WO2017136400A1 - Vlp-based vaccines for targeting staphylococcus aureus secreted virulence factors - Google Patents

Abstract

The present invention is directed to virus-like particles (VLPs) which are engineered to present epitopes from Staphylococcus aureus (SA), preferably autoinducing peptides (AIPs) which regulate quorum-sensing dependent virulence in this pathogen, or epitopes from SA toxins and leukocidins. These VLPs may be used to provide immunogenic compositions and efficacious vaccines. In a mouse model of SA dermonecrosis, vaccination with AIP-containing VLPs or SA toxin-containing VLPs induces protective immunity to limit the pathogenesis of SA infection and promote bacterial clearance.

Description

VLP-foased Vaccines for a getin Siaphyhcf/ee aurem Secreted Virulence Factors

Related Applications and Grant Support

This applicat ion claims the benefit of priority o f Uni ted States provisional application US62/290.,0 2 of identical title., filed February 2 , 20.16., the entire contents of which

application is incorporated by reference herein.

This invention was made with government support under grant nos. AI091917, AO 14706 and ΑΪ083305 awarded by the National Institutes of Health, The government has certain rights in the invention.

Field of the Invention

The present invention is directed to virus-like particles (VLPs) which are engineered to present epitopes from Staphylococcus aureus (SA) auioinducing peptides (AiPs), which regulate quorijm-sensing dependent virulence in this pathogen, or epitopes from S A toxins and leukocidins. These VLPs may be used to provide immunogenic compositions and efficacious vaccines. In a mouse model of SA dermonecrosis, vaccination with ΑΓΡ- contaming VLPs or SA toxin-containing VLPs induces protective immunity to limit the pathogenesis of SA infection and promote bacterial clearance.

Background and Overview of the invention

The Gram Positive pathogen Staphylococcus aureus (SA),. including both

methiciliin senstttve and niethicillin-resistant SA (MSSA, MRSA), is a .major cause of human disease and the primary cause of skin and soft tissue infection (SSTI) in the US.

Staphylococcus aureus is a Gram-positive bacterium well known for what is commonly known as staph infections. More serious forms of this infection can progress to

bacterial pneumonia and bacteria in the bloodstream. These conditions sometimes can be fataL With the advent of antibiotics, over time certam strains of S. aureus became resistant to antibiotics. Drug-resistant, including methicillin-resistant S. aureus (MRSA) infections began to appear. Today, MRSA is viewed as any strain of S aureus that has developed resistance to β-lactaras and other antibiotics, which include the penicillins, erythromycin, methicillui, dicloxacillin, nafcilHn, oxacillin, the cephalosporins and others. Resistance does render M SA infections far more difficult to treat with standard antibiotics. MRSA is a dangerous infection and poses serious health problems to the genera! public especially in hospitals, prisons, and nursing homes, but also in various community settings. People who are immunocompromised (for example, those with diabetes) or have immune systems that are weakened are at much greater risk of infection than the general public. MRSA causes a range of diseases from skin and wound infections to pneumonia and bloodstream infections that can cause sepsis and death.

Both community acquired MRSA (CA-MRSA) and hospital acquired MRSA (HA- MRSA) are resistant to traditional ami -staphylococcal p aetam antibiotics.

