Abstract:
The invention relates to an antigenic polypeptide referred to as DivlB and variants thereof, vaccines comprising said polypeptide and the use of the vaccine in protecting subjects from microbial infection.

Description:
REFERENCE TO RELATED APPLICATIONS 
     This application is the US national phase entry of International Patent Application No. PCT/GB2010/001722, filed Sep. 13, 2010, which claims priority to GB Patent Application No. 0917685.0, filed Oct. 9, 2009. 
     FIELD OF THE INVENTION 
     The invention relates to an antigenic polypeptide, vaccines comprising said polypeptide and the use of the vaccine in protecting subjects from microbial infection. 
     BACKGROUND 
     Vaccines protect against a wide variety of infectious diseases. Many vaccines are produced by inactivated or attenuated pathogens which are injected into a subject. The immunised subject responds by producing both a humoral (e.g. antibody) and cellular (e.g. cytolytic T cells) responses. For example, some influenza vaccines are made by inactivating the virus by chemical treatment with formaldehyde. For many pathogens chemical or heat inactivation while it may give rise to vaccine immunogens that confer protective immunity also gives rise to side effects such as fever and injection site reactions. In the case of bacteria, inactivated organisms tend to be so toxic that side effects have limited the application of such crude vaccine immunogens (e.g. the cellular pertussis vaccine) and therefore vaccine development has lagged behind drug-development. Moreover, effective vaccine development using whole cell inactivated organisms suffers from problems of epitope masking, immunodominance, low antigen concentration and antigen redundancy. This is unfortunate as current antibiotic treatments are now prejudiced by the emergence of drug-resistant bacteria. 
     Many modern vaccines are therefore made from protective antigens of the pathogen, isolated by molecular cloning and purified from the materials that give rise to side-effects. These latter vaccines are known as ‘subunit vaccines’. The development of subunit vaccines has been the focus of considerable research in recent years. The emergence of new pathogens and the growth of antibiotic resistance have created a need to develop new vaccines and to identify further candidate molecules useful in the development of subunit vaccines. Likewise the discovery of novel vaccine antigens from genomic and proteomic studies is enabling the development of new subunit vaccine candidates, particularly against bacterial pathogens. However, although subunit vaccines tend to avoid the side effects of killed or attenuated pathogen vaccines, their ‘pure’ status means that subunit vaccines do not always have adequate immunogenicity to confer protection. 
     An example of a pathogenic organism which has developed resistance to antibiotics is  Staphylococcus aureus. S. aureus  is a bacterium whose normal habitat is the epithelial lining of the nose in about 20-40% of normal healthy people and is also commonly found on people&#39;s skin usually without causing harm. However, in certain circumstances, particularly when skin is damaged, this pathogen can cause infection. This is a particular problem in hospitals where patients may have surgical procedures and/or be taking immunosuppressive drugs. These patients are much more vulnerable to infection with  S. aureus  because of the treatment they have received. Antibiotic resistant strains of  S. aureus  have arisen since their wide spread use in controlling microbial infection. Methicillin resistant strains are prevalent and many of these resistant strains are also resistant to several other antibiotics. 
     Currently there is no effective vaccination procedure for  S. aureus.    
       S. aureus  is therefore a major human pathogen capable of causing a wide range of diseases some of which are life threatening diseases including septicaemia, endocarditis, arthritis and toxic shock. This ability is determined by the versatility of the organism and its arsenal of components involved in virulence. At the onset of infection, and as it progresses, the needs and environment of the organism changes and this is mirrored by a corresponding alteration in the virulence determinants which  S. aureus  produces. At the beginning of infection it is important for the pathogen to adhere to host tissues and so a large repertoire of cell surface associated attachment proteins are made. The pathogen also has the ability to evade host defenses by the production of factors that reduce phagocytosis or interfere with the ability of the cells to be recognised by circulating antibodies. Often a focus of infection develops as an abscess and the number of organisms increases.  S. aureus  has the ability to monitor its own cell density by the production of a quorum sensing peptide. Accumulation of the peptide, associated with physiological changes brought about by the beginning of starvation of the cells, elicits a switch in virulence determinant production from adhesins to components involved in invasion and tissue penetration. 
     SUMMARY 
     This disclosure relates to the identification of an antigenic polypeptide, DivlB, isolated and characterized from the gram positive bacterium  S. aureus . DivlB is an integral membrane protein comprising an intracellular domain [amino acids 1-171] and intermembrane domain [amino acids 172-192] and an extracellular domain [amino acids 193-439]. This is schematically illustrated in  FIG. 1 . DivlB and fragments thereof, is shown to provide protection from at least an  S. aureus  challenge and represents a novel vaccine candidate. DivlB homolgues are referred to as FtsQ in gram negative bacteria. 
     According to an aspect of the invention there is provided a polypeptide selected from the group consisting of:
         i) a polypeptide encoded by a nucleotide sequence as represented in  FIG. 2   a ,  3   a , or  4   a  (SEQ ID NO: 1, 3 or 5), or an antigenic fragment thereof;   a polypeptide encoded by a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);   iii) a polypeptide comprising an amino acid sequence wherein said sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in  FIG. 2   b ,  3   b  or  4   b  (SEQ ID NO: 2, 4 or 6), wherein said polypeptide is for use as a vaccine.       

