIED IMMUNO-STIMULATIVE B CELL EPITOPES OF C. SUIS AND C.

ABORTUS STRAINS

Results from the B-cell epitope mapping for C. suis strains can be found in Table 4 and for C. abortus strains in Table 5.

Table 4. C. suis MOMP B cell epitopes

Table 5. C. abortus MOMP B cell epitopes

IN VIVO EXPERIMENT FOR RECOMBINANT MOMP T CELL EPITOPE MAPPING

For T-cell epitope identification, overlapping synthetic peptides of 15 amino acids with 14 amino acids overlap of conserved regions (CR) 1 to 5 of the MOMP sequences of C. suis and C. abortus strains were produced, in duplo, by Pepscan systems (The Netherlands) with an amount of 1 mg peptide/well in 96 well plates. The MOMP CR sequences of the strains being used can be found in Tables 6 and 7. In the first set of plates, the peptides served as stimulation agents for T-cells in a T-cell proliferation assay. The second set of plates served for flow cytometric identification of the CD4⁺ and/or CD8⁺ proliferating T cell subpopulations and for the ELISA-based detection of cytokines namely IFN-gamma for CD8⁺ cytotoxic T-cells and activated CD4⁺Th1-cells and IL-4 for activated CD4⁺Th2-cells in the supernatant. Table 6. MOMP CR sequences of C. abortus used for T cell epitope mapping

P.S. MOMP sequence of the herein provided C. abortus strains is identical (except strain 4873p, sequence unknown). Here strain S26 is taken as a model.

Table 7. MOMP sequences used for T cell epitope mapping

IDENTIFIED IMMUNO-STIMULATIVE T CELL EPITOPES OF C. SUIS AND C.

ABORTUS STRAINS

Peptides eliciting one or more, and preferably all, of following characteristics are categorized as suitable for vaccine design: a) Counts per minute (cpm) in proliferation assay for: i) CD8 > 10000 ii) CD4Th1 > 10000 iii) Cluster of CD8 and CD4Th1 > 10000 iv) CD4Th2 > 5000 v) Cluster of CD8 and CD4Th2 > 5000 b) %CD4 > 10, c) %CD8 > 10, d) IFN-g > 20 pg/ml, and e) IL-4 > 50 pg/ml.

The peptides identified to comprise a T-cell epitope are displayed in Tables 8 to 10.

Table 8. C. abortus T-cell epitopes

Table 9. C. suis H7 T-cell epitopes

for C. suis strain S45

Table 10. C. suis R24 T cell epitopes

2. Use of B- and T-cell epitopes in a mucosal vaccine construct in in vivo experiment in pigs IN VITRO CONSTRUCTION OF THE POLYEPITOPE

A recombinant vaccine was designed comprising the following peptides including (clusters of) B- and T-cell epitopes of the major outer membrane protein of C. suis and C. abortus.

Table 11.

The amino acid sequence and encoding nucleic acid sequence of the constructs of the invention are provided in Figure 1 and Figure 2. The polyepitope CsCa was cloned in a constitutive non-invasive Lactococcus lactis MG1363 vector (Wegmann et al. 2007) with pT1 FX plasmid (VIB vzw - BCCM accession no. LMBP 10260). A design of a polyepitope construct plus signal sequence and NLuc tag used for cloning in pT 1 FX and subsequent transformation of L lactis is given in Figure 1.

In the design of an alternative polyepitope construct, one or more of the following T-cell epitopes represented by SEQ ID NO 126-128, SEQ ID NO 140-141 , SEQ ID NO 146-148, SEQ ID NO 152-153, SEQ ID NO 165-167, SEQ ID NO 171-174, SEQ ID NO 179-180, SEQ ID NO 186, or a variant thereof, can be added to the epitopes as provided in Table 11 ; or one or more of the T-cell epitopes as provided in Table 11 can be replaced by one or more of the following T-cell epitopes represented by SEQ ID NO 126-128, SEQ ID NO 140-141 , SEQ ID NO 146-148, SEQ ID NO 152-153, SEQ ID NO 165-167, SEQ ID NO 171-174, SEQ ID NO 179-180, or SEQ ID NO 186, or a variant thereof.