Staphylococcus ati eus is the leading cause of skin and soft tissue infections (SSTIs) in the United States, Mounting antibiotic resistance requires innovative treatments such as ones thai inhibit S. aureus pathogenicity and support innate immune clearance. aureus coordinates virulence factor expression through the density-dependent accessory gene regulator (cigr) operon via secretion of cyclic autoitiducing peptides (AIPs). «V, aureus lacking agr fails to cause dermonectosis in mouse models of SSTI and is more readi ly cleared compared to agr positive isolates. Therefore, the inventors hypothesized that vaccination against 8. aureus AIP could generate protective immunity against subsequent SSTI challenge. Because S. aureus AIPs are too small to stimulate a natural immune response (7-9 amino acids), the inventors engineered a viras-like-particle (PP7-VLPs) for surface presentation of a modified autoinducing peptide sequence (AIP1.S). VLP-based vaccines allow multivalent presentation of target antigens and are highly unmunogenic due to their repetitive, vires-like structure. As expected, vaccination with PP7-AIP1S induced AIP 1 -specific antibodies, and transcriptional analysis of skin from vaccinated and challenged mice showed that PP7-AI IS vaccination limits %^rr-activation in vivo. Most importantly, in a challenge model of >$'. aureus SSTI, PP7-A1P1S vaccinated mice showed significantly reduced deraioiieerosis and increased bacterial clearance compared to control vaccinated mice, demonstrating the efficacy of this vaccination approach. To the best of our knowledge, this is the first report of an efficacious, VLP-based vaccine which induces immune control of 8. aureus AIP1- regu!ated virulence. To date, no vaccine against SA has been successful in clinical trials. However, these data suggest that VLP-based vaccination, in particular, PP7-AJP1S

vaccination could be an effective tool to limit..S. aureus pathogenesis during SSTI. Brief Description of the Invention

Pursuant to the present invention, the inventors used VLPs to present epitopes from SA autoinducmg peptides (AiPs), which regulate quorum-sensing dependent virulence in tins pathogen, or epitopes from SA toxins and leukoeidins, as efficacious vaccines, in a. mouse model of SA derrnonecrosis, vaccination with A IP- VLPs or SA toxin- VLPs induces protective immunity to limit the pathogenesis of SA infection and promote bacterial clearance.

The development, and commercialization of vaccines for bacterial infections,

especially vaccines for Staphylococcus aureus infections including MRSA, would be a significant public health breakthrough towards the goal of controlling and eradicating Staphylococcus aureus infections, especially MRSA infections, given how rapidly bacterial resistance occurs in these microbes.

The present invention provides immunotherapeutic and prophylactic bacteriophage viral-like particles (VLPs) which are useful in the treatment and prevention of

Staphylococcus aureus (SA) infections, especially MRSA and related disorders. Related compositions (e.g. vaccines), nucleic acid constructs, and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses against Staphyloc ts aureus and protect against SA challenge in vivo. VLPs, VLP- containing compositions, and therapeutic methods of the invention indisce an immunogenic response against SA infection,, confer immunity against SA infection, protect against SA infection, and reduce the likelihood of infection by and/or inhibit SA infection, especially including MRSA infection.

Because antibodies that are specific for epitopes of AiPs which are thiolactone.

(cyclic), peptides may be necessary for antibody-mediated neutralization of Staphylococcus aureus. AlPi or A!Pl S (also referred to as AIP1C4S) targeting VLPs and related

compositions (e.g. vaccines) of the invention provide a more comprehensi ve protection against infection by Staphylococcus aureus, especially including MRSA. Surprisingly, these do not require the presence of the thiolactone in the epitopic peptide in order to provide excellent immunogenicity. Thus, the invention provides immunoiherapeutic and prophylactic bacteriophage viral-like particle (VLPs) which are useful in the prevention of Staphylococcus aureus (SA), including MRS A, infections and related disease states and conditions, including persistent infections associated with SA. Related compositions (e.g. vaccines), nucleic acid constructs, and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses against S. aureus and protect against S. aureus challenge in viva, VLPs, VLP~eontamiiig compositions, and therapeutic methods of the invention induce an immunogenic response against SA infection, confer immunity against SA infection, protect against SA infection, and reduce the likelihood of infection by SA.