     A modified polypeptide or variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies. 
     In one embodiment, the variant polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the full length amino acid sequences illustrated herein. 
     In a preferred embodiment of the invention said polypeptide is encoded by a nucleotide sequence as represented in  FIG. 2   a  (SEQ ID NO: 1). 
     In an alternative preferred embodiment of the invention said polypeptide is represented by the amino acid sequence in  FIG. 2   b  (SEQ ID NO: 2), or antigenic part thereof. 
     In a preferred embodiment of the invention said polypeptide is encoded by a nucleotide sequence as represented in  FIG. 3   a  (SEQ ID NO: 3). 
     In an alternative preferred embodiment of the invention said polypeptide is represented by the amino acid sequence in  FIG. 3   b  (SEQ ID NO: 4), or antigenic part thereof. 
     In a preferred embodiment of the invention said polypeptide is encoded by a nucleotide sequence as represented in  FIG. 4   a  (SEQ ID NO: 5). 
     In an alternative preferred embodiment of the invention said polypeptide is represented by the amino acid sequence in  FIG. 4   b  (SEQ ID NO: 6), or antigenic part thereof. 
     According to a further aspect of the invention there is provided a vaccine composition for use in the vaccination against a microbial infection, comprising a polypeptide selected from the group consisting of:
         i) a polypeptide encoded by a nucleotide sequence as represented in  FIG. 2   a ,  3   a  or  4   a  (SEQ ID NO: 1, 3, or 5), or an antigenic fragment thereof;   ii) a polypeptide encoded by a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);   iii) a polypeptide comprising an amino acid sequence wherein said sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in  FIG. 2   b ,  3   b  or  4   b  (SEQ ID NO: 2, 4 or 6): wherein said composition optionally includes an adjuvant and/or carrier.       

