# Patent

## Title
P97 protein and uses thereof as vaccine adjuvant

## Abstract

Immunogenic compositions comprising a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, and a heterologous antigen are disclosed. Uses of the p97 adhesin adjuvant polypeptide or nucleic acid, or immunogenic compositions comprising same, for inducing an immune response against a heterologous antigen in a subject are also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. provisional application Ser. No. 61/639,127, and Canadian application No. 2,776,119, both filed Apr. 27, 2012, which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to vaccines, and more particularly to vaccine adjuvants that potentiate the immune response against antigens.
REFERENCE TO SEQUENCE LISTING
Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named “782 16045.2 ST25.txt”, that was created on Nov. 13, 2014 and having a size of  ̃140 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Vaccines are considered to be one of the most successful and cost-effective medical interventions against infectious diseases (Hilleman M. R., Vaccine 18: 1436-1447, 2000). A vaccine is used to evoke an antigen-specific effectors and memory immune response against a pathogen, with minimal adverse reactions and it should lead to a specific long-term protection against this pathogen.
Traditional live anti-viral and anti-bacterial vaccines typically require no immunological adjuvants. Similarly, live microbial attenuated vaccines are generally much more immunogenic than killed pathogen or subunit protein vaccines and can be effective with no adjuvant or with adjuvants that have limited ability to stimulate immune responses. Recently developed killed pathogens, several vector types or subunit protein vaccines, while offering significant advantages over the traditional vaccines in terms of safety and cost of production, generally have limited immunogenicity compared to natural pathogens. As a result, these vaccines typically require adjuvants with significant immunostimulatory capability to reach their full potential in preventing disease.
A vaccine adjuvant is more precisely a particulate, solid or soluble agent that increases the specific immune responses to an antigen. Vaccine adjuvants can enhance the immune response to vaccine antigens in various ways. When weak antigens are available, they are very useful for augmenting the immunogenicity of these molecules, thereby enhancing their vaccinal potency. They are also used to enhance the speed, vigor, and persistence of the immune response to a strong antigen. They can also modify the nature of the immune response. Depending on which adjuvant is used to stimulate a protective immune response, humoral or cell-mediated immunity can be selected. An adjuvant can modulate antibody specificity, as well as its quantity, isotype and subclass distribution. When used in direct contact with mucous membrane (e.g., intranasal) it can effectively induce mucosal immunity. Adjuvants are also useful for potentiating the immune responses in immunologically immature, immunosuppressed or senescent individuals, acting as an immunological booster. Also, an adjuvant can effectively decrease the dose of antigen and/or the frequency of injection necessary to provide protection.
Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Some of these adjuvants are toxic, however, and can cause undesirable side effects, making them unsuitable for use in humans and many animals. Indeed, only few adjuvants are routinely used in human and veterinary vaccines. Also, currently available adjuvants and vaccines fail to induce a proper immune response capable of protecting against or treating certain infectious diseases, for instance those associated to Human immunodeficiency virus (HIV) or Hepatitis C virus (HCV).
Therefore, there is a need for the development of novel vaccine adjuvants and immunogenic compositions.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
More specifically, in accordance with the present invention, there is provided an immunogenic composition comprising a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, and a heterologous antigen.
In another aspect, the present invention provides the use of an immunogenic composition comprising a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, and a heterologous antigen, for inducing an immune response against said heterologous antigen in a subject.
In another aspect, the present invention provides the use of an immunogenic composition comprising a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, and a heterologous antigen, for the preparation of a medicament for inducing an immune response against said heterologous antigen in a subject.
In another aspect, the present invention provides a method of inducing an immune response against a heterologous antigen in a subject, the method comprising administering to said subject an effective amount of an immunogenic composition comprising a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, and said heterologous antigen.
In another aspect, the present invention provides the use of a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, as an adjuvant for a vaccine.
In another aspect, the present invention provides the use of a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, for the preparation of a vaccine.
In an embodiment, the above-mentioned p97 adhesin adjuvant polypeptide is (i) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7; (ii) a polypeptide comprising a sequence that is at least 60% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 7 and having adjuvant properties; or (iii) a fragment of (i) or (ii) having adjuvant properties.
In a further embodiment, the above-mentioned p97 adhesin adjuvant polypeptide is (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 7; (ii) a polypeptide comprising a sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1 or 7 and having adjuvant properties; or (iii) a fragment of (i) or (ii) having adjuvant properties.
In a further embodiment, the above-mentioned p97 adhesin adjuvant polypeptide comprises a sequence corresponding to residues 534 to 1093 of the polypeptide of SEQ ID NO: 7.
In another embodiment, the above-mentioned p97 adhesin adjuvant polypeptide comprises a sequence corresponding to residues 899 to 1108 of the polypeptide of SEQ ID NO: 1.
In an embodiment, the above-mentioned heterologous antigen is an antigen from a human pathogen or an antigen of human origin.
In an embodiment, the above-mentioned heterologous antigen is a polypeptide.
In an embodiment, the above-mentioned p97 adhesin adjuvant polypeptide and the heterologous antigen polypeptide are linked together. In a further embodiment, the above-mentioned p97 adhesin adjuvant polypeptide is N-terminal relative to the heterologous antigen polypeptide.
In an embodiment, the above-mentioned immunogenic composition or vaccine further comprises one or more pharmaceutically acceptable excipients.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
FIGS. 1A to 1E show the expression of recombinant adenoviruses (rAdVs) in vitro. Western Blot analysis of A549 cells lysates mock-infected or infected with rAdVs (MOI:10) expressing GFP, M-GP5 (FIG. 1A), Cap-GP5 (FIG. 1B), P97c-GP5 (FIG. 1C), Cap (FIG. 1D), P97c (FIG. 1E) or P97c and Cap (FIGS. 1D and 1E). Immunoblot was done with a convalescent Porcine reproductive and respiratory syndrome virus (PRRSV)-specific pig antiserum (1:5,000, FIG. 1A), a convalescent Porcine circovirus type 2b (PCV2b)-specific pig antiserum (1:10,000, FIGS. 1B and 1D) or mouse monoclonal Mycoplasma hyopneumoniae anti-P97c antibody (1:5,000, FIGS. 1C and 1E), as primary antibodies, and an anti-pig IgG-HRP (FIGS. 1A, 1B and 1D) or anti-mouse IgG-HRP (FIGS. 1C and 1E) as secondary antibody (1:10,000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunostaining was used as a loading control.
FIGS. 2A to 2C show the antibody responses in sera of mice vaccinated with the recombinant adenoviruses expressing M-GP5, Cap-GP5 or P97c-GP5. IgGs specific to GP5 (FIG. 2A), P97c (FIG. 2B) or Cap (FIG. 2C) were detected in serum samples of mice (n=5 per group) at various time points by indirect ELISA. Data are expressed as mean OD of three independent ELISA tests.
FIGS. 3A and 3B show the antibody responses against P97c in sera of mice vaccinated with the recombinant adenoviruses. FIG. 3A: IgG specific to P97c were detected in sera sample of mice (n=6) at various time points by indirect ELISA in triplicate. Data are shown as mean+SD. FIG. 3B: Serum P97c-specific antibody isotype production in mice (n=6) at 56 days post primary immunization determined by indirect ELISA. Data are shown as mean OD+SEM. When 2 sets of data are labeled with superscripts of different letters it indicates that these sets of data are statistically different (P<0.05).
FIGS. 4A and 4B show the antibody responses against Cap in sera of mice vaccinated with the recombinant adenoviruses. FIG. 4A: IgG specific to Cap were detected in sera sample of mice (n=6) at various time points by indirect ELISA in triplicate. Data are shown as mean+SD. FIG. 4B: Serum Cap-specific antibody isotype production in mice (n=6) at 56 days post primary immunization determined by indirect ELISA. Data are shown as mean+SEM. When 2 sets of data are labeled with superscripts of different letters it indicates that these sets of data are statistically different (P<0.05) When 2 sets of data are labeled with superscripts of different letters it indicates that these sets of data are statistically different (P<0.05);
FIG. 5 shows the mean serum neutralizing antibody (Nab) titer of mice immunized with recombinant adenoviruses (rAdVs). Mice were immunized at days 0 and 14 with rAdVs expressing GFP, Cap, P97c-Cap or Cap-P97c. Neutralizing antibody titers were detected in sera sample of mice (n=6) at 56 dpi. The neutralizing antibody titers are expressed as the reciprocal of endpoint sera dilution that inhibits more than 50% of the PCV2b replication, and are expressed as log2 mean titer+SD. When 2 sets of data are labeled with superscripts of different letters, it indicates that these sets of data are statistically different (P<0.05);
FIGS. 6A to 6G show the amino acid sequences of Mycoplasma hyopneumoniae strain 232 p97 protein (FIG. 6A, SEQ ID NO:1), a p146 adhesin like-protein p97 paralog (FIG. 6B, SEQ ID NO:2), a p102 paralog (FIG. 6C, NCBI Reference Sequence: YP—115695.1, SEQ ID NO:3), a p97 cilium adhesin paralog (FIG. 6D, SEQ ID NO:4), a p159 paralog (FIG. 6E, SEQ ID NO:5), a p216 paralog (FIG. 6F, SEQ ID NO:6) and Mycoplasma hyopneumoniae strain ATCC 25934 p97 (FIG. 6G, SEQ ID NO:7);
FIGS. 7A to 7G show nucleotide sequences encoding the Mycoplasma hyopneumoniae strain 232 p97 (FIG. 7A, SEQ ID NO:10), the p146 adhesin like-protein p97 paralog (FIG. 7B, SEQ ID NO:11), the p102 paralog (FIG. 7C, NCBI Reference Sequence: YP—115695.1, SEQ ID NO:12), the p97 cilium adhesin paralog (FIG. 7D, SEQ ID NO:13), the P159 paralog (FIG. 7E, SEQ ID NO:14), the p216 paralog (FIG. 7F, SEQ ID NO:15) and the Mycoplasma hyopneumoniae strain ATCC 25934 p97 (FIG. 7G, SEQ ID NO:16) of FIGS. 6A to 6G;
FIGS. 8A and 8B show the nucleotide (SEQ ID NO:8) and amino acid (SEQ ID NO:9) sequences, respectively, of the C-terminal portion of the P97 protein from Mycoplasma hyopneumoniae strain 232. The nucleotides encoding the R1 domain (FIG. 8A), and the residues corresponding to the R1 domain (FIG. 8B), are in bold and italics. The nucleotides encoding the R2 domain (FIG. 8A), and the residues corresponding to the R2 domain (FIG. 8B), are underlined;
FIGS. 9A and 9B show the nucleotide (SEQ ID NO:18) and amino acid (SEQ ID NO:19) sequences, respectively, of the rGP5 (PRRSV IAF-Klop, Genbank accession No. U64928) used in the experiments described herein;
FIGS. 10A and 10B show the nucleotide (SEQ ID NO:20) and amino acid (SEQ ID NO:21) sequences, respectively, of the rCAP(PCV2b, strain FMV-06-1717, nucleotides 120 to 708 and amino acids 40 to 236) used in the experiments described herein;
FIGS. 11A and 11B show the nucleotide (SEQ ID NO:22) and amino acid (SEQ ID NO:23) sequences, respectively, of the rP97c (M. hyopneumoniae, strain 25934, nucleotides 1603 to 3279 and amino acids 534 to 1093) used in the experiments described herein;



DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Terms and symbols of genetics, molecular biology, biochemistry and nucleic acid used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. All terms are to be understood with their typical meanings established in the relevant art.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
The p97 adhesin adjuvant polypeptide, or nucleic acid encoding same, of the present invention can be used to enhance the immunogenicity of a wide variety of antigens including, but not limited to, antigenic lipids, polypeptides, polysaccharides and polynucleotides that encode antigenic polypeptides. The adjuvant of the present invention can further be used in combination with other adjuvant formulations to further enhance the immunogenicity. Accordingly, the p97 adhesin adjuvant polypeptide and/or the p97-encoding nucleic acid is/are incorporated into a composition, e.g., an immunogenic, a vaccine or an immunomodulatory composition, together with an antigen, such as a heterologous antigen. In another aspect, the present invention provides the use of a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, as an adjuvant for a vaccine (immunogenic composition). In another aspect, the present invention provides the use of a p97 adhesin adjuvant polypeptide, or a nucleic acid encoding said p97 adhesin adjuvant polypeptide, as an adjuvant for the preparation of a vaccine. In an embodiment, the vaccine further comprises an antigen, in a further embodiment a heterologous antigen.
In another aspect, the present invention provides an immunogenic, a vaccine or an immunomodulatory composition comprising a fusion construct, the fusion construct comprising a p97 adhesin adjuvant polypeptide covalently linked to an antigen, or a nucleic acid encoding said fusion construct.
“p97 adhesin adjuvant polypeptide” refers to a polypeptide comprising a region of the Mycoplasma hyopneumoniae strain 232 or strain 25934 p97 polypeptide (FIG. 7A or 7G, SEQ ID NO: 1 or 7), or of a paralog thereof such as the p146 adhesin like-protein p97 paralog (FIG. 7B, SEQ ID NO: 2), the p102 paralog (FIG. 7C, SEQ ID NO: 3) the p97 cilium adhesin paralog (FIG. 7D, SEQ ID NO: 4), the P159 paralog (FIG. 7E, SEQ ID NO: 5), or the p216 paralog (FIG. 7F, SEQ ID NO: 6), or a variant thereof having adjuvant/immunostimulatory activity. In an embodiment, the Mycoplasma hyopneumoniae is Mycoplasma hyopneumoniae strain 232 or 25934. “p97-encoding nucleic acid” refers to a nucleic acid comprising a nucleotide sequence encoding the above-mentioned p97 adhesin adjuvant polypeptide.
The P97 adhesin protein is a proteolytically processed protein encoded by mhp183, the first gene in a two gene operon with the gene encoding the p102 paralog. It is expressed as a 125 kDa protein that undergoes a post-translational cleavage to yield the functional 97 kDa p97 protein, and is involved in the adherence of M. hyopneumoniae to the host respiratory cilia. It contains a 17 amino acid N-terminal hydrophobic region. It also contains two functional repeats designated R1 and R2. R1 is a domain comprising several repeats of the amino acid motif A(T)-A(T)-K-P-E(V) (SEQ ID NO: 24) (corresponding to residues 814-888 in FIG. 7A, underlined, or residues 814-858 in FIG. 7G), and is involved in the binding to cilia. The R2 region is a C-terminal domain comprising repeats (typically 3 to 5) of the amino acid motif G-A(E,S,T)-P-N(S)-Q-G-K-K-A-E (SEQ ID NO: 25) (corresponding to residues 991-1020 in FIG. 7A, italicized, or residues 955-1004 in FIG. 7G).
In an embodiment, the p97 adhesin adjuvant polypeptide comprising a region of a M. hyopneumoniae p97 polypeptide or the p102 paralog, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In a further embodiment, the p97 adhesin adjuvant polypeptide comprises a region of a M. hyopneumoniae p97 polypeptide, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In an embodiment, the p97 adhesin adjuvant polypeptide comprises the R1 region of a p97 adhesin protein or a paralog thereof. In another embodiment, the p97 adhesin adjuvant polypeptide comprises the R2 region of a p97 adhesin protein or a paralog thereof. In another embodiment, the p97 adhesin adjuvant polypeptide comprises the R1 and R2 regions of a p97 adhesin protein or a paralog thereof.
In embodiments, the p97 adhesin adjuvant polypeptide comprises a region or fragment of at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 amino acids of the M. hyopneumoniae p97 polypeptide or paralog thereof, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In an embodiment, the region is located in the C-terminal portion of the M. hyopneumoniae p97 polypeptide, e.g. about the last 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 amino acids. In another embodiment, the p97 adhesin adjuvant polypeptide comprises the full sequence of the M. hyopneumoniae p97 polypeptide or paralog thereof, or a variant thereof having adjuvant/immunostimulatory activity. In another embodiment, the p97 adhesin adjuvant polypeptide comprises the full sequence of the M. hyopneumoniae p97 polypeptide or paralog thereof, but lacking at least the N-terminal hydrophobic region, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In another embodiment, the p97 adhesin adjuvant polypeptide comprises the amino acid sequence of SEQ ID NO: 23 (FIG. 11B). In another embodiment, the p97 adhesin adjuvant polypeptide comprises residues corresponding to about residues 799 to 1108 of the Mycoplasma hyopneumoniae p97 polypeptide of FIG. 7A (SEQ ID NO: 1). Amino acid numbering for p97 adhesin adjuvant polypeptides described herein uses numbering based on the reference M. hyopneumoniae p97 polypeptide of FIG. 7A, with residue 1 corresponding to the first methionine in this sequence. It will be understood that amino acid numbering can thus be shifted in situations where the residues corresponding to those referred to herein are within a polypeptide having more or fewer amino acids N-terminal to the region(s) where the residues reside (e.g., a different paralog), relative to the reference M. hyopneumoniae strain 232 p97 polypeptide (FIG. 7A), thereby resulting in different amino acid numbering. The corresponding positions in other paralogs may be easily identified, for example by aligning the sequence of a given paralog polypeptide with that depicted in FIG. 7A (e.g., using software/tools for sequence alignment such as Clustal W or EMBOSS Needle).
“Variant” as used herein refers to a p97 adhesin adjuvant polypeptide in which one or more of the amino acids of the native hyopneumoniae p97 polypeptide or paralog thereof has/have been modified, but which retains adjuvant, immunostimulatory and/or immunopotentiating activity. The modification may be, for example, a deletion of one or more consecutive or non-consecutive amino acids, a substitution of amino acids, one or more substitution(s) of a naturally occurring amino acid (L-amino acid) by a corresponding D-amino acid, an extension of the sequence by e.g., one, two, three or more amino acids. In an embodiment, the above-mentioned substitution(s) are conserved amino acid substitutions. As used herein, the term “conserved amino acid substitutions” (or sometimes “conservative amino acid substitutions”) refers to the substitution of one amino acid for another at a given location in the p97 adhesin adjuvant polypeptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the p97 adhesin adjuvant polypeptide by routine testing.
In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about −1.6 such as Tyr (−1.3) or Pro (−1.6) are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554,101): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (O); Pro (−0.5); Thr (−0.4); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4).
In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).
In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, H is; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.
Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cystic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.
In other embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J. Mol. Biol. 179: 125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically, encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys.
Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents.
An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held, equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile.
A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln.
An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.
The above classifications are not absolute and an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.
Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid.
