USRE45467E1 - Streptococcus suis polypeptides and polynucleotides encoding same and their use in vaccinal and diagnostic applications - Google Patents
Streptococcus suis polypeptides and polynucleotides encoding same and their use in vaccinal and diagnostic applications Download PDFInfo
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- USRE45467E1 USRE45467E1 US13/866,780 US200613866780A USRE45467E US RE45467 E1 USRE45467 E1 US RE45467E1 US 200613866780 A US200613866780 A US 200613866780A US RE45467 E USRE45467 E US RE45467E
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
-
- 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
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
-
- 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
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
Definitions
- the present invention relates to the field of Streptococcus. More specifically, the present invention relates to the identification of polypeptides and polynucleotide sequences encoding the same which are involved in the pathogenic mechanism of S. suis. The present invention also relates to the use of such polypeptides in compositions and methods for the prevention, the treatment and diagnosis of S. suis-associated diseases and infections caused by S. suis.
- Streptococcus suis is an important swine pathogen that causes many pathological conditions such as arthritis, endocarditis, meningitis, pneumonia and septicemia (13, 14). It is also an important zoonotic agent for people in contact with contaminated pigs or their by-products, causing meningitis and endocarditis (1, 36). Thirty-three serotypes (types 1 to 31, 33 and 1/2) based on capsular antigens are currently known (9-11, 15, 17, 31). Type 2 is considered the most virulent and prevalent type in diseased pigs. The mechanisms involved in the pathogenesis and virulence of S. suis are not completely understood (13) and attempts to control the infection are hampered by the lack of effective vaccines.
- An object of the invention is to fulfill the above-mentioned need. More specifically, the object is achieved by providing an isolated polypeptide comprising at least 15 contiguous amino acids in the N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1.
- Another object of the invention also concerns an polynucleotide encoding a polypeptide as defined above.
- the present invention is further concerned with an antibody which specifically binds to a polypeptide of the invention.
- a further object of the invention is to provide a vector comprising the polynucleotide as defined above.
- Yet another object of the invention is to provide a composition for preventing or treating Streptococcus suis-associated diseases or infection caused by S. suis, comprising an acceptable carrier and at least one of the following elements:
- Another object of the invention concerns a method for treating and/or preventing a Streptococcus suis-associated disease or infection in an animal, the method comprising the step of administering to the animal a composition as defined above.
- a further object concerns a method for detecting the presence or absence of a Streptococcus suis strain in a sample, comprising the steps of:
- Another object of the invention concerns a method for detecting the presence or absence of antibodies raised against a Streptococcus suis strain in a sample, comprising the steps of:
- the present invention also provide in another object a diagnostic kit for the detection of the presence or absence of antibodies indicative of Streptococcus suis strain, comprising:
- Yet another object is to provide a diagnostic kit for the detection of the presence or absence of antibodies indicative of Streptococcus suis strain, comprising:
- an isolated polypeptide comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO 11 or functional derivative thereof, and an isolated polynucleotide encoding said polypeptide, and their use in a composition and/or a method for treating and/or preventing a Streptococcus suis-associated disease or infection in an animal.
- FIG. 1 Schematic representation and partial restriction map of a preferred polynucleotide of the invention, namely the DNA insert of recombinant plasmid pSS735. Numbers indicate the distance (in base pairs) from the 5′ end.
- FIG. 2 Nucleotide sequence (SEQ ID NO: 5)(SEQ ID NO: 20) and deduced amino acid sequence (SEQ ID NO: 1)(SEQ ID NO: 19) of the gene encoding a preferred polypeptide of a first embodiment of the invention, namely the SP1 (or Sao) protein of S. suis.
- the Shine-Dalgarno sequence is in italic letters and underlined.
- the initiation codon, ATG, and the stop codon, TAA, are shown in bold type.
- the two hydrophobic segments at the both N- and C-terminal ends of SP 1 are underlined.
- the vertical arrow indicates the cleavage site of potential signal peptidase.
- R1 to R10 indicate the beginning of the repeating units.
- the potential cell wall-associated region is underlined with dash line.
- the LPVTG (SEQ ID NO: 10) membrane anchor motif is boxed, and the charged C-terminal tail is indicated.
- FIG. 3 Amino acid sequence alignment of the region Lys 319 to Val 601 of SP1 (SEQ ID NO: 18) with the AvrXa7 avirulence factor of Xanthomonas oryzae pv. oryzae (SEQ ID NO: 17).
- the vertical lines indicate positions with identical residues. Double dots represent conserved substitutions and single dots represent functional substitutions.
- FIG. 4 Expression of MBP-SP1 fusion protein in E. coli XL1-Blue and purification of the recombinant mature SP1.
- the Coomassie-stained gel (A) and Western blot analysis (B) of the corresponding samples probed with convalescent swine serum show E. coli whole cell lysate before (lane 1) and after (lane 2) induction of IPTG, extract of cytoplasm (lane 3), affinity purified MBP-SP1 fusion protein (lane 4), SP1 and MBP cleaved by factor X (lane 5) and recombinant SP1 devoid of MBP purified using ion-exchange chromatography (lane 6).
- the molecular masses of standard proteins are indicated on the left.
- FIG. 5 Immunoelectron microscopy of S. suis (4500 ⁇ ). The surface location of SP1 on S. suis is demonstrated using a monospecific SP1 antiserum and a gold-conjugated secondary antibody (B). No labeling was found in the control bacterial cell (A). Bars, 200 nm.
- FIG. 6 Antibody responses after vaccination with the SP1 in piglets.
- A Total SP1-specific IgG in sera was measured by ELISA, showing that single injection of SP1 elicited a significant IgG response that was obviously enhanced by the booster.
- B ELISA for serum IgG isotypes in SP1 immunized pigs showed that IgG1 levels were consistently higher than IgG2 levels. The results are expressed as the means of absorbances and standard errors. *: p ⁇ 0.05.
- FIG. 7 SP1-specific total humoral IgG titres in mice immunized with Quil A and Quil A plus SP1.
- FIG. 8 IgG subclasses in sera from mice immunized with recombinant SP1.
- FIG. 9 Vaccination with recombinant SP1 protects mice against S. suis challenge infection.
- FIG. 10 Vaccination with recombinant SP1 protects mice from S. suis death.
- FIG. 11 Nucleotide sequence (SEQ ID NO: 8) of a preferred functional polynucleotide fragment of the invention, namely the SP1A gene fragment and the deduced amino acid sequences (SEQ ID NO: 4).