In a first embodiment, the invention provides a VLP comprising a bacteriophage single chain coat polypeptide dimer and an epitopic S. aureus heterologous peptide ("SA peptide"), wherein the epitopic SA peptide is displayed on the VLP in the A-B loop (in the downstream or upstream A-B loop, preferably the downstream A-B loop), or at the amino or carboxy! terminal ends of the dimer, and wherein vaccination with the V LP is prophylactic for & cmre -mdiKed disorders. In embodiments of the invention, the epitopic SA heterologous peptide is a SA autoinducing peptide (AIP), which regulates quorum-sensing dependent virulence in SA or is an epitopic peptide from SA toxins and lukocidins as otherwise described herein. In preferred embodiments, the epitopic S A heterologous peptide is the peptide AIP1 (YSTCDFIM, SEQ. ID NO; I) or the peptide ASP I S (YSTSDFSM SEQ. ID NO:2), which are set forth in figure 10 hereof (note that the thioSaetone is not expressed on the VLP) . In preferred embodiments of the invention, the expressed epitopic peptide on the VLP does not contain a thioiactone group, in alternative preferred embodiments, the SA heterologous peptide is A1P2 GVNACSSLF (SEQ ID NO: 3) or A1P2S GVNASSSLF (SEQ ID NO: 4) AIP3 INCDFLL (SEQ ID NO: 5} or A1P3S INSDFLL (SEQ ID NO: 6)

AIP YSTGYFIM (SEQ IB NO: 7) or AIP4S YSTSYP!M (SEQ ID NO: 8). In certain embodiments, the VLP expresses two of the above heterologous epitopic peptides.

In another aspect, the invention provides a composition comprising a VLP comprising a bacteriophage single chain coat polypeptide dimer and an epitopic SA peptide, wherein the epitopic SA peptide is displayed on the VLP, and wherein the composition is prophylactic for SA-indoced disorders, especially including SA infections, including M SA and related disease states and/or conditions. Certain aspects of the invention reflect that the single-chain dimer of PP7 (as well as MS2) coat protein can tolerate the insertion of a wide variety of peptides, including peptides deri ved from cyclic autoinducing peptides AIPs and are highly immunogenic, even though the AIPs tend to be of sm all size and the thioiactone bond has heretofore hindered vaccine development.

In addition to heterologous peptides based upon AIPs, other SA toxin and leukocidin peptide sequences may be used and are described in greater detail in the detailed description of the invention which follows.

In another aspect, the invention provides a composition comprising a VLP comprising a bacteriophage single chain coat polypeptide dimer and a SA epitopic peptide as otherwise described herein (preferably, a AIP peptide, e.g. AiPl, AIP1S. AIP2, AIP2S, AIPS, AJP3S, ΑΣΡ4 or AIP4S, especially A IP I or AIP I S as otherwise described herein), wherein the heterologous peptide is displayed on the VLP, preferably in an unconstrained conformation, and preferably eneapsidates bacteriophage mRNA, and wherein the composition is

imunotherapeutic and prophylactic for SA-induced disorders. The AIP peptide, when incorporated into the VLP does not contain a. thioiactone or is displayed without the thioiactone (the earboxylic acid of the methionine is incorporated as a peptide bond into the VLP structure), while still providing excellent immunogemeity.

In certain embodiments, VLPs and VLP-eontaining compositions (e.g. vaccines) of the invention are comprised of VLPs comprising AIP peptides, heterologous peptides from SA toxins and/or lukocidins. In other aspects, VLPs and VLP-containing compositions of the invention comprise hybrid V LPs that displa SA epitopic peptide sequences preferably in an unconstrained conformation derived from several AIPs (e.g. AIP I , AIP1S, AIP2, AIP2S, ΑΪΡ3, AIP3S, ΑΪΡ4 or AIP4S).