     In a preferred embodiment of the invention said composition includes an adjuvant and/or carrier. 
     In a preferred embodiment of the invention said adjuvant is selected from the group consisting of: cytokines selected from the group consisting of GMCSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin 2, interleukin 1, TGF, TNFα, and TNFβ. 
     In a further alternative embodiment of the invention said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly I:C and derivatives thereof. 
     In a preferred embodiment of the invention said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehalose dicorynomycolate (TDM). 
     An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, agonistic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, liposomes. An adjuvant is therefore an immunomodulator. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the “carrier” protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen. 
     In a preferred embodiment of the invention said microbial infection is caused by a bacterial species selected from the group consisting of:  Staphylococcus  spp,  Enterococcus faecalis, Mycobacterium tuberculosis, Streptococcus  group B,  Streptococcus pneumoniae, Helicobacter pylori, Neisseria gonorrhoea, Streptococcus  group A,  Borrelia burgdorferi, Coccidiodes immitis, Histoplasma capsulatum, Klebsiella edwardii, Neisseria meningitidis  type B,  Proteus mirabilis, Shigella flexneri, Escherichia coli, Haemophilus influenzae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Francisella tularensis, Pseudomonas aeruginosa, Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Burkholderia mallei  or  B. pseudomallei.    
     In a preferred embodiment of the invention said bacterial species is selected from the group consisting of:  S. epidermidis, S. aureus, S. hominis, S. haemolyticus, S. wameri, S. capitis, S. saccharolyticus, S. auricularis, S. simulans, S. saprophyticus, S. xylosus, S. hyicus, S. caprae, S. gallinarum, S. intermedius.    
     In a further preferred embodiment of the invention said staphylococcal cell is  S. aureus  or  S. epidermidis.    
     The vaccine compositions of the invention can be administered by any conventional route, including injection, intranasal spray by inhalation of for example an aerosol or nasal drops. The administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or intradermally I. The vaccine compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a vaccine composition that alone or together with further doses, produces the desired response. In the case of treating a particular bacterial disease the desired response is providing protection when challenged by an infective agent. 
     In a preferred embodiment of the invention said vaccine composition is adapted for administration as a nasal spray. 
     In a preferred embodiment of the invention said vaccine composition is provided in an inhaler and delivered as an aerosol. 
     The amounts of vaccine will depend, of course, on the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used sufficient to provoke immunity; that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. 
     The doses of vaccine administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. 
     In general, doses of vaccine are formulated and administered in effective immunizing doses according to any standard procedure in the art. Other protocols for the administration of the vaccine compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. Administration of the vaccine compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep or goat. 
     In a preferred embodiment of the invention there is provided a vaccine composition according to the invention that includes at least one additional anti-bacterial agent. 
     In a preferred embodiment of the invention said agent is a second different vaccine and/or immunogenic agent (for example a bacterial polypeptide and/or polysaccharide antigen). 
     According to a further aspect of the invention there is provided a polypeptide as herein described for use in the treatment of microbial infections or conditions that result from microbial infections. 
     In a preferred embodiment of the invention said microbial infection is a staphyloccal infection. 
     In a preferred embodiment of the invention said condition that results from a microbial infection is selected from the group consisting of: tuberculosis, bacteria-associated food poisoning, blood infections, peritonitis, endocarditis, osteomyelitis, sepsis, skin disorders, meningitis, pneumonia, stomach ulcers, gonorrhoea, strep throat, streptococcal-associated toxic shock, necrotizing fasciitis, impetigo, histoplasmosis, Lyme disease, gastro-enteritis, dysentery, shigellosis, and arthritis. 
     According to a further aspect of the invention there is provided a method to immunize a subject comprising vaccinating said subject with an effective amount of the polypeptide, nucleic acid molecule or vaccine composition according to the invention. 
     In a preferred method of the invention said subject is a human. 
     In an alternative preferred method of the invention said subject is an animal, preferably a livestock animal, for example cattle. 
     In a preferred method of the invention said live stock animal is vaccinated against bacterial mastitis caused by staphylococcal bacterial cells. 
     In a preferred method of the invention said life stock animal is a caprine animal (e.g. sheep, goat). 
     In a preferred method of the invention said life stock animal is a bovine animal (e.g. a cow). 
     Staphylococcal mastitis is a serious condition that affects live stock and can result in considerable expense with respect to controlling the disease through administration of antibiotics and in terms of lost milk yield. The vaccine according to the invention provides cost effective control of bacterial, in particular staphylococcal mastitis. 
     Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. 
     Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. 
     Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An embodiment of the invention will now be described by example only and with reference to the following figures: 
         FIG. 1  The DivlB protein is predicted to be a membrane proteins with the majority model topological distribution indicated. The N-terminal of the protein (aminoacids 1 through 171) is located inside of the cell, while the C-terminal of the protein (aminoacids 193 through 439) is exposed on the outside of the membrane; the predicted external portion of the  S. aureus  DivlB (aminoacids 193 through 439) corresponds to the fragment termed DivlB-1; 
         FIG. 2   a  illustrates the full DivIB nucleotide sequence from  S. aureus 8325(SEQ ID NO: 1); 
         FIG. 2   b  illustrates the amino acid sequence from  S. aureus 8325(SEQ ID NO: 2) and corresponds to the GeneBank ID number ABD30258.1; 
         FIG. 3   a  illustrates the nucleotide sequence (SEQ ID NO: 3) and  FIG. 3   b  the amino acid sequence (SEQ ID NO: 4) of the extramembranous fragment of the  S. aureus  DivIB (DiviB-1) that encompasses amino acids 193 through 439; 
         FIG. 4   a  illustrates the nucleotide sequence (SEQ ID NO: 5) and  FIG. 4   b  the amino acid sequence (SEQ ID NO: 6) of DivIB-2; and 
         FIG. 5  and  FIG. 6  illustrate the protection against infection conferred by DivlB-2 vaccination. 
     