- FIG. 12 Schematic representation and partial restriction map of the 6.3 kb insert of recombinant phage. Numbers indicate the distance (in base pairs) from the 5′ end.
- FIG. 13 Nucleotide sequence (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO: 11) of the gene encoding a preferred polypeptide of another embodiment of the invention, namely the SP2 protein of S. suis.
- the positive charge cluster at N-terminal end of SP2 is underlined.
- the potential N-terminal signal sequence is underlined with dash line.
- the LysM domain is boxed, and the arrows indicate the beginning of the repeating units.
- FIG. 14 Distribution of SP2 gene in different S. suis serotypes.
- the SP2 genes were amplified by PCR from 31 of the 33 S. suis serotype reference strains.
- FIG. 15 Expression of Trx-His-SP2 fusion protein in E. coli and purification of the recombinant mature SP2.
- the Coomassie-stained gel shows E. coli whole cell lysate after induction of IPTG, affinity purified Trx-His-SP2 fusion protein, SP2 and Trx-His cleaved by enterokinase, separated mature SP2 and Trx-His tag by an anion-exchange chromatography. The molecular masses are indicated on the left.
- FIG. 16 Immunogenic and IgG-binding activity of recombinant SP2.
- SP2-specific rabbit serum reacts with the cell preparation of S. suis S735.
- Recombinant SP2 reacts with the convalescent swine serum.
- Recombinant SP2 binds to human (c) and pig (d) IgG.
- FIG. 17 Antibody response after vaccination with recombinant SP2 in mice. SP2-specific IgG in sera was measured by ELISA.
- FIG. 18 Vaccination with recombinant SP2 alleviates clinical signs of the mice challenged with a virulent S. suis strain.
- FIG. 19 Vaccination with recombinant SP2 protects mice from S. suis death.
- FIG. 20 Body temperature of pigs vaccinated with the composition according to a preferred embodiment of the invention, after challenge.
- FIG. 21 Clinical disease of pigs vaccinated with the composition according to a preferred embodiment of the invention, after challenge.
- FIG. 22 Survival of pigs vaccinated with the composition according to a preferred embodiment of the invention, after challenge.
- FIG. 23 Serum total IgG titers of pigs vaccinated with the composition according to a preferred embodiment of the invention.
- FIG. 24 IgG subclasses induced from pigs vaccinated with the composition according to a preferred embodiment of the invention.
- FIG. 25 Amino acid sequence alignment between two SP1 polypeptides according to preferred embodiments of the invention, namely SEQ ID NO 1 and 2.
- FIG. 26 Amino acid sequence alignment between two SP1 polypeptides according to preferred embodiments of the invention, namely SEQ ID NO 1 and 3.
- FIG. 27 Amino acid sequence alignment between two SP1 polypeptides according to preferred embodiments of the invention, namely SEQ ID NO 2 and 3.
- FIG. 28 Amino acid sequence alignment between three SP1 polypeptides according to preferred embodiments of the invention, namely SEQ ID NO 1, 2 and 3.
- the inventors have surprisingly found two novel S. suis polypeptides and polynucleotides encoding same that are involved during the S. suis pathogenic mechanism.
- the present invention specifically relates to their identification and to the use of said polypeptides or polynucleotides in compositions and methods for the prevention, the treatment and the diagnosis of Streptococcus suis-associated diseases or infection caused by S. suis.
- Streptococcus suis-associated diseases which the methods of the invention may be useful for, includes those, such as arthritis, endocarditis, meningitis, pneumonia and septicemia.
- isolated is meant to describe a polynucleotide, a polypeptide or an antibody that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs.
- animal refers to any animal susceptible to be infected by a Streptococcus strain, such as S. suis. Specifically, such an animal may be, but not limited to, mice, pig, sheep, horse and human. More specifically, the animal consists of a pig.
- treating refers to a process by which the symptoms of an infection or a disease associated with a Streptococcus strain are alleviated or completely eliminated.
- preventing refers to a process by which symptoms of an infection or a disease associated with a Streptococcus strain are obstructed or delayed.
- protection response means prevention of onset of a Streptococcus suis-associated disease or an infection caused by S. suis. or lessening the severity of such a disease existing in an animal.
- the level of “protective response” may be evaluated, for instance, by the assignment of clinical scores such as those defined in Example 4.
- an acceptable carrier means a vehicle for containing the compounds obtained by the method of the invention that can be administered to an animal host without adverse effects.
- Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions.
- Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
- fragment refers to a polynucleotide sequence (e.g., cDNA) which is an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art.
- cDNA polynucleotide sequence
- the present invention concerns an isolated polypeptide which consists of a surface protein, and more particularly a C-terminal-anchored surface protein of Streptococcus suis, namely called SP1 or Sao (Genbank accession number AY864331).
- SP1 or Sao Genetic accession number AY864331
- the SP1 polypeptide advantageously elicits a protective response to a Streptococcus suis strain challenge when administered to an animal, such as a pig.
- the isolated polypeptide of the first embodiment of the invention comprises at least 15 or even preferably at least 25 or even more at least 35 contiguous amino acids in the N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1.
- N-terminal region in the context of the present invention when referring to the Sao protein, preferably consists of the region spanning from amino acid residue 1 to 293 of the amino acid sequence set forth in SEQ ID NO: 1.
- the isolated polypeptide of the first embodiment may further comprises at least one repetitive amino acid sequence such as shown in FIG. 2 .
- a repetitive amino acid sequence contemplated by the present invention consists of the amino acid sequence shown in SEQ ID NO 9,
- the preferred SP1 polypeptide of the invention may comprises only one of said repetitive sequence, whereas in some other cases, the preferred SP1 polypeptide of the invention may comprises at least two repetitive sequences or even more than ten of such repetitive sequences.
- the isolated SP1 polypeptide advantageously comprises at least 15 or even preferably 25 or even more preferably 35 contiguous amino acids in the C-terminus region of the amino acid sequence set forth in SEQ ID NO: 1.
- the C-terminus region comprises a membrane anchor motif, such as the one consisting of the amino acid sequence as set forth is SEQ ID NO 10, namely Leu Pro Val Thr Gly.
- C-terminus region in the context of the present invention when referring to the SP1 protein, preferably consists of the region spanning from amino acid residue 593 to 670 of the amino acid sequence set forth in SEQ ID NO: 1.
- a SP1 polypeptide of the invention comprises an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOS 1 to 3 or functional derivative thereof. Most preferably, a SP1 polypeptide of the invention consists of an amino acid sequence substantially identical to the sequence shown in SEQ ID NO 1, or a functional derivative thereof.