In another aspect, the invention provides a composition comprising a VLP displaying SA epitopic peptides from two or more peptides on the same VLP, preferably in an unconstrained conformation, and wherein the composition is immunotherapeutic and prophylactic for SA-induced disorders. i embodiments, the invention provides a VLP, or a composition comprising VLP, wherein the V LP is made by transforming a prokaryote with a nucleic acid construct

comprising either:

(1) (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of a bacteriophage (e.g., PP7 of MS2, preferably a PP7) single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to: (i) define a first restriction site which is located in the upstream or downstream (preferably upstream) portion of the coat polvpeptide dimer coding sequence and which is either

positioned 5' to, or located within, the sequence which defines the coat polypeptide dimer A- B loop, N-termmus or carboxy-terminus, and (ii) contain a nucleotide sequence encoding a SA epitopic peptide; (b) a second restriction site positioned 3' to the coat polypeptide dimer coding sequence; (c) an antibiotic resistance gene which is operably associated with the promoter; and (d) a replication origin for replication in a prokaryotic cell; or

(2) (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of bacteriophage (e.g. PP7 or MS2 single chain coat polypeptide dimer, wherein the coat, polypeptide dimer coding sequence is modified to (i) define a codoii sequence positioned 5' to that portion of the sequence which defines the coat polypeptide dimer A-B loop, N- terminus or carboxy-terminus, and (ii) contain a nucleotide sequence encoding a SA epitopic peptide; (b) a restriction site positioned 3^* to the coat polypeptide dimer coding sequence; (c) a PCR primer positioned 3' to the second restriction site; id) repressor to resistance to a first antibiotic, wherein the repressor is operably associated with the promoter; (e) a helper phage gene modified to contain a gene conferring resistance to a second antibiotic, and (f) a replication origin for replication in a prokaryotic cell.

In certain aspects, the invention provides a VLP, or a composition comprising a VLP, wherein the VLP is made by transforming a prokaryote with a nucleic acid construct

comprising either:

(!) (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of a bacteriophage (preferably PP7 or S2, more preferably PP7) single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to: (i) define a first restriction site which is located i the downstream portion of the coat polypeptide dimer coding sequence and which is either positioned 5' to, or located within, the sequence which defines the coat polypeptide dimer AB loop, and (ii) contain a nucleotide sequence encoding a SA epitopic peptide, preferably a AIP epitopic peptide, such as ΑίΡί or AIP1 S;

(b) a second restrictio site positioned 3' to the coat polypeptide dimer coding sequence;

(c) an antibiotic resistance gene which is operabl y associated with the promoter; and

(d) a replication origin for replication in a prokaryotic. cell; or

(2) (a) a bacterial or bacteriophage promoter which is operabiy associated with a coding sequence of a bacteriophage (preferably PP7 or S2, more preferably PP7), single chain coat polypeptide dimer, wherein the coat polypeptide dimer codin sequence is modified to:

(i) define a first restriction site which is located in the downstream portion of the coat polypeptide dimer coding sequence and winch is either positioned 5' to, or located within (preferably within), the sequence which defines the coat polypeptide dimer AB loop, and

(ii) contain a nucleotide sequence encoding a SA epitopic peptide, preferably a AIP epitopic peptide, such as A!Pl or AIP IS;

(b) a second restriction site positioned 3' to the coat polypeptide dimer coding sequence;

(c) a PCR primer positioned 3⁵ to the second restriction site;

(d) an antibiotic resistance gene which is operabiy associated with the promoter; and

(e) a replication origin for replication in a prokaryotic cell; or

(3) (a) a bacterial or bacteriophage promoter which is operabiy associated with a coding sequence of a bacteriophage (preferably PP7 or MS2, more preferably PP?) singl e chai coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to (i) define a cotton sequence positioned 5' to that portion of the sequence which defines the coat polypeptide dimer AB loop, aad (ii) contain a nucleotide sequence encoding a SA epitopic peptide, preferably a AIP epitopic peptide, such as AIP I or AIPIS;

(b) a restriction site positioned 3' to the coat polypeptide dimer coding sequence;

(c) a PCR primer positioned 3' to the second restriction site;

(d) an antibiotic resistance gene for resistance to a first antibiotic, wherein the resistance gene is operabiy associated with the promoter;

(e) a helper phage gene modified to contain a second antibiotic resistance gene conferring resistance to a second antibiotic, and

(f) a replication origin for replication in a prokaryotic cell in alternative embodiments, die present invention provides a VLP, or a composition comprising a VLP, wherein the V LP is made by transforming a prokaryote with a nucleic acid construct comprising either;