In addition to the substitutions outlined above, synthetic amino acids providing similar side chain functionality can also be introduced into the p97 adhesin adjuvant polypeptide. For example, aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or L-phenylglycine, D- or L-2-thienylalanine, D- or L-1-, 2-, 3-, or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylalanine D- or L-p-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylalanines wherein the alkyl group is selected from the group consisting of substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, and iso-pentyl.
Non-carboxylate amino acids can be made to possess a negative charge, as provided by phosphono- or sulfated (e.g., —SO3H) amino acids, which are to be considered as non-limiting examples.
Other substitutions may include unnatural alkylated amino acids, made by combining an alkyl group with any natural amino acid. Basic natural amino acids such as lysine and arginine may be substituted with alkyl groups at the amine (NH2) functionality. Yet other substitutions include nitrile derivatives (e.g., containing a CN-moiety in place of the CONH2 functionality) of asparagine or glutamine, and sulfoxide derivative of methionine. In addition, any amide linkage in the peptide may be replaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide, thioamide or reversed amide moieties, (e.g., (—C═O)—CH2—), (—CHOH)—CH2—), (CH2—CH2—), (—C═S)—NH—), or (—NH—(—C═O) for (—C═O)—NH—)).
Other modifications are also included within the definition of variant of the p97 adhesin adjuvant polypeptide of the present invention. For example, the size of the p97 adhesin adjuvant polypeptide can be reduced by deleting one or more amino acids, and/or amino acid mimetics or dipeptide mimics containing non-peptide bonds may be used. Examples of using molecular scaffolds such as benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, β-turn dipeptide cores and β-aminoalcohols for these purposes are known to peptide chemists and are described in for example Peptidomimetic protocols (Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 & 2, A. Abell (Ed).
Covalent modifications of the p97 adhesin adjuvant polypeptide are thus included within the scope of the present invention. Such modifications may be introduced into the p97 adhesin adjuvant polypeptide for example by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation.
Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters, e.g. methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues is typically performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosinyl residues per se is well-known, such as for introducing spectral labels into tyrosinyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidazole and tetranitromethane may be used to form O-acetyl tyrosinyl species and 3-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues may be frequently deaminated to the corresponding glutamyl and aspartyl residues. Other modifications of the peptides in the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.
Covalent attachment of fatty acids (e.g., C6-C18) to the peptides may confer additional biological properties such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc. The above description of modification of the p97 adhesin adjuvant polypeptide does not limit the scope of the approaches nor the possible modifications that can be engineered.
In an embodiment, the variant and/or fragment has an identity or similarity of at least 60% with a native M. hyopneumoniae p97 polypeptide or a paralog thereof, or with a fragment of the M. hyopneumoniae p97 polypeptide or a paralog thereof, and retain adjuvant, immunostimulatory and/or immunopotentiating activity. In further embodiments, the variant and/or fragment has a similarity or identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with a native M. hyopneumoniae p97 polypeptide or a paralog thereof, or with a fragment of the M. hyopneumoniae p97 polypeptide or a paralog thereof, and retain adjuvant, immunostimulatory and/or immunopotentiating activity. In other embodiments, the variant and/or fragment has an identity or similarity of at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the native M. hyopneumoniae strain 232 or 25934 p97 polypeptide of SEQ ID NO:1 or 7, or a fragment thereof.
“Similarity” and “identity” refers to sequence similarity/identity between two polypeptide molecules. The similarity or identity can be determined by comparing each position in the aligned sequences. A degree of similarity or identity between amino acid sequences is a function of the number of matching or identical amino acids at positions shared by the sequences. As the term is used herein, an amino acid sequence is “similar” or “identical” to another sequence if the two sequences are substantially similar or identical (shares at least 60% similarity or identity) and the functional activity of the sequences is conserved. As used herein, a given percentage of similarity or identity between sequences denotes the degree of sequence identity in optimally aligned sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with a sequence described herein.
Optimal alignment of sequences for comparisons of similarity or identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence similarity or identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information web site (http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. ScL USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
In an embodiment, the adjuvant is a nucleic acid encoding the above-mentioned p97 adhesin adjuvant polypeptide, fragment thereof and/or variant thereof as defined above. In an embodiment, the nucleic acid comprises a region or fragment of the M. hyopneumoniae p97 nucleic acid (FIG. 7A, SEQ ID NO: 10, or FIG. 7G, SEQ ID NO: 16) or a p102 paralog (FIG. 7C, SEQ ID NO: 12), or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In a further embodiment, the p97 adhesin adjuvant polypeptide comprising a region of the M. hyopneumoniae p97 nucleic acid, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity.
In embodiments, the p97 adhesin adjuvant nucleic acid comprises a fragment of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500 or 3000 nucleotides of the M. hyopneumoniae p97 nucleic acid or paralog thereof, or a variant thereof having adjuvant/immunostimulatory activity. In an embodiment, the nucleic acid fragment encodes a region located in the C-terminal portion of the M. hyopneumoniae p97 polypeptide, e.g. about the last 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 amino acids. In another embodiment, the p97 adhesin adjuvant nucleic acid comprises the full sequence of the M. hyopneumoniae p97 nucleic acid or paralog thereof (FIGS. 8A to 8G), or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In another embodiment, the p97 adhesin adjuvant nucleic acid comprises the full sequence of the M. hyopneumoniae p97 nucleic acid or paralog thereof, but lacking the nucleotide sequence encoding at least the N-terminal hydrophobic region, or a variant thereof having adjuvant, immunostimulatory and/or immunopotentiating activity. In another embodiment, the p97 adhesin adjuvant nucleic acid comprises a sequence encoding residues corresponding to about residues 534 to 1093 of the Mycoplasma hyopneumoniae p97 polypeptide of FIG. 7G, SEQ ID NO: 7). In an embodiment, the nucleic acid comprises a sequence corresponding to about nucleotides 1603 to 3279 of the Mycoplasma hyopneumoniae p97 nucleic acid of FIG. 8G, SEQ ID NO: 16). In another embodiment, the p97 adhesin adjuvant nucleic acid comprises a sequence encoding residues corresponding to about residues 799 to 1108 of the Mycoplasma hyopneumoniae p97 polypeptide (FIG. 7A, SEQ ID NO: 1). In an embodiment, the nucleic acid comprises a sequence corresponding to about nucleotides 2395 to 3327 of the Mycoplasma hyopneumoniae p97 nucleic acid (FIG. 8A).
“Variant” as used herein refers to a p97 adhesin adjuvant nucleic acid in which one or more of the nucleotides of the native hyopneumoniae p97 nucleic acid or paralog thereof has/have been modified, but which retains adjuvant, immunostimulatory and/or immunopotentiating activity. The modification may be, for example, a deletion of one or more consecutive or non-consecutive nucleic acids, a substitution of one or more nucleotide(s), or an extension of the sequence by e.g., one, two, three or more nucleotide(s).
In an embodiment, the variant and/or fragment has an identity or similarity of at least 60% with a native M. hyopneumoniae p97 nucleic acid or a paralog thereof, or with a fragment of the M. hyopneumoniae p97 nucleic acid or a paralog thereof, and retain adjuvant/immunostimulatory activity. In further embodiments, the variant and/or fragment has a similarity or identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with a native M. hyopneumoniae p97 nucleic acid or a paralog thereof, or with a fragment of the M. hyopneumoniae p97 nucleic acid or a paralog thereof, and retain adjuvant, immunostimulatory and/or immunopotentiating activity. In other embodiments, the variant and/or fragment has an identity or similarity of at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the native M. hyopneumoniae p97 strain 232 or 25934 nucleic acid of SEQ ID NO:10 (FIG. 7A) or SEQ ID NO: 16 (FIG. 7G), or a fragment thereof. In an embodiment, the p97 adhesin adjuvant nucleic acid comprises the nucleotides sequences of FIG. 11A (SEQ ID NO:22). In another embodiment, the p97 adhesin adjuvant nucleic acid comprises the nucleotides sequences of FIG. 9A (SEQ ID NO:8).