    
    
     DETAILED DESCRIPTION 
     Materials and Methods 
     Construction of plasmid for the overexpression of the DivlB-1 Fragment from  S. aureus  in  E. coli    
     Fragment DivlB-1 was PCR amplified from the chromosome of strain  S. aureus  SH1000 (Horsburgh M J, Aish J L, White I J, Shaw L, Lithgow J K, Foster S J: sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from  Staphylococcus aureus  8325-4 . J Bacteriol  2002, 184:5457-5467) using primers 5′GLUSh341C and 3′ GLUSh341C (corresponding to sequences primer 1 and primer 2 respectively) and the following PCR reaction conditions: 1 initial denaturation cycle of 94° C. for 4 min; 30 amplification cycles of denaturation 94° C. for 30 seconds, annealing 45° C. for 30 seconds, and extension at 72° C. for 2.5 seconds; finally, ongoing amplification rounds were allow to complete at 72° C. for 4 min. Two restrictions sites were engineered within primers 5′GLUSh341C and 3′ GLUSh341C, NcoI and XhoI, respectively (underlined in the sequence). Fragment DivlB-1 digested with NcoI and XhoI was cloned into the NcoI and XhoI sites from pET-21d (+) from Novagen (Cat. No. 69743-3) resulting in the overexpression plasmid named pGL601 generating a 6×His-tagged form of the DivlB-1 fragment. The latter was transferred into  E. coli  BL21 for overexpresion of the recombinant protein fragment. 
     
       
         
               
               
             
           
               
                 Primer 1 (5′GLUSh341C) 
                   
               
               
                 ATAATA CCATGG CTCCACTTAGTAAAATTGCGCATG 
                 SEQ ID NO: 7 
               
               
                   
               
               
                 Primer 2 (3′GLUSh341C) 
                   
               
               
                 ATAATA CTCGAG ATTATTCTTACTTGATTGTTTG 
                 SEQ ID NO: 8 
               
             
          
         
       
     
     The cloning of the PCR amplified fragment indicated above into the recipient pET21d(+) recipient plasmid vector at the NcoI and XhoI sites entailed the addition of two aminoacids (methionine and alanine) upstream of the DivlB-1 sequence and eight aminoacids (leucine, glutamate and six histidines) downstream of the DivlB-1 sequence. This whole region encompasses the protein fragment to be produced from the ATG translational start codon to the TGA translational stop codon (indicated in bold within the sequence), and named DivlB-2. The DNA ( FIG. 4   a ) and protein ( FIG. 4   b ) sequences of DivlB-2 are indicated below and the supplementary nucleotides to the DivlB-1 fragment are underlined. 
     EXAMPLE 
     Vaccinations with DivlB-2 Protect Balb/C Mice Against  S. aureus  Infections 
     In each experiment, a group of 10 female Balb/C 6 to 12 weeks old were vaccinated with DivlB-2 according to the following protocol. Each animal was primed with 100 microliters of a solution made up of a mixture 50 micrograms of recombinant DivlB-2 (approximately 98% purity) in 50 microliters endotoxin-free PBS (Phosphate Buffer Saline pH 7.4) and 50 microliters of Complete Freund&#39;s adjuvant. Two weeks later the animals were boosted with 100 microliters of a solution made up of a mixture 50 micrograms of recombinant DivlB-2 (approximately 98% purity) in 50 microliters of endotoxin-free PBS and 50 microliters of Incomplete Freund&#39;s adjuvant. A week later the animals received an identical boost. In each experiment, a control group of 10 animals were treated following an identical protocol except for the fact that instead of the DivlB-2 recombinat protein the mixture contained commercially available KLH protein (Keyhole limpet hemocyanin). 
     Priming and boost injections were performed intradermally in the back of the neck of the animals. 
     One week after the second boost each animal was infected with an i.v. (tail vein) injection of 100 microliters of endotoxin-free PBS containing 1.1×10 7  (±0.3×10 7 ) cells of  S. aureus  strain Newman. The latter were prepared from cultures growing to early stationary phase in Brain Heart Infusion medium (BHI), which was then washed three times with the same volume of PBS. 
     After 10 to 14 days the animals were sacrificed according to Schedule 1 cervical dislocation. The pair of kidneys from each animal was extracted in aseptic conditions, and homogenized in sterile PBS. Serial dilutions of the kidney homogenates were carried out in PBS and plated on BHI agar plates. Plates containing between 10 to 150 staphylococcal colonies were counted and dilution corrected. The number of viable cells in the kidneys was inferred from the number of colony forming units (CFU) on the plates. Evaluation of the possible protection against infection conferred by vaccination with DivlB-2 was determined from difference in the number of  S. aureus  cells in the kidneys of animals vaccinated with KLH and those vaccinated with DivlB-2. The statistic significance of the difference was calculated using the Mann-Whitney test. A significantly higher (p&lt;0.05) number of  S. aureus  in KLH vaccinated animals compared to the DivlB-2 vaccinated animals was concluded as protection. 
     Examples of the experiments described above and illustrating the protection against infection conferred by DivlB-2 vaccination are shown in  FIG. 5  and  FIG. 6 . The mean for each group and the statistically significant difference between the control and the vaccinated group are indicated.