IN VIVO EVALUATION OF THE IMMUNOGENICITY OF THE RECOMBINANT VACCIN

CONSTRUCT

Six Chlamydia- seronegative and PCR negative female piglets of 6 weeks old were divided into two groups. The first group was immunized vaginally using 10¹¹ CFU of L lactis MG1363 transformed with pT1 FX-polyCsCa (polyepitope Chlamydia suis and Chlamydia abortus), while the control group was immunized using the same dose of L. lactis MG1363 transformed with the empty vector. The vaccine was administered during 3 consecutive days as a primo vaccination (days 1-3) and booster vaccination (days 21-23). Serum antibody responses against rMOMP S45 (C. suis) and rMOMP S26/3 (C. abortus) were monitored. T-cell proliferation assays were performed after the primo and after the booster immunization using peripheral blood monomorphonuclear cells (PBMC’s) in vitro re stimulated with recombinant MOMP S45 (C. suis) or recombinant MOMP S26/3.

Antibody ELISA’s

Serum antibody responses were monitored on a weekly basis using two in-house developed recombinant MOMP-based ELISA’s. The serum samples were heat inactivated, kaolin treated and subsequently stored at -80°C until further analysis. In short, 96 well plates were coated with recombinant MOMP of the C. suis reference strain S45 or of the C. abortus reference strain S26/3 (3h, 37°C), subsequently washed (3x, PBS) and blocked using PBS + 5% BSA (ON, 4°C). The next morning, all plates were washed (3x) and serum was added in dilution series (1/30-1/960) in dilution liquid (PBS +3% BSA +0.05% Tween20) during 1 h, 37°C. After washing (3x), HRP labeled goat anti-pig IgG (H + L) antibody (1/1000) was added (1 h, 37°C), followed by washing (3x). ABTS substrate/chromogen solution was added and the OD 405 was measured.

The results for the rMOMP S45 and S26/3 ELISA’s are shown in Figure 3. Statistical analysis was performed using 2-way ANOVA (Tukey’s multiple comparisons test). The serum antibody titres (ELISA) against recombinant S45 MOMP and against recombinant S26/3 MOMP significantly augmented for the animals that were immunised compared to the non-immunized controls (Figure 3). Augmented serum antibodies were already present following a primo immunization.

T cell proliferations assays

PBMC’s were isolated from fresh blood on heparin. In short, blood was added dropwise on lymfoprep and centrifugated. The PBMC’s were collected and residual red blood cells were lysed. PBMC’s were counted and seeded in 96 well plates at 5.10^s cells/well. Cells were stimulated with ConA (positive control), rMOMP C. suis, rMOMP C. abortus and C. suis or C. abortus elementary bodies. Cells were microscopically analysed on daily basis and at the time of blasting, the cells were pulsed using 1 pCi ³H-thymidin/well during 16 hours and harvested. The radioactive signal was counted using the scintillation counter. Stimulation indexes (SI) were calculated based upon ratio of the measured cpm for the stimuli on the corresponding negative control medium. Statistical analysis was performed using a Mann Whitney U test was performed to calculate the significant differences between groups (p< 0.05).

T-cell proliferation after in vitro re-stimulation with recombinant S45 MOMP or recombinant S26/3 MOMP showed a significant increase in stimulation index (SI) after the booster immunization when comparing the immunised animals to the controls (Figure 4 and 5).

Conclusions

ELISA results and results on the T cell proliferation assays proof that a polyepitope (polyCsCa) based upon: i) B cell epitopes of the MOMP of C. suis, ii) B cell epitopes of the MOMP of C. abortus, iii) CD4⁺ Th1 , CD4⁺ Th2 and CD8⁺ T cell epitopes of the MOMP of C. suis and iv) CD4⁺ Th1 , CD4⁺ Th2 and CD8⁺ T cell epitopes of the MOMP of C. suis C. abortus MOMP, is capable of inducing a humoral and cellular immune response in pigs when mucosally (vaginally) administered as transformed L lactis.

CHALLENGE STUDY

1. Experimental setup

Twenty 4-weeks old piglets, which were seronegative for Chlamydia (C.) suis-specific antibodies (recombinant Major Outer Membrane Protein) and PCR-negative for C. suis were housed in our animal units at the Faculty of Veterinary medicine (Merelbeke) in four different groups (n=5) as shown in the overview. Starting from the day of arrival, they orally received collistin during 4 consecutive days as well as enrofloxacine (10% IM, 3 consecutive days, 1 ml/10kg) and were allowed to adapt to the new environment.