- a “functional derivative”, as is generally understood and used herein, refers to a protein/peptide sequence that possesses a functional biological activity that is substantially similar to the biological activity of the whole protein/peptide sequence. In other words, it preferably refers to a polypeptide or fragment(s) thereof that substantially retain(s) the capacity of eliciting an immune response, such as a protective response to a S. suis strain challenge when said functional derivative is administered to an animal.
- a preferred functional derivative contemplated by the present invention comprises an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO 4. More specifically, a preferred functional derivative consists of a 315-amino acids fragment (S 28 -K 342 ) of SEQ ID NO 1.
- SP1A fragment or polypeptide
- FIG. 11 Such a fragment or polypeptide is designed as SP1A and its nucleotide and amino acid sequences are shown in FIG. 11 .
- SP1A strongly reacted with a convalescent swine serum in immunoblots and immunization with recombinant SP1A elicits significant humoral antibody responses in pigs and mice, demonstrating that SP1A is highly immunogenic. (See Example 5)
- the present invention relates to another isolated S. suis polypeptide, namely called SP2, which advantageously elicits a protective response in an animal.
- the isolated polypeptide of the second embodiment of the invention comprises an amino acid sequence substantially identical to a sequence as set forth in SEQ ID NO: 11 or functional derivative thereof.
- polypeptide of the present invention preferably has an amino acid sequence having at least 75% homology, or even preferably 85% homology, or even more preferably 95% homology to part or all of the sequence shown in SEQ ID NOS 1 to 4 and 11.
- “Homology” in this context means identical or similar to the referenced sequence while straightforward replacements/modifications of any of the amino acids provided, are included as well.
- a homology search in this respect can be performed with the BLAST-P (Basic Local Alignment Search Tool), a program well known to those of skill in the art.
- BLAST-P Basic Local Alignment Search Tool
- homology is referred to the BLASTX and BLASTN programs known in the art.
- the present invention also concerns an isolated polynucleotide encoding a preferred SP1 or a preferred SP2 polypeptide of the invention.
- the isolated polynucleotide of the invention comprises a nucleotide sequence substantially identical to the sequence shown in SEQ ID NOS 5 to 7 when referring to SP1 and SEQ ID NO 12 when referring to SP2 and their respective functional fragments thereof.
- the polynucleotide of the invention preferably has a nucleic acid sequence which is at least 65% identical, more particularly 80% identical and even more particularly 95% identical to part or all of the sequence shown in SEQ ID NOS 5 to 7 and 12 or functional fragments thereof.
- a “functional fragment”, as is generally understood and used herein, refers to a nucleic acid sequence that encodes for a functional biological activity that is substantially similar to the biological activity of the whole nucleic acid sequence. In other words, and within the context of the present invention, it preferably refers to a nucleic acid or fragment(s) thereof that substantially retains the capacity of encoding a polypeptide/protein which elicits an immune response, and more preferably a protective response, to a Streptococcus suis strain challenge when administered to an animal.
- a fragment is the polynucleotide shown in SEQ ID NO 8, which codes for the SP1A polypeptide as defined above.
- the invention is further directed to vector (e.g., cloning or expression vector) comprising a polynucleotide of the invention as defined above.
- vector e.g., cloning or expression vector
- vector refers to a polynucleotide construct designed for transduction/transfection of one or more cell types.
- Vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector.
- vectors suitable for stable transfection of cells and bacteria are available to the public (e.g., plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.
- the invention features antibodies that specifically bind to the polypeptides of the invention. More specifically, the antibody is a purified polyclonal or monoclonal antibody that specifically binds to the preferred S. suis polypeptides as defined above.
- the antibodies of the invention may be prepared by a variety of methods known to one skilled in the art.
- the polypeptides of the invention may be administered to an animal in order to induce the production of polyclonal antibodies.
- antibodies used as described herein may be monoclonal antibodies, which are prepared using known hybridoma technologies (see, e.g., Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., 1981; Charland, N., M. Jacques, S. Iacouture and M. Gottschalk. 1997. Characterization and protective activity of a monoclonal antibody against a capsular epitope shared by Streptococcus suis serotypes 1, 2 and 1 ⁇ 2. Microbiology 143 (Pt 11): 3607-14).
- the term “specifically binds to” refers to antibodies that bind with a relatively high affinity to one or more epitopes of the SP1 or SP2 polypeptide of the invention, but which do not substantially recognize and bind molecules other than the SP1 or SP2 polypeptides of the invention.
- the term “relatively high affinity” means a binding affinity between the antibody and the SP1 or SP2 polypeptides of at least 10 6 M ⁇ 1 , and preferably of at least about 10 7 M ⁇ 1 and even more preferably 10 8 M ⁇ 1 to 10 10 M ⁇ 1 . Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which are common knowledge to one skilled in the art.
- SP1 and SP2 polypeptides, polynucleotides encoding same and antibodies of the invention may be used in many ways in the treatment and/or prevention of Streptococcus suis-associated diseases or infection caused by S. suis.
- the SP1 and/or SP2 polypeptides of the invention may be used as immunogens for the production of specific antibodies for the treatment and/or prevention of Streptococcus suis infection.
- Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against Streptococcus suis infection in a test model. Examples of an animal model are the mouse and pig models described in the examples herein.
- polynucleotides encoding polypeptides of the invention or derivatives thereof may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in vivo.
- polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells.
- the vector is injected intramuscularly.
- a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles [Wolf et al. H M G (1992) 1: 363, Turnes et al., Vaccine (1999), 17: 2089, Le et al., Vaccine (2000) 18: 1893, Alves et al., Vaccine (2001)19: 788], injection of plasmid DNA with or without adjuvants [Ulmer et al., Vaccine (1999) 18: 18, MacLaughlin et al., J. Control Release (1998) 56: 259, Hartikka et al., Gene Ther.
- a suitable delivery method or system such as direct injection of plasmid DNA into muscles [Wolf et al. H M G (1992) 1: 363, Turnes et al., Vaccine (1999), 17: 2089, Le et al., Vaccin
- a further aspect of the invention is the use of the antibodies directed to the polypeptides of the invention for passive immunization.
- composition of the present invention advantageously comprises an acceptable carrier and a SP1 and/or SP2 polypeptide(s) of the invention.
- composition of the invention can comprise an antibody and/or a polynucleotide and/or an expression vector of the invention.
- the composition of the invention further comprises an adjuvant.
- adjuvant means a substance added to the composition of the invention to increase the composition's immunogenicity. The mechanism of how an adjuvant operates is not entirely known. Some adjuvants are believed to enhance the immune response (humoral and/or cellular response) by slowly releasing the antigen, while other adjuvants are strongly immunogenic in their own right and are believed to function synergistically.