By “antigen” is meant a molecule that is capable of stimulating a host's immune system to make a cellular antigen-specific immune response and/or a humoral antibody response when the antigen is presented/administered. It refers to any natural or synthetic compound or chemical entity (lipids, phospholipids, glycolipids, saccharides, nucleic acids, etc.) capable of stimulating a immune response in a host. In an embodiment, the antigen is a polypeptide (e.g., a protein or peptide derived from a pathogen or a tumor cell). A polypeptide antigen may contain one or more epitope(s). Normally, an epitope will include between about 3-15, generally about 5-15, amino acids. Epitopes of a given protein can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance (NMR). See, e.g., Epitope Mapping Protocols, supra. “Antigen” also refers to any natural or synthetic compound or chemical entity (lipids, phospholipids, glycolipids, saccharides, nucleic acids, etc.) capable of stimulating an immune response in a host.
The term “antigen” as used herein denotes both subunit antigens, i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature, as well as killed, attenuated or inactivated bacteria, viruses, parasites or other microbes, or tumor cell lysates. Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide that expresses an immunogenic protein, or antigenic determinant in vivo, such as in nucleic acid immunization applications, is also included in the definition of antigen herein. The antigenic polynucleotide can be delivered through two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid (DNA vaccine). Viral and bacterial vaccine vectors are well known in the art (see New Generation Vaccines, 3rd edition, 2004 and Vaccine Protocols, 2nd edition, Humana Press, 2003) and include, for example, Poxvirus, adenovirus, Measles virus, alphavirus, Yellow Fever virus, Semliki Forest virus, poliovirus, herpex simplex virus, vesicular stomatitis virus, Listeria monocytogenes, Salmonella and Shigella. The vaccine vector contains a polynucleotide antigen that is placed under the control of elements required for expression.
“Heterologous antigen” as used herein refers to an antigen that is derived from a species that is different from the p97 adhesin adjuvant polypeptide, i.e. that is not derived from Mycoplasma hyopneumoniae. In an embodiment, the antigen is not derived from a pathogen affecting pigs, or is not of pig origin. In an embodiment, the antigen is not derived from a pathogen affecting farm animals (pig, cow, horse, poultry, etc.), or is not of farm animal origin. In another embodiment, the antigen is not derived from a pathogen affecting non-human animals. In an embodiment, the antigen is derived from a human pathogen (e.g., a bacteria or a virus affecting humans), or is from human origin (such as a human polypeptide or a fragment thereof). In another embodiment, the antigen is not derived from PRRSV, PCV2, pseudorabies virus, swine influenza virus, Salmonella cholerasuis, Salmonella typhimurium, Erysipelothrix rhusiopathiae, Lawsonia intracellulars, Haemophilus parasuis, Bordetella bronchiseptica, Streptococcus suis, Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida, Clostridium perfringens type A and type C, bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncitial virus (BRSV), parainfluenza virus, Pasteurella multocida, Haemophilus somnus, Mycoplasma mycoides, Mycoplasma bovis, Mycoplasma agalactiae, Mycoplasma californicum, Mycoplasma bovirhinis, Mycoplasma dispar, Mycoplasma canis, or Manheimia haemolytica. 
Further, for purposes of the present invention, antigens (e.g., polypeptides or other biomolecules) can be derived from any of several known pathogens, such as viruses, bacteria, parasites and fungi, as well as any of the various tumor antigens. The antigen may also be an antigen involved in diseases or conditions for which vaccination may be useful, e.g., certain allergies and/or immune/inflammation disorders.
The immunogenic or vaccine compositions of the present invention contains an antigen capable of eliciting an immune response against a pathogen, such as an animal or human pathogen, which antigen may be derived from Human Immunodeficiency virus (HIV), such as Tat, Nef, Gag, Pol, gp120 or gp160, human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus (such as gB or derivatives thereof), Rotavirus, Epstein Barr virus (such as gp350 or derivatives thereof), Varicella Zoster Virus (such as gpl, II and IE63), or from a hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen or a derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as paramyxoviruses: Respiratory Syncytial virus (such as F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18, etc.), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenza virus (whole live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10: 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof).
Antigens can also be derived from bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PiIC, adhesins); S. pyogenes (for example M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans: H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C, Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (for example shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp., including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp., including P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani (for example tetanus toxin and derivative thereof), C. botulinum (for example botulinum toxin and derivative thereof), C. difficile (for example clostridium toxins A or B and derivatives thereof); Bacillus spp., including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC. DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp. including C. trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), C. psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum (for example the rare outer membrane proteins), T. denticola, T. hyodysenteriae; or derived from parasites such as Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leishmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; Schisostoma spp., including S. mansoni, or derived from yeast such as Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans, Streptococcus spp., including S. pneumoniae (for example capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25: 337-342), and mutant detoxified derivatives thereof (WO 90/06951; WO 99/03884), antigens derived from Haemophilus spp., including H. influenzae type B (for example PRP and conjugates thereof), non typeable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464) or multiple copy variants or fusion proteins thereof.
The vaccine composition of the present invention may also comprise a tumor antigen and be useful for the prevention or immunotherapeutic treatment of cancers. For example, the adjuvant formulation finds utility with tumor rejection antigens such as those for prostate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary antigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens, PRAME, BAGE, Lage (also known as NY-Eos-1) SAGE and HAGE or GAGE. Indeed these antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma and bladder carcinoma.
Other tumor-specific antigens are suitable for use in the vaccine composition of the present invention and include, but are not restricted to tumor-specific gangliosides such as GM2, and GM3 or conjugates thereof to carrier proteins; or said antigen may be a self-peptide hormone such as whole length Gonadotrophin hormone releasing hormone, a short 10 amino acid long peptide, useful in the treatment of many cancers, or in immunocastration.
Prostate antigens can also be utilized, such as Prostate specific antigen (PSA), PAP, STEAP, PSCA, PCA3, PSMA or Prostase.
Other tumor-associated antigens (TAA) useful in the context of the present invention include: Carcinoembryonic antigen (CEA), KSA (also known as EpCAM), gp100, Plu-1, HASH-1, HasH-2, Cripto, Criptin. Additionally, antigens particularly relevant for vaccines in the therapy of cancer also comprise tyrosinase and survivin).
Other antigens include Mucin-derived peptides such as Muc1, for example Muc1-derived peptides that comprise at least one repeat unit of the Muc1 peptide, preferably at least two such repeats and which is recognized by the SM3 antibody. Other mucin-derived peptides include peptides from Muc5.