At the age of 7 weeks, all animals received a primo immunisation using 1011 CFU/ animal in PBS (0,5 ml total volume) of pT1 FX with or without polyCsCa vaginally. The animals were sedated using Zoletyl/Xylazin during the vaginal administration. After the administration the animals were positioned on a pillow with backside up to avoid vaginal discharge. At the age of 10 weeks, the animals of groups 1 and 3 received a booster vaginally (sedation). At the age of 13 weeks all animals were challenged with 107 TCID50/ml of the S45 strain (1ml intravaginally, under sedation).

Overview of groups in animal experiment:

Unit 1

- Group 1 : pT1 FX-polyCsCa vaginally + booster vaginally + challenge (n=5)

= LL-polyCsCa primo + booster

Unlt 2

- Group 2: pT1 FX-polyCsCa vaginally + challenge (n=5)

= LL-polyCsCa primo

Unit 3

- Group 3: pT 1 FX-empty vaginally + booster vaginally + challenge (n=5)

= LL-empty primo + booster

- Group 4: pT1 FX-empty vaginally + challenge (n=5)

= LL-empty primo

Blood for serum was be collected on weekly basis, while more blood on heparin was be collected at day 14, 28 and day 47 for isolation of PBMC’s (T cell proliferation and characterization). PBMC’s were restimulated with C. suis rMOMP and live Chlamydia suis (S45 reference strain). The excess of cells at day 47 was be frozen for characterization of PBMC’s. Vaginal swabs for detection of mucosal IgA (in PI buffer) were collected at day 0, 5, 7, 14, 21 , 23, 26, 28, 35, 42 and 47. Vaginal swabs to monitor excretion (in TM buffer) was be taken on day 35, 37, 40, 42, 44 and 47.

At the time of euthanasia, PBMC’s were be isolated from blood, spleen and local draining lymph nodes. At euthanasia, macroscopic lesions were scored at euthanasia for vagina, cervix, uterus, uterin tubes, oviducts, ovaries, ligamentum latum uteri, mesovarium, mesosalpinx, spleen, liver, pelvic lymph nodes, cervical lymph nodes. Tissue pieces for histopathology (in 10% formalin) and cryosections (in methocel) were collected from vagina, cervix, corpus uteri, uterine horn L and R, oviduct L and R and urethra. 2. Results

Serum antibody responses (Figure 6) against rMOMP showed that animals in both immunized groups became seropositive following a primo immunization, whereas control groups became only seropositive after challenge. An effect of the booster vaccination can be observed at day 35. Mean serum antibody titres were the highest in the LL-polyCsCa primo + booster group, albeit not significantly different from the LL-polyCsCa primo group. Significant differences between the LL-polyCsCa primo + booster and its control (LL- empty primo + booster) were found at day 35 and 42 post first immunization.

Mucosal IgA antibody responses (Figure 7) against rMOMP were only detected in the immunized groups following booster vaccination and in the control groups following challenge. Mucosal IgA titer were overall higher in the LL-polyCsCa primo + booster group albeit not significantly different from the LL-polyCsCa primo group. Significant differences between the immunized groups and its controls were observed at day 47 post first immunization.

Cellular immune responses were measured using PBMC’s which were stimulated with rMOMP of C. suis or C. suis strain S45 (Figure 8). Averages for each group are represented in Figure 5. The stimulation indexes (SI) against rMOMP are always lower compared to the live Chlamydia. This might be caused by the immune suppressive sequences which are present in rMOMP. At day 14 no significant effects could be observed. Day 28 shows significant higher SI for both immunized groups compared to the control groups with the highest SI for the LL-polyCsCa primo + booster group. At euthanasia, no significant differences could be observed, which might be the effect of too late sampling point as immune cells might have left the local immune sites and can be circulating in the whole body.

Characterization of the cellular immune responses was performed using flow cytometry (Figure 9). The amount of T helper cells and lgM+ B cells were significantly higher in de LL-polyCsCa primo group compared to its control. A significant higher amount of mature B cells and monocytes was found when comparing the LL-polyCsCa primo + booster with its control.

Vaginal excretion of C. suis was monitored by collecting vaginal mucus. Chlamydial growth was analysed at 6 days post inoculation using DIF staining (FITC-labelled antibody against Chlamydial LPS). All slides were examined and scored using immunofluorescence microscopy (BX41 Olympus, 600x). The average values of the different groups (+ SD) are represented in Figure 10. Statistically significant differences (p<0.05) have been observed between the LL-polyCsCa primo and its control group on day 42 and 44 and between LL-polyCsCa primo + booster and its control group on day 44 and 47.

