adjuvants ( immune potentiators or immunomodulators ) have been used for decades to improve the immune response to vaccine antigens . the incorporation of adjuvants into vaccine formulations is aimed at enhancing , accelerating and prolonging the specific immune response to vaccine antigens . advantages of adjuvants include the enhancement of the immunogenicity of weaker antigens , the reduction of the antigen amount needed for a successful immunisation , the reduction of the frequency of booster immunisations needed and an improved immune response in elderly and immunocompromised vaccines . selectively , adjuvants can also be employed to optimise a desired immune response , e . g . with respect to immunoglobulin classes and induction of cytotoxic or helper t lymphocyte responses . in addition , certain adjuvants can be used to promote antibody responses at mucosal surfaces . aluminium hydroxide and aluminium or calcium phosphate has been used routinely in human vaccines . adjuvants can be classified according to their source , mechanism of action and physical or chemical properties . the most commonly described adjuvant classes are gel - type , microbial , oil - emulsion and emulsifier - based , particulate , synthetic and cytokines . more than one adjuvant may be present in the final vaccine product according to the invention . they may be combined together with a single antigen or all antigens present in the vaccine , or each adjuvant may be combined with one particular antigen . the origin and nature of the adjuvants currently being used or developed is highly diverse . for example , aluminium based adjuvants consist of simple inorganic compounds and plg is a polymeric carbohydrate . mdp is derived from bacterial cell walls ; saponins are of plant origin , squalene is derived from shark liver and recombinant endogenous immunomodulators are derived from recombinant bacterial , yeast or mammalian cells . there are several adjuvants licensed for veterinary vaccines , such as mineral oil emulsions that are too reactive for human use . similarly , complete freund &# 39 ; s adjuvant , although being one of the most powerful adjuvants known , is not suitable for human use . 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 . 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 . 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 . the ratio of antigens may be varied in pair wise fashion . the ratio of each antigen may be 1 : 1 , 2 : 1 , 3 : 1 , 4 : 1 , 5 : 1 , 6 : 1 , 7 : 1 , 8 : 1 , 9 : 1 or 10 : 1 to optimize the response of the subject to particular combinations of antigen . for example the ratio of divib and ydie may be varied as described above . 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 composition comprising a nucleic acid molecule [ s ] comprising or consisting of nucleotide sequences of two or more different staphylococcal genes and encoding immunogenic polypeptides selected from the group consisting of : i ) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in seq id no : 1 or 6 ; ii ) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in seq id no : 2 or 7 ; iii ) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in seq id no : 3 or 8 ; iv ) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in seq id no : 4 or 9 ; v ) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in seq id no : 5 or 10 ; vi ) a nucleic acid molecule comprising or consisting of a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in i - v above ; or vii ) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in seq id no : 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 or 10 and wherein said nucleic acid molecule encodes a staphylococcal antigenic polypeptide . hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other . the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids , the nature of the hybridization method , and the composition and length of the nucleic acid molecules used . calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in sambrook et al ., molecular cloning : a laboratory manual ( cold spring harbor laboratory press , cold spring harbor , n . y ., 2001 ); and tijssen , laboratory techniques in biochemistry and molecular biology - hybridization with nucleic acid probes part i , chapter 2 ( elsevier , new york , 1993 ). the t m is the temperature at which 50 % of a given strand of a nucleic acid molecule is hybridized to its complementary strand . the following is an exemplary set of hybridization conditions and is not limiting : very high stringency ( allows sequences that share at least 90 % identity to hybridize ) wash twice : 2 × ssc at room temperature ( rt ) for 15 minutes each high stringency ( allows sequences that share at least 80 % identity to hybridize ) wash twice : 1 × ssc at 55 ° c .