The present invention is also useful in combination with breast cancer antigens such as her2/Neu, mammaglobin. Preferably the Her2/neu comprises the entire extracellular domain (comprising approximately amino acids 1-645) or fragments thereof and at least an immunogenic portion of or the entire intracellular domain approximately the C-terminal 580 amino acids. In particular, the intracellular portion should comprise the phosphorylation domain or fragments thereof.
The compositions may comprise antigens associated with tumor-support mechanisms (e.g. angiogenesis, tumor invasion), for example Angiopoietin (Ang)-1 and -2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie)-2 as well as vascular endothelial growth factor (VEGF).
The vaccine or immunogenic composition of the present invention may be used for the prophylaxis or therapy of allergy. Such composition would comprise allergen specific (for example Der p1 and Der p5) and allergen non-specific antigens (for example peptides derived from human IgE, including but not restricted to the Stanworth decapeptide. Other antigens include for example antigens derived from Aspergillus fumigatus. 
The vaccine or immunogenic composition of the present invention may also be used for the prophylaxis or therapy of chronic disorders others than allergy, cancer or infectious diseases. Such chronic disorders are diseases such as inflammatory and autoimmune diseases, atherosclerosis, and Alzheimer. Antigens relevant for the prophylaxis and the therapy of patients susceptible to or suffering from Alzheimer neurodegenerative disease are, in particular, the N-terminal 39-43 amino acid fragment (Abeta) of the amyloid precursor protein (APP) and smaller fragments.
In embodiments, the p97 adhesin adjuvant polypeptide or nucleic acid may be covalently linked to the antigen either directly (e.g., through a peptide bond) or via a suitable linker moiety, e.g., a linker of one or more amino acids (e.g., a polyglycine linker) or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc. (see, e.g., Hermanson (1996) Bioconjugate techniques). Accordingly, in another aspect, the present invention provides a vaccine or immunogenic composition comprising a fusion polypeptide, the fusion polypeptide comprising a p97 adhesin adjuvant polypeptide linked to an antigen, in a further embodiment a heterologous antigen.
In an embodiment, one or more additional peptides or polypeptides may be inserted (1) between the p97 adhesin adjuvant polypeptide and the antigen (2) N-terminal to the p97 adhesin adjuvant polypeptide/antigen construct, and/or (3) C-terminal to the p97 adhesin adjuvant polypeptide/antigen construct. In an embodiment, the p97 adhesin adjuvant polypeptide and the antigen are covalently linked through a peptide bond (as a fusion protein). In an embodiment, the p97 adhesin adjuvant polypeptide is N-terminal relative to the antigen. In an embodiment, the p97 adhesin adjuvant nucleic acid is 5′ relative to the nucleic acid encoding the antigen.
In an embodiment, the N and/or C-terminal end of the p97 adhesin adjuvant polypeptide/antigen construct is modified. The N- and/or C-terminal amino acids may be modified by amidation, acetylation, acylation or other modifications known in the art. In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end of the polypeptide) of the p97 adhesin adjuvant polypeptide/antigen construct is modified (e.g., for protection against degradation). In an embodiment, the modification is acylation with a C2-C16 acyl group, in a further embodiment, the modification is an acetylation.
In an embodiment, the carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the polypeptide) of the p97 adhesin adjuvant polypeptide/antigen construct is modified (e.g., for protection against degradation). In an embodiment, the modification is an amidation.
In an embodiment, the “immunogenic composition” or “vaccine” comprises a plurality (2, 3, 4, 5 or more) of repeats of the p97 adhesin adjuvant polypeptide or nucleic acid.
“Immunogenic composition” or “vaccine” as used herein refers to a composition or formulation comprising one or more polypeptides or a vaccine vector. Vaccination methods for treating or preventing infection in a mammal comprise use of a vaccine or vaccine vector to be administered by any conventional route known in the vaccine field, e.g., via a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g., via a transdermal delivery system such as a patch).
“Adjuvant” refers to a substance which, when added to an immunogenic agent such as an antigen, nonspecifically enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. “Adjuvant”, “immunostimulatory” and “immunopotentiating” activity as used refers to an increase in the immune response/reaction to an antigen due to the p97 adhesin adjuvant polypeptide, i.e. relative to the immune response/reaction when the antigen is used alone.
In another aspect, the present invention also provides a nucleic acid encoding the above-mentioned p97 adhesin adjuvant polypeptide/heterologous antigen construct. The invention also provides a vector comprising the above-mentioned nucleic acid. In yet another aspect, the present invention provides a cell (e.g., a host cell) comprising the above-mentioned nucleic acid and/or vector. The invention further provides a recombinant expression system, vectors and host cells, for the expression/production of encoding the above-mentioned p97 adhesin adjuvant polypeptide/heterologous antigen construct, using for example culture media, production, isolation and purification methods well known in the art.
Such vectors comprise a nucleic acid sequence capable of encoding such a p97 adhesin adjuvant polypeptide/heterologous antigen construct operably linked to one or more transcriptional regulatory sequence(s). In an embodiment, the p97 adhesin adjuvant polypeptide/heterologous antigen construct further comprises a domain which facilitates its purification (e.g., His-tag, GST-tag). Nucleic acids may be introduced into cells for expression using standard recombinant techniques for stable or transient expression.
The term “vector” refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”.
A recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for selectable markers and reporter genes are well known to persons skilled in the art.
A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing DNA into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.
“Transcriptional regulatory sequence/element” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably linked. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.
The p97 adhesin adjuvant polypeptide/heterologous antigen construct of the invention can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify the p97 adhesin adjuvant polypeptide/heterologous antigen construct will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art. For affinity chromatography purification, any antibody which specifically binds the p97 adhesin adjuvant polypeptide/heterologous antigen construct, or an affinity tag attached thereto, may for example be used.
In an embodiment, the above-mentioned p97 adhesin adjuvant polypeptide/heterologous antigen construct is substantially pure. A product is “substantially pure” when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, preferably over 90% and more preferably over 95, 96, 97, 98 or 99% by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived. A substantially pure compound can be obtained, for example, by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
The vaccine or immunogenic composition of the present invention may also further comprise one or more pharmaceutically acceptable carrier or excipient. As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds or multiple heterologous antigens can also be incorporated into the compositions.
The vaccine or immunogenic composition of the present invention may also further comprise one or more additional adjuvants (in addition to the p97 adhesin adjuvant polypeptide), for example adjuvants currently used in the field of vaccines such as (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and/or (6) inert vehicles, such as gold particles.
The composition of the present invention may be used for both prophylactic and therapeutic purposes. Accordingly, there is provided the use of a p97 adhesin adjuvant polypeptide, in combination with a heterologous antigen, in the manufacture of an immunogenic composition (e.g., a vaccine) for the prophylaxis and/or the treatment of viral, bacterial, fungal, parasitic infections, allergy, cancer and other disorders in which the heterologous antigen may be useful. Accordingly, the present invention provides for a method of treating a mammal susceptible to or suffering from an infectious disease or cancer, or allergy, or autoimmune disease using the above-mentioned composition or vaccine (e.g., by administering an effective amount of the composition to a subject in need thereof). In a further aspect of the present invention, there is provided a vaccine or immunogenic combination, comprising a p97 adhesin adjuvant polypeptide and a heterologous antigen, as herein described, for use as a medicament. Immunogenic/vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978.