Presence of C. suis in tissues was examined in cryosections of the vagina, cervix, the corpus uteri, the left and right oviduct (Figure 11). Statistically significant differences in were found when comparing the means (+ SD) of immunized groups with their respective controls. Both immunized groups resulted in a lower amount of Chlamydial EB’s and inclusions in the vagina and uterus as well as at the more distal parts of the genital tract (both uterine horns). In the cervix, only a significant lower amount of Chlamydia was observed when comparing the primo groups. This is probably due to the large spread in the LL-polyCsCa primo + booster group.

In Table 12, the average scores for macroscopic lesions in all groups, determined at necropsy are shown. Summarized, the urethra and vagina were significantly more congested in the empty groups compared to the vaccinated groups. At the corpus uteri, significant more congestion and hypertrophy were observed in the control group compared to the polyCsCa primo group. Congestion of the ligamentum latum uteri, mesosalpinx and mesovarium was significantly higher in the control groups compared to the immunized groups. Also, spleen and lymph nodes were significantly more congested in the control groups compared to the vaccinated groups. Table 12. Mean scores + standard deviations for macroscopic lesions in the LL- polyCsCa primo and/or booster and respective control groups

LL-polyCsCa LL-empty

Tissue LL-polyCsCa LL-empty primo + primo +

Macroscopic lesion primo primo booster booster Vu Iva Congestion 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45 0,0 + 0,00

Serous exudate 0,2 + 0,45 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Urethra Congestion 0,0 + 0,00^a 1,4+ 1,14 0,4 + 0,55^b 2,0+ 1,22

Vagina Congestion 0,0 + 0,00 0,6+ 1,34 0,2 + 0,45^b 1,2+ 1 ,64

Cervix Congestion 0,0 + 0,00 0,0 + 0,00 0,6 + 0,55 0,0 + 0,00

Hyperthrophy 0,0 + 0,00 0,8 + 0,45 0,0 + 0,00 0,6 + 0,55

Mucopurulent 0,0 + 0,00 0,8+ 1,09 0,0 + 0,00 0,0 + 0,00

Corpus Uteri Congestion 0,0 + 0,00^a 1,0+ 1,41 0,4 + 0,55 0,2 + 0,45

Hyperthrophy 0,0 + 0,00^a 1,0 + 0,71 0,6 + 0,55 1,0 + 0,71

Oedema 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45 0,0 + 0,00

Serous exudate 0,4 + 0,55 0,0 + 0,00 0,2 + 0,45 0,6 + 0,55

Uterus horn Hyperthrophy 0,0 + 0,00 0,8 + 0,45 0,4 + 0,55 0,4 + 0,71 Left Oedema 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Serous exudate 0,0 + 0,00 0,0 + 0,00 0,4 + 0,89 0,6 + 0,55

Congestion 0,0 + 0,00 0,2 + 0,45 0,0 + 0,00 0,0 + 0,00

Uterus horn Right Hyperthrophy 0,0 + 0,00 0,8 + 0,45 0,4 + 0,55 1,0 + 0,77

Oedema 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Serous exudate 0,0 + 0,00 0,0 + 0,00 0,4 + 0,89 0,96+ 1,34

Ovarium Left Congestion 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Petechia 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,6 + 0,89

Ovarium Right Petechia 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Hyperthrophy 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Serous exudate 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,2 + 0,45

Lig latum uteri Congestion 0,0 + 0,00^ac 2,8 + 0,45 1,2 + 0,45^b 2,2 + 0,84 Mesosalpinx Congestion 0,0 + 0,00^a 1,2 + 0,45 0,2 + 0,45 1,0 + 0,71 Mesovarium Congestion 0,0 + 0,00^a 1,0 + 0,00 0,2 + 0,45 1,0 + 0,71 Spleen Congestion 0,2 + 0,45^a 1,4+ 1,14 0,2 + 0,45^b 1,4+ 1,14