- adjuvants include, but are not limited to, oil and water emulsions (for example, complete Freund's adjuvant and incomplete Freund's adjuvant), Corytzebactei-ium parvuin, Quil A, cytokines such as IL12, Emulsigen-Plus®, Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto Adjuvant, certain synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, paraffin oil, and muramyl dipeptide.
- oil and water emulsions for example, complete Freund's adjuvant and incomplete Freund's adjuvant
- Corytzebactei-ium parvuin Quil A
- cytokines such as IL12, Emulsigen-Plus®
- Bacillus Calmette Guerin aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alg
- Adjuvants also encompass genetic adjuvants such as immunomodulatory molecules encoded in a co-inoculated DNA, or as CpG oligonucleotides.
- the coinoculated DNA can be in the same plasmid construct as the plasmid immunogen or in a separate DNA vector.
- a further embodiment of the present invention is to provide a method for treating and/or preventing a Streptococcus suis-associated disease or infection in an animal.
- the method of the invention comprises the step of administering to the animal a composition according to the invention.
- composition of the invention may also comprise agents such as drugs, immunostimulants (such as ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), and interleukin 2 (IL2)), antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives.
- immunostimulants such as ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), and interleukin 2 (IL2)
- antioxidants such as antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives.
- surfactants such as ⁇ -interferon, ⁇ -interferon, ⁇ -inter
- the amount of the components or the elements of the composition of the invention is preferably a therapeutically effective amount.
- a therapeutically effective amount of the contemplated component is the amount necessary to allow the same to perform their immunological role without causing overly negative effects in the host to which the composition is administered.
- the exact amount of the components to be used and the composition to be administered will vary according to factors such as the type of condition being treated, the type and age of the animal to be treated, the mode of administration, as well as the other ingredients in the composition.
- composition of the invention may be given to an animal through various routes of administration.
- the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions.
- sterile injectable preparations such as sterile injectable aqueous or oleaginous suspensions.
- suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
- the sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os.
- Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the animal to be treated. Any other methods well known in the art may be used for administering the composition of the invention.
- SP1 and/or SP2 polypeptides, polynucleotides encoding same and antibodies of the invention may also be used in different ways in the detection and diagnosis of Streptococcus suis-associated diseases or infections caused by S. suis.
- the present invention provides a method for detecting the presence or absence of a Streptococcus suis strain in a sample, comprising the steps of:
- sample refers to a variety of sample types obtained from an animal and can be used in a diagnostic or detection assay.
- the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue culture or cells derived therefrom.
- the present invention provides a method for detecting the presence or absence of antibodies raised against a Streptococcus suis strain in a sample, comprising the steps of:
- this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay, essentially to determine whether antibodies specific for the protein (such as SP1 and/or SP2) are present in an organism.
- an immunological test such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay, essentially to determine whether antibodies specific for the protein (such as SP1 and/or SP2) are present in an organism.
- ELISA enzyme-linked immunosorbent assay
- radioimmunoassay essentially to determine whether antibodies specific for the protein (such as SP1 and/or SP2) are present in an organism.
- kits for use within any of the above diagnostic methods.
- Such kits typically comprise two or more components necessary for performing a diagnostic assay.
- Components may be compounds, reagents, containers and/or equipment.
- one container within a kit may contain an antibody or fragment thereof that specifically binds to a SP1 or SP2 polypeptide of the invention.
- One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
- the present invention also provides a diagnostic kit for the detection of the presence or absence of antibodies indicative of Streptococcus suis strain, comprising:
- Another diagnostic kit preferably contemplated is a kit for the detection of the presence or absence of polypeptides indicative of Streptococcus suis strain, comprising:
- SP1 Streptococcus suis surface protein reacting with a convalescent serum from pigs clinically infected by S. suis type 2 was identified.
- the apparent 110 kDa protein designated SP1 exhibits typical features of membrane-anchored surface proteins of Gram-positive bacteria such as a signal sequence and a LPVTG (SEQ ID NO:10) membrane anchor motif. Moreover, a conserved avirulence domain that often found in plant pathogens has been detected. Electron microscopy using a SP1-specific antiserum has confirmed the surface location of SP1 protein on S. suis.
- the SP1-specific antibody reacts with the cell lysates of most S. suis serotypes and type 2 isolates in immunoblots, demonstrating its high conservation in S. suis species.
- Immunization of piglets with the recombinant SP1 by intramuscular route elicits a significant total immunoglobulin G (IgG) antibody response.
- IgG immunoglobulin G
- the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.
- Reference strain S735 of S. suis serotype 2 was used for the genomic library construction. Reference strains of the thirty-three serotypes (types 1 to 31, 33 and 1/2), 26 field strains of serotype 2 from different origin as well as five other Gram-positive organisms are listed in Table 1. Phage Lambda Zap II and Escherichia coli XL1-Blue MRF strain were obtained from a commercial source (Stratagene, La Jolla, Calif). S. suis were grown in Todd-Hewitt broth (THB, Difco, Detroit, Mich.) or agar plates (Quelab Laboratories, Montreal, Canada) at 37° C.
- E. coli was grown in either Luria-Bertani (LB) medium alone or LB medium supplemented with 2 g of maltose/liter at 37° C. Where appropriate, E. coli was grown in the presence of 50 ⁇ g of ampicillin/ml and 0.8 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
- pMalTM-p vector (New England BioLabs) was used for generating the MBP-SP1 fusion protein.
- Convalescent swine sera were collected from pigs clinically infected with S. suis type 2 strain S735. Monospecific anti-SP1 serum was obtained by immunizing New Zealand White rabbits intravenously with 230 ⁇ g of purified SP1 emulsified with 0.5 ml of Freud's incomplete adjuvant. The rabbits received two booster injections with the same dose of the SP1 at 2-week intervals and then were bled 10 days after the last booster immunization. Sera were stored at ⁇ 20° C. until used.
- Chromosomal DNA from S. suis S735 strain was isolated as previously described (33). Purified chromosomal DNA was partially digested with the restriction enzyme EcoRI, and the resulting fragments were electrophoresed in 1% agarose gel. Fragments in the 6- to 10-kb size range were extracted from the gel and ligated to the EcoRI arms of ⁇ ZAPII vector, and the vector was encapsidated using the Gigapack II packaging extract (Stratagene). The recombinant phages were used to infect E. coli XL1-Blue MRF′, which was then plated onto LB agar.