As used herein, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, (i) prevention, that is, causing the clinical symptoms not to develop, e.g., preventing disease/infection from occurring and/or developing to a harmful state; (ii) alleviation or amelioration of one or more symptoms, (iii) diminishment of extent of disease, (iv) stabilizing (i.e., not worsening) state of disease, (v) preventing spread of disease, (vi) delay or slowing of disease progression, (vii) amelioration or palliation of the disease state, and (viii) remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine or vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age, gender, and the like).
An “effective amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering a vaccine composition of the present invention, an effective amount is, for example, an amount sufficient to achieve a modulation (quantitative and/or qualitative) of the immune response as compared to the immune response obtained when the antigen is administered alone (without a p97 adhesin adjuvant polypeptide). An effective amount can be administered in one or more administration(s).
The present invention is illustrated in further details by the following non-limiting examples.
EXAMPLE 1
Material and Methods
Viruses, Cells and Synthetic Genes:
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) IAF-Klop strain (Genbank accession No U64928) was propagated and titrated in MARC-145 cells in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with 8% of fetal bovine serum (FBS; PAA Laboratories, Inc., Etobicoke, Ontario) at 37° C. in a humidified atmosphere of 5% CO2 (Kheyar, A., et al., Vaccine, 2005. 23(31): 4016-22).
Porcine circovirus type 2b (PCV2b) FMV-06-1717 strain (Gagnon, C. A., et al., J Vet Diagn Invest, 2008. 20(5): p. 545-58) was propagated and titrated in PK15A cells, a subclone of PCV non-infected PK15 cells in Earle's minimal essential medium (EMEM; Wisent, St-Bruno, Québec, Canada), supplemented with 10% FBS, penicillin (100 U/ml)/streptomycin (100 μg/ml) (Invitrogen), 0.1 mM nonessential amino acids (Wisent), 1 mM sodium pyruvate (Wisent), 2.5 mg/ml of amphotericin B (Invitrogen), and 10 mM HEPES buffer (Wisent) at 37° C. in a humidified atmosphere (Gagnon, C. A., et al., Can Vet J, 2007. 48(8): 811-9).
Recombinant adenoviruses (rAdVs) were propagated and titrated in the AD-293 cells line (Agilent Technologies, Santa Clara, Calif.) in DMEM supplemented with 8% of FBS at 37° C. in a humidified atmosphere of 5% CO2. A549 cells were propagated in DMEM supplemented with 8% of FBS and maintained at 37° C. in a humidified atmosphere of 5% CO2. These cells were E1-deficient and were used to confirm protein expression from the rAdVs (Imler, J. L., et al., Gene Ther, 1996. 3(1): 75-84).
The codons most frequently used by Sus Scrofa cells were used to generate synthetic open reading frame: ORF5 and ORF6 of PRRSV on the basis of PRRSV IAF-Klop sequence (Genbank accession No. U64928); ORF2 of PCV2b gene on the basis of PCV2b FMV-06-1717 sequence (Gagnon, C. A., et al., J Vet Diagn Invest, 2008. 20(5): 545-58), P97c gene (933 C-terminal nucleotides) on the basis of M. hyopneumoniae VR232 sequence (Genbank accession No. U50901.1). The constructs were synthesized by the GeneArtTM Gene Synthesis Services (Invitrogen).
Fusion of Genes of Interest:
Different genes of interest were amplified by PCR and were cloned into pBluescriptTM KS(+) (Stratagene, La Jolla, Calif.). When necessary, genes of interest were linked with the nucleotide sequence GTTACCACC (GTT, Jiang W, et al. Vet Immunol Immunopathol 2006; 113(1-2): 169-80). The open reading frames (ORF) encoding P97c, Cap, M-GP5, Cap-GP5, P97c-GP5, P97c-Cap or Cap-P97c were validated by DNA sequencing through the McGill University Sequencing Services (Montreal, Québec, Canada).
Construction of Recombinant Adenoviruses:
The rAdVs used in the studies described herein were a replication-defective E1- and E3-deleted human serotype 5. The different amplicon previously cloned into pBluescriptTM KS(+) were digested with Bg/II/XhoI (New England Biolabs, Ipswich, Mass.) and subsequently cloned into the corresponding restriction sites into the transfer vector pShuttle-IRES-hrGFP-1 (Agilent Technologies). All constructs were confirmed by DNA sequencing through the McGill University Sequencing Services. The recombinant plasmids were linearized with Pmel (New England Biolabs) and rescued into the genome of the pAdEasy-1 vector (Agilent Technologies) by homologous recombination in E. Coli BJ5183 bacteria cells (MP biomedicals, Irvine, Calif.) by electroporation (2.5 kV, 200 Ohms and 25 μF). The integrity of the recombinant rAdVs genome was confirmed by PCR and restriction enzyme digestion. To produce rAdVs, plasmids were linearized by PacI digestion and AD-293 cells were transfected with 2 μg per well of plasmids of a 6-well plate using PolyFectTM transfection reagent (Qiagen, Valencia, Calif.). The transfected cells were 24 h later overlayed with agarose (Invitrogen) (0.45% in DMEM supplemented with 5% FBS) and monitored daily until the appearance of viral plaques. After three rounds of viral plaque purification, rAdVs were propagated at high titers and purified by double cesium chloride gradient, as previously described (Bourbeau D, et al., Cancer Res 2007; 67(7): 3387-95). The titers of rAdVs were determined in AD-293-infected cells and titers were expressed in tissue culture infectious dose 50 per ml (TCID50/ml).
Western Blot Assay:
A549 cells were seeded in 6-well tissue culture plates and infected with rAdVs (MOI: 10). 24 h post-infection, cells from six wells were lysed and total cell protein concentrations were quantified with the DC protein assay kit (Bio-Rad, Mississauga, ON, CA). For each sample, 50 μg of total cell extract prepared as described (Gomez Corredor A and Archambault D. J Virol 2009; 83(24): 12842-53) was electrophoretically separated onto 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in phosphate buffered saline (PBS) containing 0.05% TweenTM-20 (PBS-T) in presence of 5% non-fat dry milk for 1 h at room temperature and then incubated overnight at 4° C. with convalescent PRRSV-specific pig antiserum (1/5000), PCV2b-specific pig antiserum (1/10000), mouse monoclonal anti-P97c antibody (MAb 8H4-G6) (1/1,000) (Okamba, F. R., et al., Clin Vaccine Immunol, 2007. 14(6): p. 767-74) or GAPDH 1/10,000 (Sigma-Aldrich, St. Louis, Mo.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunostaining (Sigma-Aldrich, St-Louis, Mo.) was used as a loading control. Detection of the proteins was performed as described in Gomez Corredor A and Archambault D., J Virol 2012; 86(9):4892-905.
Recombinant Protein Production:
Plasmid pGEX-4T1 (Pharmacia Biotech, Piscataway, N.J.) encoding GP5 of PRRSV IAF-Klop isolate or P97c (amino acids 534 to 1093) of M. hyopneumoniae 25934 isolate (GenBank accession # AY512905) were kindly provided by Dr. Carl Gagnon and has been described in Okamba, F. R., et al., 2007, supra. Cap protein (lacking the 39 N-terminal amino acids; Zhou, J. Y., et al., J Biotechnol, 2005. 118(2): p. 201-11) of PCV2b FMV-06-1717 isolate was cloned into pGEX-4T1. The recombinant GP5 protein (rGP5), P97c protein (rP97c) or Cap protein (rCap) were produced in BL21(DE3)pLysS competent E. coli cells (Promega, Madison, Wis.) upon induction at OD(600 nm) of 1.2 with 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) for 4 h at 37° C. Cells were lysed and separated onto a 12% SDS-PAGE. The band corresponding to recombinant proteins was excised from the gel and electroeluted. The purified proteins were dialyzed against PBS and stored at −80° C. for further use. The protein concentration was quantified with the DC protein assay kit as described above. The nature of the eluted proteins was confirmed by Western blot as described above. These proteins were used in the ELISA.
Mice Immunization:
Animal protocols were approved by the University's Animal Protection Institutional Committee according to the regulations of the Canadian Council for Animal Care. 6 weeks old female BALB/c mice were obtained from Charles River Laboratory (St-Constant, Quebec, Canada).