White pulpa pron. 0,6 + 0,55 0,6 + 0,55 1,0 + 0,00 0,6 + 0,55

Lymphnodes Congestion 0,6 + 0,55^a 1,6+ 1,14 0,0 + 0,00 0,0 + 0,00

Enlarged 2,0 + 1,0 2,4 + 0,55 2,6 + 0,55 2,4+ 1,34

Hyperthrophy 0,0 + 0,00 0,0 + 0,00 0,0 + 0,00 0,4 + 0,89

Significant differences between LL-polyCsCa primo and LL-empty primo are indicated with (a); LL-polyCsCa primo + booster vs LL-empty primo + booster with (b); LL-polyCsCa primo vs LL- polyCsCa primo + booster with (c). Histopahological scores were determined by the histological severity grade (Table 13) assigned by the pathologist for the observed lesions. Sums for pathological observations as well as inflammatory processes (Table 14) were calculated per group and total sums were calculated over the complete tissues (Table 15). In the same way sums and total sums for inflammation were calculated (Table 16).

Table 13. Histological severity grade scoring table used to score histopathological lesions

Severity grade Related pathology

0 Absent

1 Minimal

2 Slight

3 Moderate

4 Marked

5 Severe Table 14. Overview of pathological lesions and inflammatory processes observed during the determination of the histological severity grade

The observations clearly show that the LL-polyCsCa primo + booster group has less pronounced pathological lesions and less inflammation compared to its control group but also compared to both primo groups. The cervix which is an important site of Chlamydia replication shows clearly less severe pathology and the ascending infection until the oviducts is clearly less pronounced in the LL-polyCsCa primo + booster group compared to the other groups. Interestingly, perhaps there is also an adjuvant effect of L lactis as LL-empty primo + booster showed less histopathological lesions and inflammatory lesions as compared to LL-empty primo, which is not unlikely as PRR’s might be stimulated by L. lactis stimulating the innate immune response, which also controls the adaptive immune response. Indeed, L lactis cell wall components have been successfully used to enhance the immunogenicity of vaccines.

Table 15. Total sum for pathological observation scores (intraluminal proteinaceous fluid; superficial layer of exfoliated epithelial cells and/or inflammatory cells in the lumen; degeneration and/or apoptosis of epithelial cells in the mucosa-epithelium; infiltration of polymorphonuclear inflammatory cells in the lamina propria) per tissue per group

Organ LL-polyCsCa primo LL-empty primo LL-polyCsCa primo + booster LL-empty primo + booster

Urethra 0 0 0 o

Vagina 1 3 1

Cervix 6 15 1

Uterus corpus 0 2 0

Uterus horn L 5 8 5 1

Uterus horn R 5 5 2 3

Oviduct L 13 9 3 9

Oviduct R 12 10 4 10

Total sum 42 52 16 28

Table 16. Total sum for inflammatory lesion scores (interepithelial inflammatory cells; infiltration of mononuclear inflammatory cells in the lamina propria) per tissue per group

Organ LL-polyCsCa primo LL-empty primo LL-polyCsCa primo + booster LL-empty primo + booster

Urethra 7 3 4 1

Vagina 12 11 5 8

Cervix 12 15 7 9

Uterus corpus 10 12 10 9 Uterus horn L 16 23 20 13

Uterus horn R 13 19 15

Oviduct L 10 15 5

Oviduct R 8 19 5

Total sum 88 117 71 72

3. Discussion

Humoral immune response

Serum IgG (H + L) antibody titres were highest in the LL-polyCsCa primo + booster group compared to the control group with significant differences at day 35 and day 42. Mucosal IgA antibody responses followed a similar trend as the IgG serum titres and were significantly higher at day 47 compared to the control groups.

Cellular immune response

T cell responses showed interesting results as a significant increase in stimulation index was observed at day 28 (one-week post booster immunization) between each vaccinated group and its respective control for the live C. suis strain. The LL-polyCsCa primo + booster group showed the highest stimulation index for T cells. At day 47, no significant differences were observed, which might be due to the timepoint of sampling. Characterization of the immune cell populations at euthanasia showed a significantly higher amount of T helper and lgM+ B cells in the LL-polyCsCa primo group and a significantly higher amount of mature B cells and monocytes for the LL-polyCsCa primo + booster group compared to its controls.

Efficacy of the vaccine

Vaginal excretion was significantly lower in the LL-polyCsCa immunized groups compared to their controls from day 42 (7 days post infection) onwards..

The macroscopic lesions caused by the infection with C. suis were significantly more pronounced in the control groups. Significant differences were observed at urethra and vagina, corpus uteri, ligamentum latum uteri, mesosalpinx and mesovarium. The spleen and lymph nodes were congested in the control group, indicative for an active infection, while local draining lymphnodes in LL-polyCsCa immunized animals were enlarged but not congested, indicative for immunostimulation.