- the resulting plaques were lifted onto nitrocellulose membranes (Bio-Rad, Mississauga, Ontario, Canada).
- the membranes were blocked using Tris-saline buffer (TBS) with 2% skim milk and sequentially incubated with the convalescent swine serum from S. suis serotype 2 infection, peroxidase-conjugated rabbit anti-swine immunoglobulin G (IgG) antisera (Jackson Immuno Research Laboratories, Inc., West Grove, Pa.), and O-phenylenediamine.
- TBS Tris-saline buffer
- IgG peroxidase-conjugated rabbit anti-swine immunoglobulin G
- the positive plaques were purified to homogeneity.
- the recombinant pBluescript plasmids were excised with ExAssist helper phage (Stratagene) according to the manufacturer's instructions.
- the sequence of the insert was determined using T3 and T7 promoters as primers in DNA Sequencing Facility, University of Maine (Orono, Me., USA).
- the nucleotide and amino acid sequences deduced from open reading frames (ORFs) were analyzed using programs available on the internet.
- the sequence coding for mature SP1 was amplified from purified chromosomal DNA of strain S735 by PCR primers P1 (5′-ATGGATCCATTGAAGGCCGCTCGGCACAAGAAGTAAAA-3′; SEQ ID NO 13) and P2 (5′-CCAAGTCGACTTATAATTTACGTTTACGTGTA-3′; SEQ ID NO 14), which contained BamHI and Sal I restriction sites, respectively.
- the PCR was performed with 5 min at 94° C., followed by 30 cycles of 1 min at 94° C., 30 s at 56° C., and 1 min at 72° C.
- the resulting PCR fragment was cloned into Bam HI and Sal I sites of pMAL-p expression vector.
- the recombinant plasmid containing the sp1 gene was named pORF3.
- the purified plasmid pORF3 was used to transform E. coli XL1-Blue strain by electroporation with Genepulse II apparatus (Bio-Rad) following the manufacturer's recommendations.
- This recombinant strain was grown in LB medium plus 2 g of glucose/L and 50 ⁇ g of ampicillin/ml.
- the culture was inoculated from an overnight culture with its starting OD 600 adjusted to 0.1.
- the culture was incubated with agitation until OD 600 of approximately 0.8, and then IPTG was added in order to induce production of the MBP-SP1 fusion protein.
- the fusion protein was found in the bacterial periplasm as well as in the cytoplasm. It was decided to use extracts of the bacterial lysates for purification of the SP1 protein.
- the fusion protein was purified by affinity chromatography using an amylose resin (New England BioLabs) following the manufacturer's instructions.
- the E. coli cell pellet was suspended in the affinity column binding buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4) and cells were lysed using the French Pressure Cell Press (SLM Instruments, Inc.). After filtration with a 0.45 ⁇ m membrane, the supernatant was subjected to the amylose resin.
- the MBP-SP1 fusion protein was eluted with 1% maltose in the binding buffer and protein-containing fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- the purified fusion protein was cleaved with protease Factor Xa (New England BioLabs) at a concentration of 20 ⁇ g/mg protein, and applied to a mono-Q column (Amersham Pharmacia Biotech, Baie d'Urfee, Canada).
- the recombinant SP1 devoid of MBP carrier was eluted from the column by using a linear NaCl gradient (0 to 0.4 M NaCl in 20 mM Tris-HCl, pH 7.4).
- the SP1-containing fractions were combined and dialyzed against PBS buffer.
- the purity of the recombinant SP1 was evaluated by SDS-PAGE, and the concentration of the protein was determined by the Bradford protein assay (Bio-Rad) according to the manufacturer's instructions.
- SDS-PAGE and western immunoblotting were performed as described by Laemmli (21). Total cell extract or purified protein was separated on a 10% acrylamide gel and the gel was then stained with Coomassie brilliant blue R250 (Sigma, St. Louis, Mo.). Prestained low molecular mass markers (Bio-Rad) were used to determine the apparent molecular weights of proteins.
- Western blotting of proteins transferred to nitrocellulose membranes was performed essentially as described by Burnette (5).
- S. suis S735 strain was grown in 5 ml of THB overnight, centrifuged, and resuspended in 500 ⁇ l of PBS (pH8.0). 20 ⁇ l of the bacterial suspension was placed on nickel-formvar grids (INRS, Institut Armand Frappier, Laval, Canada) and allowed to partially air dry. After blocking for 30 min with 10% normal donkey serum in dilution buffer (PBS-1% bovine albumin-1% Tween 20, pH8.0), the grids were soaked in 50 ⁇ l of SP1-specific rabbit serum or control rabbit anti-MBP serum (New England BioLabs) diluted 1/25 in the dilution buffer for 2 h at room temperature.
- PBS nickel-formvar grids
- the grids were washed three times in PBS-1% Tween20, and then transferred into 50 ⁇ l of 12 nm colloidal gold-affinipure donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) diluted 1/30 in the dilution buffer and incubated for 1 h at room temperature. After three washes with PBS-1% tween20 and one wash with distilled water, bacteria were stained with 1% phosphotungstic acid and examined with an electron microscope (Philips 201) at an accelerating voltage of 60 kV.
- the pigs were injected intramuscularly twice at a 3-week interval with 1 ml of either 100 ⁇ g purified SP1 mixed with 30% Emulsigen-Plus (MVP Laboratories, Ralston, Nebr.) adjuvant or 30% Emulsigen-Plus in physiological saline as a control. Eleven days after the second injection, the immunized and control animals were challenged by aerosol of 1 ml (4.6 ⁇ 10 6 CFU) of a log-phase culture of S. suis virulent strain 166, which has been confirmed to be highly virulent (3). Blood samples were collected prior to each injection, challenge and the end of the experiment for determination of antibody responses. Pigs were monitored daily for clinical signs, body temperature and mortality for ten days after challenge. All pigs were examined postmortem for gross pathology and blood was cultured to detect the presence of S. suis bacteremia.
- Emulsigen-Plus MVP Laboratories, Ralston, Nebr.
- Serum SP1-specific total IgG and IgG isotypes (IgG1 and IgG2) of immunized piglets were determined by enzyme-linked immunosorbent assay (ELISA).
- Polysorb plates (Nunc-Immunoplates, Rochester, N.Y., USA) were coated overnight at 4° C. with 100 ⁇ l per well of the purified recombinant SP1 at a concentration of 0.3 ⁇ g/ml in carbonate buffer. After three washes with PBS containing 0.05% Tween20 (PBST), the plates were blocked with 5% skim milk in PBST for 1 h at 37° C.