In a first experiment (Experiment 1), mice were randomly divided into 4 groups of 5 mice and immunized intramusculary (IM) at days 0 and 14 with 109 TCID50 of rAdVs expressing M-GP5, Cap-GP5, P97c-GP5 or GFP as negative control. Blood samples were collected from the mandibular vein at 0, 14, 28 and 35 days post-immunization (dpi). At each time point, serum samples were pooled for each group of mice. On 49 dpi, blood was obtained from cardiac blood puncture in mice under anaesthesia with isoflurane (Fisher Scientific, Ville St-Laurent, Qc, Canada). Following clotting, samples were centrifuged (600×g at room temperature for 10 min) and the resulting sera were stored at −20° C. until analysis.
In a second experiment (Experiment 2), mice were randomly divided into 5 groups of 6 mice and immunized IM at days 0 and 14 with 109 TCID50 of rAdVs expressing P97c, Cap, P97c-Cap, Cap-P97c or GFP as negative control. Blood samples were collected from the mandibular vein at 0, 14, 28, 35 and 49 dpi. At each time point, serum samples were pooled for each group of mice. On 56 dpi, blood was obtained from cardiac blood puncture and sera were prepared as described above.
Antibody Response:
Indirect ELISA.
The presence of serum GP5, Cap or P97c-specific antibodies was evaluated by an indirect ELISA using ImmulonTM 2HB 96-well microtiter plates (Thermo Labsystems, Franklin, Mass.). Plates were coated with 0.1 μg of recombinant proteins (rGP5, rCap or rP97c) or with 1 μg of PRRSV GP5 peptide (SSSQLQSIYNLTIC, SEQ ID NO: 15) per well diluted in 0.05 M sodium carbonate buffer (pH 9.6) to a final volume of 100 μl. Following an overnight incubation at 4° C., plates were washed 4 times with PBS-T and then saturated with 150 μl of PBS-T with 1% BSA overnight at 4° C. One hundred microliters of mice serum, diluted to 1/100 (or 1/50 for IgG isotype detection) in PBS-T with 1% BSA, were added into wells and incubated for 2 h at 37° C. Plates were then washed as previously described and HRP-conjugated goat anti-mouse total IgGs (1:10,000), IgG1 (1:5,000), IgG2a (1:2,500), IgG2b (1:2,500) or IgG3 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, Calif.) in PBS-T with 1% BSA, were added for 1 h at 37° C. Plates were washed and HRP signal was detected by adding 100 μl of tetramethylbenzidine (TMB, Sigma-Aldrich) per well. Reaction was stopped after 20 minutes by adding 50 μl 1M H2SO4 to each well at room temperature, and optical density (OD) was determined at a wave length of 450 nm (using TecanTM Infinite M1000 reader, Tecan Group Ltd, Mannedorf, Switzerland). For each serum sample, O.D. was corrected by subtracting O.D. value of uncoated wells from O.D. value obtained with antigen-coated wells.
Virus-Specific Neutralization Assay (NT).
Two-fold serial dilutions of each serum sample (inactivated for 30 min at 56° C.) were done in cell culture medium. The PRRSV-specific NT was performed by a viral cytopathic effect inhibition method using four wells of indicator cells per serum dilution and an incubation time of 96 h (Dea, S., et al., J Clin Microbiol, 1996. 34(6): 1488-93). The neutralizing Ab (NAb) titer was expressed as the reciprocal of the highest sample dilution neutralizing 100 TCID50 of the virus. For the PCV2b-specific NT, 25 μl of serum two-fold serial dilutions was incubated with 25 μl of 100 TCID50 of the virus for 1 h at 37° C. The mixture was then transferred to PK15A cells plated in a 96-well plate (four wells per specimen dilution, 8×104 cells/well). The inoculum was removed 1 h later and 50 μl of fresh cell culture medium was added into each well. Cells were fixed in PBS-formaldehyde 4% after 96 h, permeabilized with 1:1 acetone/methanol solution for 15 min at −20° C., and blocked with 5% BSA in PBS for 1 h at 37° C. Cells were incubated with rabbit FITC-labeled anti-porcine secondary Ab (1/250) (MP Biomedicals) for 45 min at 37° C. Nuclei were counterstained with propidium iodide (Sigma-Aldrich). Images were taken using an inverse microscope (Nikon® TE-300) coupled to the confocal Bio-Rad® MRC-1024ES system, and analyzed with Image-J® software. For each well, the number of PCV2b-infected cells was compared to that of PK15A cells inoculated with PCV2b exposed to serum from non-immunized mice (control). NAb titers were expressed as the reciprocal of the highest dilution of the serum that inhibited more than 50% of PCV2b infection.
Statistical Analysis:
Where indicated, one-way analysis of variance (ANOVA) followed by Tukey's post-test were carried out for statistical analyses between multiple groups using GraphPadTM Prism 5 software (Windows Version 5.0, Lajolla, Calif., USA).
EXAMPLE 2
Experiment 1
Expression of Recombinant Adenoviruses
Expression of proteins was detected by western-blot 24 h after infection of A549 cells with rAdVs expressing GFP, M-GP5, Cap-GP5 or P97c-GP5. As shown in FIGS. 1A-1E, M-GP5 (FIG. 1A), Cap-GP5 (FIG. 1B), P97c-GP5 (FIG. 1C), Cap (FIG. 1D), P97c (FIG. 1E), P97c-Cap and Cap-P97c (FIGS. 1D and 1E) proteins were expressed at the expected molecular weights. No immunoreactive bands were detected with extracts from uninfected control cells or from cells infected with AdVs expressing GFP.
Antibody (Ab) Response in Mice Following Immunization
In a first experiment, the ability of AdVs expressing Cap-GP5 or P97c-GP5 fusion proteins to induce GP5-specific Abs in mice was compared to that of AdVs expressing PRRSV M-GP5. The latter group of mice was included because expression of the M-GP5 fusion protein from AdVs induces a higher GP5-specific Ab response when compared to that of GP5 alone (Jiang W et al., Vet Immunol Immunopathol 2006; 113(1-2):169-80; Zheng Q, et al. Virus Genes 2007; 35(3):585-95). All groups of mice produced GP5-specific Abs from 14 dpi (FIG. 2A). The highest Ab level was obtained in mice of the P97c-GP5 group at 14, 35 and 49 dpi whereas Ab levels between the M-GP5 and Cap-GP5 groups were similar at any time point of the experiment. No GP5-specific NAbs were detected in any groups of mice. P97c- and Cap-specific Ab responses were also determined in these groups of mice. Anti-P97c Abs were detected at 14 dpi in mice of the P97c-GP5 group with a peak level from 28 dpi (FIG. 2B). Mice of the Cap-GP5 group developed a low level of PCV2 Cap-specific Abs (FIG. 2C) even though PCV2-specific NAbs with a mean titer of 3.4 were detected at 49 dpi in mice of this group.
EXAMPLE 3
Experiment 2
Antibody (Ab) Response Against P97c in Mice Following Immunization
IN a second set of experiments, mice were immunized at days 0 and 14 IM with 109 TCID50 with rAdVs expressing either P97c protein, Cap protein or both proteins in fusion. Specific antibody response was determined by indirect ELISA. As shown in FIG. 3A, all groups that received rAdVs expressing P97c developed a strong antibody response against P97c at 14 dpi. This strong antibody response was maximal for all groups at 14 dpi except for the Cap-P97c group in which the response was maximal at 28 dpi. IgG isotyping was performed at 49 dpi in order to define the immune profile response; IgG1 are preferentially induced when a T helper 2 (Th2)-type response is elicited whereas the presence of IgG2a, IgG2b and IgG3 is indicative of a T helper 1 (Th1)-type response. As shown in FIG. 3B, all immunized groups have developed IgG1, IgG2a, IgG2b and IgG3 against P97c. For all isotypes, there was no significant difference between groups that have received P97c-Cap or Cap-P97c (P>0.05). Groups having received P97c alone have developed a significantly higher IgG1 (P<0.05) and IgG3 (P<0.01) responses than groups who had received P97c in fusion with Cap protein. As for IgG2a, mice from the P97c group have developed a significantly higher antibodies response than those of the P97c-Cap group, and the IgG2b level was significantly higher in mice of the P97c group relative to those of the Cap-P97c group (P<0.05).
Antibody (Ab) Response Against Cap in Mice Following Immunization
Mice immunized with rAdVs expressing Cap protein developed specific antibody against Cap at 28 dpi. This antibody response was higher with the P97c-Cap group than in Cap or Cap-P97c groups throughout the experiment. At 56 dpi, Cap and Cap-P97c groups developed the same levels of antibody (FIG. 4A). As shown in FIG. 4B, IgG1 specific to Cap protein was not significantly different between the different groups tested. However, mice form the P97c-Cap group developed significant amounts of IgG2b and IgG3.
The PCV2-specific NT was performed to determine the NAb production at 56 dpi. Mice of the Cap-P97c group showed the highest NAb response (P<0.001) with a mean NAb titer of 6.5 (Log 2) as compared to the mean NAb titer of 4.16 (Log 2) from the group of mice given the Cap protein alone (FIG. 5). Only one mouse of the P97c-Cap developed a low level of PCV2-specific NAbs with a resulting mean NAb titer of 0.33 (Log 2).
Thus, fusion of the GP5 or Cap with P97c expressed from rAdVs allowed higher immunogen-specific Ab responses when compared to those obtained in mice immunized with rAdVs expressing each immunogen alone, indicating a P97c-associated immunopotentiation/adjuvant effect.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