Histopathological scoring showed that pathologic lesions and inflammation were least present in the LL-polyCsCa primo + booster group, followed by the LL-polyCsCa primo group.

In tissues, significantly less Chlamydiae were detected in LL-polyCsCa immunized groups compared to control groups. The data obtained during this in vivo immunization and challenge study in SPF pigs showed a significant protective effect of vaccination with LL-polyCsCa against C. suis vaginal infection.

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Claims

Claims

1. A composition comprising one or more peptides each of said peptides comprising one or more epitopes selected from the list consisting of a B-cell epitope, a CD4+ Th2 cell epitope, a CD4+ Th1 cell epitope and a CTL epitope; wherein said composition comprises at least a B-cell epitope, a CD4+ Th2 cell epitope, a CD4+ Th1 cell epitope and a CTL epitope, and wherein the B-cell epitope consists of an amino acid sequence selected from the group consisting of: SEQ ID NO 46-75, SEQ ID NO 200 and SEQ ID NO 207-211 , or a variant thereof having at least 80% identity; and wherein the CD4+ Th2 cell epitope, the CD4+ Th1 cell epitope and the CTL epitope independently consists of an amino acid sequence selected from the group consisting of SEQ ID NO 124-196, or a variant thereof having at least 80% identity; and wherein the composition does not comprise the full-length Chlamydia suis and/or Chlamydia abortus major outer membrane protein (1VIOMP).

2. The composition according to claim 1 , comprising at least two of the following peptides: PTGTAAANYKTP (SEQ ID NO 200), GSSIAADQLP (SEQ ID NO 74), AAVLNLTTWNPTLL (SEQ ID NO 75), NTGNTTSPTQL (SEQ ID NO 70), TTAAQATA (SEQ ID NO 71), KVEDKGSA (SEQ ID NO 72), MKKLLKSALLFAATGSALSLQ (SEQ ID NO 197), NGYFKASSAAFNLVG (SEQ ID NO 130),

QGIVEFYTDTTFSWSVGARGALWECGCATLGAEFQYAQSNPKIEMLNVVSSPAQ (SEQ ID NO 198),

SATIKYHEWQVGLALSYRLNMLVPYISVNWSRATFDADAIRIAQPKLA (SEQ ID NO 199), SVLVFAALGSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPC (SEQ ID NO 201), IWDRFDVFCTLGATNGYLKGNSAAFNLVGL (SEQ ID NO 202), VSLSQSVIELYTDTAFAWSVGARAALWE (SEQ ID NO 203), TKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKLATAVFDVT (SEQ ID NO 205),

MQIVSMQINKMKSRKSCGLAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF (SEQ ID NO 206), or variants thereof having at least 80% identity.

3. The composition according to claims 1 or 2, wherein part or all of the peptides are present as a mixture or are part of a polyepitope construct.

4. The composition according to claim 3, wherein the polyepitope construct comprises the sequence as provided in SEQ ID NO 212, 214, 216 or 218, or a variant thereof having at least 80% identity.

5. A composition comprising a nucleic acid sequence encoding the peptide(s) as provided in any one of claims 1 to 4.

6. A composition according to any one of claims 1 to 5, further comprising an antigen delivery system.

7. A vector comprising a nucleic acid sequence as provided in claim 5, and optionally a linker sequence, a signal sequence, a promotor sequence, a terminator fragment, an enhancer sequence and/or a marker sequence.

8. The vector according to claim 7, wherein said vector is a plasmid.

9. A cell comprising the vector according to claims 7 or 8.

10. The cell according to claim 9, wherein the cell is a micro-organism, in particular a Lactic acid bacterium.

11. A composition, a vector or a cell according to any one of claims 1 to 10, further comprising or in combination with an adjuvant.

12. A composition, vector or cell according to any one of claims 1 to 10, for use as a medicament.

13. A composition, vector or cell according to any one of claims 1 to 10, for use in inducing an immune response in a subject against an infection with a species of the genus Chlamydia, in particular Chlamydia suis and/or Chlamydia abortus.

14. A composition, vector or cell according to any one of claims 1 to 10, for use as a vaccine against an infection with a species of the genus Chlamydia, in particular Chlamydia suis and/or Chlamydia abortus.