- 70 ° c . for 30 minutes each low stringency ( allows sequences that share at least 50 % identity to hybridize ) wash at least twice : 2 ×− 3 × ssc at rt to 55 ° c . for 20 - 30 minutes each . in a preferred embodiment of the invention said composition is a vaccine composition and includes and includes at least one carrier and / or adjuvant . the nucleic acid or dna combination vaccines comprise nucleic acid molecules that encode antigenic polypeptides as herein disclosed . the specific combinations of polypeptide antigens as represented by amino acid seq id can be substituted for the corresponding nucleotide seq id as herein disclosed in the manufacture of dna vaccines . according to a further aspect of the invention there is provided a combination vaccine according to the invention for use in the protection or treatment of a subject animal to a staphylococcal infection or condition that results from a staphylococcal infection . in a preferred embodiment of the invention said staphylococcal infection is caused by a staphylococcal species selected from the group consisting of : s . epidermidis , s . aureus , s . hominis , s . haemolyticus , s . warneri , s . capitis , s . saccharolyticus , s . auricularis , s . simulans , s . saprophytics , s . cohnii , s . xylosus , s . hyicus , s . caprae , s . gallinarum , s . intermedius , in a further preferred embodiment of the invention said staphylococcal species is s . aureus or s . epidermidis . in a preferred embodiment of the invention said subject is a human . in an alternative preferred embodiment of the invention said subject is a non - human animal , preferably a livestock animal , for example cattle . in a preferred embodiment of the invention said live stock animal is vaccinated against bacterial mastitis caused by staphylococcal bacterial cells . in a preferred embodiment of the invention said life stock animal is a caprine animal ( e . g . sheep , goat ). in a preferred embodiment 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 . an embodiment of the invention will now be described by example only and with reference to the following figures tables 3 and 4 illustrate the vaccination of a mouse model with a combination antigens of the extracellular domains of ydie and divib compared to individual antigen vaccinations . construction of plasmids for the overexpression in e . coli of the extramembranous fragments of the s . aureus proteins the phep selected peptide was synthesized and conjugated through a cysteine at its c terminal to the carrier protein klh to undertake as a chimeric protein used in vaccinations . the extramembranous fragments of ydie , divib , divic and ftsl were 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 oligonucleotide pairs indicated on table 1 according to 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 up to 2 . 5 minutes ; finally , ongoing amplification rounds were allow to complete at 72 ° c . for 4 min . the restrictions sites engineered within the oligonucleotides are also indicated in table 1 ( underlined ; ncoi or xhoi ). the amplified fragments were digested with the corresponding restriction enzymes ( ncoi for the 5 ′ end , and xhoi for the 3 ′ end ) and cloned into the equivalent sites of the pet - 21d (+) expression vector from novagen ( cat . no . 69743 - 3 ) and resulting in the overexpression plasmids indicated in table 1 generating a t7 - tagged ( partial , at the n - terminal ) and 6 × his - tagged ( at the c - terminal end ) form of the extramembranous fragments . in the seq ids the t7 - and his - tags are indicated in bold , and the extramembranous portion of the proteins of interest are underlined . the over expression plasmids were transferred into e . coli bl21 for over expression of the recombinant protein fragment . 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 hinge amino acids between the t7 - tag and the extramembranous fragment , and between the latter and the his - tag . these amino acids are neither bold nor underlined in the seq ids . seq id nos 25 through 28 were over expressed from plasmids pgl597 , pgl601 , palb26 , and palb27 in e . coli bl21 strain using brain heart infusion broth ( cm0225 , oxoid , united kingdom ) in the presence of 100 μg / ml ampicillin and the plac promoter gratuitous inducer iptg ( isopropyl β - d - 1 - thiogalactopyranoside , 1 mm ) for 4 to 6 hours at 37 ° c . and vigorous shaking . following harvesting of the cells by centrifugation ( 5 , 000 × g for 15 minutes at 4 ° c .) and subsequent lysis with 1 mg / ml lysozyme in phosphate buffer ( buffer a ; 0 . 1m ph7 . 2 ) containing 0 . 5m nacl ) for one hour and subsequent sonication ( 3 cycles of 10 second pulses in sonicating water bath ) the soluble and insoluble forms of the proteins of interest were separated by centrifugation at 13 , 000 × g for 10 minutes . the precipitate was then resuspended in buffer a containing 8m urea by freeze / thawing ( 3 cycles of freezing at − 80 ° c . for 10 minutes and subsequent thawing to room temperature ) and sonication ( 3 cycles of 10 second pulses in sonicating water bath ), and subsequent centrifugation for 25 minutes at 18 , 000 × g ). the over expressed proteins of interest in the supernatant and the solubilised pellet were purified by initial specific binding ( through their his - tag ) to a nickel ( niso4 )- bound sepharose chromatography column ( ni - sepharose ) and elution with an imidazole solution run through the column in the following stepwise manner : 5 % for 5 minutes , 30 % for 60 minutes , 35 % for 60 minutes , 50 % for 100 minutes and 55 % for 100 minutes . fractions from this stepwise elution were analysed in acrylamide denaturing gels with a 4 % acrylamide / bis - acrilamide stacking layer and a 12 % acrylamide / bis - acrylamide separating layer . the fractions containing the over expressed proteins of interest were pooled and dyalized against sterile phosphate buffer ( 8 g nacl , 0 . 