## Claims

1. An immunogenic composition comprising a p97 adhesin adjuvant polypeptide comprising a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9 and having adjuvant properties, covalently linked to a heterologous antigen polypeptide.

2. The immunogenic composition of claim 1, wherein the p97 adhesin adjuvant polypeptide comprises the amino acid sequence of SEQ ID NO: 9.

3. The immunogenic composition of claim 1, wherein the heterologous antigen is an antigen from a human pathogen or an antigen of human origin.

4. The immunogenic composition of claim 1, wherein the p97 adhesin adjuvant polypeptide is N-terminal relative to the heterologous antigen polypeptide.

5. The immunogenic composition of claim 1, further comprising one or more pharmaceutically acceptable excipients.

6. A method of inducing an immune response against a heterologous antigen polypeptide in a subject, the method comprising administering to said subject an effective amount of an immunogenic composition comprising a p97 adhesin adjuvant polypeptide comprising a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9 and having adjuvant properties, covalently linked to said heterologous antigen polypeptide.

7. The method of claim 6, wherein the p97 adhesin adjuvant polypeptide comprises the amino acid sequence of SEQ ID NO: 9.

8. The method of claim 6, wherein the heterologous antigen is an antigen from a human pathogen or antigen of human origin, and said subject is a human.

9. The method of claim 6, wherein the p97 adhesin adjuvant polypeptide is N-terminal relative to the heterologous antigen polypeptide.

10. The method of claim 6, wherein the immunogenic composition further comprises one or more pharmaceutically acceptable excipients.

11. The immunogenic composition of claim 1, wherein the p97 adhesin adjuvant polypeptide comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9 and having adjuvant properties.

12. The method of claim 6, wherein the p97 adhesin adjuvant polypeptide comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9 and having adjuvant properties.