- PBST PBS containing 0.05% Tween20
- swine sera from the control and vaccine groups were diluted 1/5000 in PBST and added to appropriate wells in duplicate at 100 ⁇ l per well. After incubation for 1 h at 37° C. and washing three times, bound antibodies were detected by incubation for 1 h at 37° C. with peroxidase-conjugated goat anti-swine IgG(H+L) antisera (Jackson Immuno Research Laboratories). For IgG1 and IgG2 detection, 1/500 diluted swine sera from vaccine group were added at 100 ⁇ l per well.
- Mouse anti-porcine IgG1 or IgG2 (Serotec, Kidlington, Oxford, UK) was used as the primary antibody, and peroxidase-conjugated goat anti-mouse IgG(H+L) (Serotec) was used as the secondary antibody.
- the plates were developed with TMB substrate (Zymed, S. San Francisco, USA). Absorbance was measured at 450 nm in an ELISA reader (Power Wave 340, Bio-Tek Instruments, Inc.). Results were expressed as the means ⁇ S.D. Statistical significance was determined by Student's t test.
- Nucleotide sequence accession number The sequence of the gene encoding SP1 protein of S. suis is shown in FIG. 2 and has been assigned GenBank accession number AY864331.
- the S. suis chromosomal library was constructed from the S. suis S735 strain in ⁇ ZAPII and screened using convalescent swine sera from S. suis serotype 2 infected animals.
- One clone which expressed a protein with an apparent molecular weight (MW) of 110 kDa that was strongly reactive against the convalescent swine serum, was selected for further characterization.
- the recombinant pBluescript plasmid, designated pSS735 was excised from the bacteriophage arms, and its schematic organization is presented in FIG. 1 .
- DNA sequence analysis of the 6057-bp insert of the pSS735 revealed four ORFs.
- This gene cluster was found in the partially sequenced genomes of S. suis Canadian strain 89/1591 (NZ_AAFA00000000) and European strain P1/7 (NC — 004549) with the same organization.
- the deduced amino acid sequences of both ORF1 and ORF2 showed identities ranging from 50-80% with a glycosyl transferase, and ORF4 showed identities ranging from 50-75% with a catabolite control protein A from many bacterial species, most of them belong to the genus Streptococcus.
- the ORF3 encodes a 670 amino acid protein, designed SP1, with a predicted pl of 6.0 and a calculated molecular mass of 74.8 kDa.
- SP1 is a novel C-terminal-anchored surface protein of S. suis.
- the 2010 bp of sp1 gene starts with an ATG codon which is preceded by putative Shine-Dalgarno sequence (GAAAGGA) 10 bp upstream of the start codon and terminates with a TAA codon ( FIG. 2 ).
- GAAAGGA putative Shine-Dalgarno sequence
- Analysis of the predicted SP1 amino acid sequence revealed a hydrophobic core of 15 amino acids at the N-terminus and a putative signal-peptidase cleavage site between Ala 29 and Gln 30 .
- the fusion protein was purified by using affinity matrix amylose column and eluting with maltose, and showed a single protein band of approximate 150 kDa on SDS-PAGE (lane 4).
- the purified fusion protein was proteolytically cleaved with Factor Xa, yielding the apparent 110 kDa of mature SP1 and the expected 45 kDa of MBP tag (lane 5).
- the mature SP1 devoid of MBP was obtained by subsequent purification with ion-exchange chromatography, with a purity>95% estimated by SDS-PAGE (lane 6).
- Both the MBP-SP1 fusion protein and the purified recombinant SP1 demonstrated specific reactivity in a western blot to the convalescent swine serum used for the initial screening of the genomic library ( FIG. 4B ).
- Identity of the purified SP1 was confirmed by N-terminal protein sequencing. The protein concentration was measured with Bradford protein assay and adjusted to 1 mg/ml.
- IgG isotypes demonstrated that sera from immunized pigs contained both IgG1 and IgG2 antibodies ( FIG. 6B ). However, IgG1 response dominated over IgG2, suggesting that vaccination with SP1 mainly induced the Th2-like immune response. Aerosol challenge of the pigs with S. suis 166 strain resulted in steady increases of clinical score starting from day 2 after the challenge and there was no significant effect of the vaccination. As summarized in Table 2, although fewer pigs suffered arthritis in the vaccinated group than in the control group, both groups showed similar symptoms after challenge. Three pigs from each group died or were euthanized due to high clinical scores prior to the end of the experiment. S. suis bacteremia was found in all dead pigs and was not detected in the surviving pigs.
- Emulsigen-Plus was used as an adjuvant in this study, because it was believed to be capable of creating an antigen depot at the site of inoculation from which the antigen is slowly released and thus providing prolonged stimulation to the immune system (23, 37).
- vaccine formulated with Emulsigen alone triggered predominantly an IgG1 response but very weak Th1-type immune response (19, 28).
- Th1-directing adjuvants such as CpG and interleukin-12 (IL-12) (4, 22, 24).
- IL-12 interleukin-12
- SP1 is a novel C-terminal-anchored surface protein of S. suis, as demonstrated by analysis of the molecular features and electron microscopy. Vaccination with the recombinant SP1 elicited significant humoral antibody response in piglets, along with the fact that convalescent swine sera present high titers of antibody against this protein, suggesting that SP1 is an exposed antigen of S. suis. Taken together with its wide distribution in different S. suis serotypes, these findings made the SP1 a candidate for consideration in the development of a subunit vaccine. The potential of SP1 as a vaccine candidate will be demonstrated in the following Examples.
- This study is to evaluate whether the SP1 recombinant protein is protective as a subunit vaccine candidate in a mouse model with a modified immunization route and adjuvant.
- EXPERIMENTAL PROCEDURE Mice (CD1) were randomly assigned to two groups of ten, and immunized subcutaneously twice at 2-week interval with either 20 ⁇ g of purified SP1 mixed with 20 ⁇ g of Quil A as a adjuvant or 20 ⁇ g of Quil A only as a control (Table 1).
- Ten days after the second vaccination the animals were challenged i.p. with 1 ⁇ 10 8 CFU of a S. suis virulent strain (31533). The mice were monitored twice a day for clinical signs and mortality until day 14 after the infection. Blood samples were collected prior to each vaccination and challenge for determining antibody responses.