2 g kcl , 1 . 44 g na 2 hpo 4 , 0 . 24 g kh 2 po 4 , per liter of distilled h 2 o , ph 7 . 4 ). all the proteins of interest were successfully over expressed from the indicated strains and under the indicated conditions . they were also subsequently extracted from the total cellular protein content of the over expressing e . coli strains with more than 95 % purity . examples of the purification obtained for each of the proteins are indicated below . evaluation of vaccination - mediated protection of balb / c mice against infection by s . aureus 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 . 5 × 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 were 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 divib - 2 was determined from difference in the number of s . aureus cells in the kidneys of animals vaccinated with klh and those vaccinated with divib - 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 divib - 2 vaccinated animals was concluded as protection . combination ( or polyvalent ) vaccines including variations of the antigens ( conjugated selected phep peptide , ydie , divib , divic and ftsl ) will follow an identical protocol with the following modifications . the vaccine priming and boost mixtures will contain rather than a single component 2 or more of the components . the total volume of mixed vaccine used for priming and boosting injections will fluctuate in a range of 50 - 100 microliters per animal . similarly the total amount in each of those injections may vary between 50 - 100 micrograms . the amount of each antigen to contribute to the total amount of vaccine in the priming or boosting mix will vary between 20 % to 80 % of the total . the various combinations of antigens to be evaluated as a vaccine mix will be undertaken according to the matrix in table 2 . the combinations are grouped in 3 tiers . depending on results from the 1 st tier of experiments the 2 nd tier of experiments would be undertaken accordingly , and depending on the results from the latter the 3 rd tier will be undertaken . in each tier a vaccination experiment will contain an antigen in the y axis , together with those ticked along the x axis , and labelled with the same colour . each vaccination experiment is labelled with a different colour . the experimental sample consisted in a combination antigen of the extracellular domains of ydie and divib . the amount of antigen administered to each mouse ( female balb / c , approx . 5 - 6 weeks old ) was 5 ug of ydie plus 50 ug of divib . those amounts were contained within 100 ul of eluent consisting on a 50 : 50 v : v of pbs ( phosphate buffer saline ) and complete freund &# 39 ; s adjuvant ( used for the vaccination priming ) or incomplete freund &# 39 ; s adjuvant ( used for the vaccination boost ). priming was undertaken day 0 , boost 1 at 14 days , and boost 2 at 21 days . subsequently , 7 days later , i . e ., at day 28 the animals were infected with staphylococcus aureus strain newman . each test group ( control and experimental ) had 10 animals . the bacterial dose administered to the animals ( both , control and experimental ) contained 4 × 10 6 bacteria in 100 ul of pbs . the infection period was run for 3 days , and the weight of the animals was monitored daily ( we also extracted organs to evaluate bacterial loads in organs , table 4 ). at that point the animals were sacrificed . the output of the experiment was calculated as the percentage body weight loss between day 3 and day 0 for every animal . the results of these experiments are shown in table 3 . statistical analysis of data in table 3 : test : non - parametric statistical hypothesis test — mann - whitney u selected options : two - tailed , unpaired , 95 % confidence interval . results : ( two group comparison ) ii and i : p = 0 . 622 iii and i : p = 0 . 039 * iv and i : p = 0 . 001 * ii and iii : p = 0 . 061 ii and iv : p = 0 . 008 * iii and iv : p = 0 . 158 * comparisons with statistically significant difference between the groups test : non - parametric statistical hypothesis test — mann - whitney u selected options : two - tailed , unpaired , 95 % confidence interval . results : ( two group comparison ) ii and i : p = 0 . 2 iii and i : p = 0 . 105 iv and i : p = 0 . 009 * ii and iii : p = 0 . 378 ii and iv : p = 0 . 131 iii and iv : p = 0 . 504 * comparisons with statistically significant difference between the groups
the disclosure relates to a composition comprising two or more immunogenic staphylococcal polypeptides and a multivalent vaccine composition comprising the immunogenic staphylococcal polypeptides .