(1) (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of bacteriophage PP7 single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to: (i) define a first restriction site which is located in the downstream portion of the coat polypeptide dimer coding sequence and which is either positioned 5' to, or located within, the sequence which defines the coat polypeptide dimer N-termmus, and (is) contain a -nucleotide sequence encoding a SA epitopie peptide, preferably a AIP epitopie peptide, such as Al PI or AJP1 S (b) a second restriction site positioned 3' to the coat polypeptide dimer coding sequence; (c) an antibiotic resistance gene which is operably associated with the promoter; and (d) a replication origin for replication in a prokaryotic cell; or

(2) (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of bacteriophage MS2 single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to (i) define a codon sequence positioned 5' to that portion of the sequence which defines the coat polypeptide dimer N-termraus, and (ii) contain a nucleotide sequence encoding a SA epitopie peptide, preferably a AIP epitopie peptide, such as A1P1 or AIP IS; (b) a restriction site positioned 3^* to the coat polypeptide dimer coding sequence; (e) a PGR primer positioned 3' to the second restriction site; (d) a repressor to resistance to a first antibiotic, wherein the repressor is operably associated with the promoter; (e) a helper phage gene modified to contain a gene conferring resistance to a second antibiotic, and (f) a replication origin for replication in a prokaryotic ceil.

In certain aspects, the invention provides VLPs .made by transforming a prokaryote with a SA epitopie peptide sequence-containing construct as described herein. In other aspects, VLPs and VLP-corttaining compositions (e.g. vaccines) of the invention are comprised of VLPs comprismg SA epitopie peptides derived from SA aiitoinducing peptides, which regulate quorum-sensing dependent virulence in SA or epitopie peptides from SA toxins and lukocidins. I other aspects, VLPs and VLP-contasning compositions of the invention comprise hybrid VLPs that display multiple SA epitopie sequences. in certain embodiments, the coding sequence of the bacteriophage single chain coat polypeptide dimer, especially PP7 or MS2, preferably PP7, further comprises a transcription terminator positioned 5^* to the second restriction site.

In certaia aspects, the invention provides a method of inoculating a subject^' at risk of developing a SA-related disorder, including an SA infection, including a MRSA infection, the method comprising administering to the subj ect one or more doses of composition comprising a SA epitopie peptide-containing VLP as described herein. I other aspects, the invention provides a method of treating a subject who is at risk of developing a SA-related infection, including MRSA or a disorder, the method comprising administering to the subject one or more doses of a composition comprising a SA epitopie peptide-containing VLP as described herein. In still other aspects, the invention provides a method of treating a subject who has developed a SA-related infection or disorder, including MRSA, the method comprising administering to the subject one or more doses of a composition comprising a SA epitopie peptide containing VLP as described herein.

Thus, the inventors describe the use of recombinant VLPs derived RNA

bacteriophages to induce high titer antibody responses against SA epitopie peptides that protect against SA infections, including MRSA infections and related disorders.

These and other aspects of the invention are described further in the Detailed

Description of the Invention, which follows.

Brief Description of the Figures

Figure 1 shows the cloning of the Staphylococcus ntrem (S A) auto-inducing peptide I (AIPI) with a C4S mutation into the AB loop of the PP? dimer. This peptide is also referred to as AlPiS. AIPI is the quorum sensing peptide produced by agr type 1 SA isolates and is required for agr signaling and virulence. SA isolates exist as one of four agr types (agr I-IV) with each type making a corresponding AIP (AIPI -4).

Figure 2 shows purification of PP7-AIP1S on gel filtration (right). Figure 3 shows the purity of PP7-AIP1S oft 1% agarose ge! electrophoresis eihidium bromide (EtBr) stain (left) and 4-12% SDS/PAGE CB Stain (right).

Figure 4 shows the homogeneity of PP7-AI 1 S using Malver Zetasizer Dynamic Light Scattering in PBS (top two panels) and PBS + TWEEN SO (0.2%).