- mice in control group Sixteen hours after administering the challenge infection, all mice in control group started to exhibit clinical signs (septicemia), such as the ruffled hair coat (suggesting fever) and slow response to stimuli. Starting from day 4 after the challenge, 8 of 10 mice in this group successively developed severe central nervous system symptoms (meningitis) such as running in circles and opisthotonos. All of the 8 ill mice died, or had to be euthanized due to the severity of the condition. In contrast, except for 6 of 10 mice in SP1-vaccinated group had transient clinical signs such as slight rough hair and reluctant to move during 16-40 hours after the challenge, all mice in this group remained healthy during the observation period ( FIGS. 3 and 4 ).
- IgG2a has been shown to be the most effective at activating opsonophagocytic function of leukocytes (2, 42, 43)).
- S. suis an encapsulated bacterial, is most effectively eliminated by opsonophagocytosis.
- IgG2 production contributed most to the observed protection.
- Recombinant SP2 strongly reacted with a convalescent swine serum collected from pigs clinically infected by S. suis type 2. Immunization of mice with the purified recombinant SP2 elicits a significant antibody response that conferred a partial protection against challenge infection with a virulent S. suis strain.
- a positive phage which reacted by non immune mechanism with different classes and species of Ig was identified by screening the constructed S. suis serotype 2 genomic library. Sequence of the DNA insert revealed a 6.3 kb insert which contains three ORFs coding for dehydrogenases, SP2 and dextran glycosidases (44), respectively ( FIG. 12 ). This gene cluster was found in the partially sequenced genomes of S. suis Canadian strain 89/1591 (NZ_AAFA00000000) and European strain P1/7 (NC — 004549) with the same organization. The SP2 amino acid sequence presented similarities with some streptococcal proteins usually exhibiting Ig-binding activity.
- AAL00677 An identity of 45% in a 395-amino acid stretch was observed with a conserveed hypothetical protein of Streptococcus pneumoniae (AAL00677). Other homologies were found with a putative 42 kDa protein of Streptococcus pyogenes (45% identity over 388-amino acid stretch) (AAK33481) and with a group B streptococcal surface immunogenic protein (40% identity over 434-amino acid stretch) (60) (AAG 18474).
- the 1158 bp SP2 gene encodes a 386-aa SP2 protein, with a theoretical pl of 4.40 and molecular mass of 42.5 kDa. This protein was rich in valine (15%), glutamic acid (10%), and alanine (9%).
- Charge distribution analysis of SP2 revealed one positive charge cluster (K 2 -K 26 ) at the N-terminus and one negative charge cluster (D 168 -E 242 ) in middle of the protein ( FIG. 13 ).
- the positive charge cluster was followed by a putative signal sequence of 23 amino acids.
- the amino acid sequence of SP2 contains a LysM (lysine) motif at positions 71 through 109.
- This LysM domain is found in a variety of enzymes involved in bacterial cell wall degradation and has a general peptidoglycan binding function, suggesting that SP2 may be a surface protein of S. suis.
- the N-terminal constitution of SP2 outlined a possibility that the positive charge cluster remained in the cytoplasm functions as a temporary stop and helps in formation of mature SP2 by cleaving the signal sequence and in location of SP2 on the bacterial surface via binding of LysM domain to peptidoglycan.
- six identical repeating sequences of 13 amino acids were identified in the middle part of SP2 ( FIG. 13 ).
- PCR were performed using primers covering the full-length SP2 gene. PCR was performed with an initial denaturing at 94° C. for 5 min followed by 30-cycles of 1 min at 94° C., 1 min at 52° C. and 2 min at 72° C., and a final elongation period of 10 min at 72° C.
- the forward and reverse primers used for SP2 distribution in different serotypes were respectively:
- SP2 gene was amplified from 31 of the 33 serotype reference strains with some size variations ( FIG. 14 ). Sequence analysis of selected variant fragments suggested that the number of repeats in the SP2 gene is responsible for the size variations.
- the gene coding for mature SP2 was generated by PCR from S. suis S735 chromosome and subcloned to a pET32+vector (New England BioLabs). The construct was used to transform E. coli DE3 strain by electroporation with Genepulse II apparatus (Bio-Rad) following the manufacturer's recommendations. For over-expression, the culture was inoculated from an overnight culture with its starting OD 600 adjusted to 0.1. The culture was incubated with agitation until OD 600 of approximately 0.8, and then IPTG (0.5 mM) was added in order to induce production of the Trx-His-SP2 fusion protein. After 2 hours of the induction, bacterial cytoplasm were prepared and used for purification of the SP2 protein.
- Trx-His-SP2 fusion protein was purified from the cytoplasm by affinity chromatography using Ni+ column (Amersham Pharmacia Biotech, Baie d'Urfee, Canada). The cytoplasm was filtered with a 0.45 ⁇ m membrane and subjected to the column. The fusion protein was eluted with 500 mM imidazole in binding buffer and protein-containing fractions were determined by SDS-PAGE. The purified fusion protein was cleaved by 0.001% (w/w) of enterokinase (New England BioLabs), yielding an apparent 58 kDa SP2 and the expected 20 kDa Trx-His tag ( FIG.
- SP2 is an Immunogenic Protein of S. suis and Exhibits IgG-Binding Activity
- SP2-specific antibody was generated by immunizing New Zealand White rabbits intramuscularly with 100 ⁇ g of recombinant SP2 protein emulsified with 0.5 ml of Freud's incomplete adjuvant. The rabbits received two booster injections with the same dose of the SP2 at 2-week intervals and then were bled 10 days after the last booster immunization. The SP2 specific antibody conversely recognized SP2 in S. suis cell preparation in a western blot ( FIG. 16a ). Moreover, recombinant SP2 reacted with a convalescent swine serum ( FIG. 16b ), demonstrating that the anti-SP2 antibody exists in the serum of pigs clinically infected by S. suis.
- mice were randomly assigned to two groups of eleven (vaccine group) and ten (control), and immunized subcutaneously twice at 2-week interval with either 50 ⁇ g of purified SP2 mixed with 20 ⁇ g of Quil A as a Th1 inducing adjuvant or 20 ⁇ g of Quil A only as a control.
- the animals were challenged i.p. with 1 ⁇ 10 8 CFU of a S. suis virulent strain (31533). The mice were monitored twice a day for clinical signs and mortality until day 14 after the infection. Blood samples were collected prior to each vaccination and challenge for determining antibody responses.
- SP2 is a new described S. suis immunogenic protein which shares little identity with other known sequences. Convalescent swine sera present antibody against this protein, demonstrating that SP2 is a potent antigen that is expressed during S. suis infection.
- a total of 24 crossbred piglets from S. suis disease-free herd (H & M Fast Farms Inc.) without any previous vaccination against S. suis were used.
- the pigs were kept under commercial conditions at the herd of origin from birth until they were weaned at an average weight of 7.79 kg at 23.5 days of age.
- Pigs were housed with controlled temperature (27 to 30° C.) and ventilation, on vinyl-covered metal flooring, and were provided with water via nipple waterers and had free access to commercially-prepared, nutritionally balanced, antibiotic-free feed.
- a veterinarian examined the pigs prior to the beginning of the study. All were healthy. At weaning, the piglets were randomly assigned to two groups, balanced by body weight.
- the pigs were anesthetized with halothane and challenged by aerosol of 1 ml of a suspension of S. suis 166.
- the bacteria were from a log-phase culture grown in filter sterilized Todd-Hewitt Yeast Broth to an OD 620 of 0.8 and diluted 1:100 in saline (0.85% NaCl).
- the bacterial concentration administered to pigs was later measured to be 6.8 ⁇ 10 6 CFU/ml.
- a veterinarian or trained animal care technician clinically evaluated the pigs once daily and measured body temperatures during assignment of clinical scores each morning for ten days after challenge.
- a daily clinical score (from 0 to 4) was derived as the sum of attitude and locomotion scores for each animal based upon signs of nervous, musculoskeletal or respiratory disease as follows:
- Pigs having a clinical score greater than 2 on either scale were euthanized by lethal injection. Pigs with rectal temperatures equal to or greater than 40.6° C. and a clinical score greater than 0, as well as those pigs that were dead, were recorded as sick on that day. Pigs that died or were euthanized prior to the end of the experiment on day 9 were recorded as dead for evaluation of the effect of treatment on mortality rate. All individuals making judgements about animals, evaluating clinical signs of disease, or performing laboratory assays were blind to the identity of the treatment.
- a heparin-treated blood sample was obtained by venipuncture for detection of S. suis bacteremia (by culture on days 0 and 3 after challenge and postmortem).
- IgG1 and IgG2 Titers of Sao-specific total IgG and IgG subclasses (IgG1 and IgG2) in sera were determined by ELISA.
- All pigs were examined postmortem and the following tissues were cultured for bacteria: cerebellum swab, tracheobronchial lymph node, a joint swab (an affected joint if lesions are present; otherwise a stifle joint), and blood.
- cerebellum swab cerebellum swab
- tracheobronchial lymph node a joint swab (an affected joint if lesions are present; otherwise a stifle joint)
- blood The number of S. suis bacteria that were recovered was recorded on an ordinal scale from 0 to 4 (approximating the log 10 number of colonies).
- the extent (percentage) of pulmonary involvement was estimated by visual examination.
- the significance of differences between groups in nominal data was determined using contingency table analysis and Likelihood-Ratio Fisher Exact Test.
- the significance of differences between groups in ordinal data was transformed by ranking and determined by t-test.
- the significance of differences between groups in survival curves was determined by survival analysis using the logrank test (equivalent to the Mantel-Haenszel test).
- the significance of differences among groups in continuous data was determined using t-test (after appropriate transformation to normality as required).
- the vaccine was shown to be safe since pigs that were vaccinated twice did not have any adverse reaction Immunization of pigs with Sao in combination with Quil A elicited significant IgG titres with a dominant IgG2 production, suggesting a predominant Th1-type immune response. Aerosol challenge of pigs resulted in disease with an overall mortality rate of approximately 58% in controls. The survival of vaccinated pigs after challenge was significantly better than controls (p ⁇ 0.05). Some pigs in each group became ill after challenge, and there was significantly less disease (lower clinical score) in the vaccinated pigs.
- Vaccination had no significant effect on the occurrence of gross pathology post-mortem; however acute streptococcal septicaemia can be fatal without appreciable gross signs of pathology. Less S. suis bacteria were recovered from vaccinated pigs than control pigs post mortem (p ⁇ 0.01).
- mice Three groups of 10 mice were immunized two times (Day 1 and Day 17) via i.p. with 40 ⁇ g of purified SP1A-maltose-binding protein (MBP) fusion protein, 20 ⁇ g of MBP or only PBS, using Freund Incomplete as an adjuvant.
- MBP purified SP1A-maltose-binding protein
- the sera were obtained before each immunization or 10 days after the second injection, and were 1:5000 diluted for ELISA assay. (See Table 5)
- Three groups of 3 pigs were immunized two times (Day 1 and Day 17) via i.m. with 200 ⁇ g of purified SP1A-MBP fusion protein, 100 ⁇ g of MBP or only PBS, using Emulsigen as an adjuvant.
- the sera were obtained before each immunization or 10 days after the second injection, and were 1:5000 diluted for ELISA assay. (See Table 6)
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CN101613399B (zh) * | 2008-06-25 | 2012-11-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种猪链球菌2型表面蛋白、其制备方法及用途 |
CN101613400B (zh) * | 2008-06-25 | 2012-11-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种猪链球菌2型表面细胞壁蛋白、其制备方法及用途 |
TWI410251B (zh) * | 2009-02-17 | 2013-10-01 | Univ Nat Pingtung Sci & Tech | 疫苗 |
EP2949340A1 (en) * | 2014-05-30 | 2015-12-02 | IDT Biologika GmbH | Vaccine composition against Streptococcus suis infection |
CN106995489B (zh) * | 2016-01-22 | 2018-11-06 | 华中农业大学 | 一种猪链球菌截短蛋白Sao及应用 |
CN107164273B (zh) * | 2017-06-12 | 2018-07-06 | 广东海大畜牧兽医研究院有限公司 | 一种免疫原性强的血清2型猪链球菌及其应用 |
US10968258B2 (en) | 2017-08-31 | 2021-04-06 | Boehringer Ingelheim Animal Health USA Inc. | Streptococcus suis vaccines to protect against reproductive, nursery-age, and growing pig diseases and methods of making and use thereof |
CN110646335A (zh) * | 2019-09-29 | 2020-01-03 | 广东工业大学 | 一种封闭液及其应用 |
CN112521458B (zh) * | 2019-12-27 | 2022-06-21 | 无锡市妇幼保健院 | 用于检测S.suis 2感染的合成肽Sao355~372及其应用 |
CN116199751B (zh) * | 2023-03-13 | 2024-03-19 | 华中农业大学 | 一种细菌全基因组水平高通量筛选免疫原性抗原蛋白的方法 |
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USRE47164E1 (en) * | 2005-09-02 | 2018-12-18 | Valorisation-Recherche Limited Partnership | Streptococcus suis polypeptides and polynucleotides encoding same and their use in vaccinal and diagnostic applications |
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