Figure 5 shows a schematic of a vaccination schedule. Four week old female BALB/c mice were vaccinated by IM injection with PBS control, PP7 control or PP7-AI IC4S (note that ASP1 C4S and AIPIS are equivalent peptides), A boost, was given 4 weeks later and mice were challenged with a SA skin infection 2 to 8 weeks after the boost.

Figure cS shows that PP7~Ai l.C4S vaccination protects mice against weight loss, used as a measure of morbidity, during skin infection challenge with agr type I MRS A.

Figure 7 shows that PP7-AlPlC4S vaccination protects mice against abscess formation, used as a measure of pathogenesis, during skin, infection challenge with agr type I MRSA.

Figure 8 shows PP7-AIP1C4S vaccinatio protects mice against demionecrosis (lei) during skin infection challenge with agr type Ϊ MRSA, Toxins regelated by agr are required for demionecrosis, suggestin that vaccination with PP7-AIPIC4S induces protection against AIP signaling. PP7-A1P1 C4S vaccinated mice are also better able to clear SA at the site of infection (right). This is consistent with inhibition of immune cell lytic toxins regulated by agr.

Figur 9 shows the agr pathway schematic.

Figure 10 shows the design and preparation of PP7~AI 1S VLPs. (a) Schematic of AIP.1 and amino acid sequence of AIP1-C4S (A IPI S). (b) Ribbon representation of the PP7 coat protein dimer depicting the first AB loop (indicated by arrow) and the AIPIS sequence (spheres) modeled into the second AB loop (PDB ID

using Galax Web3i. Image prepared using PyMol (PyMOL molecular graphics system, version 1.5.0.4; Schrodinger. LLC), (c) Schematic of the site of AIPIS insertion into the second AB loop of the PP7 single chain dimer . (d) Agarose gel electrophoresis of size exclusion chromatography fractions showing assembly and parity of FP7-A1PJ S based on Coomassie (protein) and et idium bromide (EtBr) staining (showing VL encapsulated nucleic acids).

Figure i 1 PP7-AIP1S vaccination induces antibodies which recognize soluble AIPL BALB/c mice were vaccinated twice (i.m.) at 4 week Intervals with 10 § of PP7-AIP1.S or PP7 wild-type (control), (a) Serum was collected at the indicated time points after the second vaccination. Serum was then pooled (n-3 mice per group), treated as described in Materials and Methods, and relative binding to PP7-AIP1S determined by ELISA. (b) PP7-A1P1 S antiserumeoileeted at eight weeks after the second vaccination was prepared as in (a), and relative AIP1S binding determined in the and absence of the indicated

concentrations of Ai l or AI 2 (n-3 mice per group; duplicate experiments performed in triplicate). Data are mean ± s.e.m.Krusfcal-Wallis ANOVA p<0.0001 with Dunn's post-test: * p<0.05; ***p<0.001.

Figure 12 shows that PP7-AIP1 S vaccination limits the severity of aureus skin infection i a mouse model of deraionecrosis. BALB/c mice were vaccinated twice (i.m.) at 4 week intervals with 10 pg of the indicated VLPs or PBS control Eight weeks after the second vaccination, mice were challenged by subcutaneous infection with 4 x I C CFU of USA300 LAC Representative (a) day 3 images of infection site and (b) daily measures of abscess area and dermonecrosis. Calculated area under the curve (AUG) values for (c) abscess area (ANOVA p<0.0042)_i (d) dennonecrosis (p~^:0,0i77) and (e) percent weight change over the six day infection, as well as (f) day 6 bacterial burden at the site of infection (p=0,0001 ) (representative of two independent experiments of n^::::6 mice per group), (g) Cytokine levels in clarified abscess tissue homogenate on day 6 postinfection (ANOVA IL-lp, p=O,0587; TNF{, p-O.0358) (n=6 mice per group). Dat are mean ± s.e.m. ewman-Keuls post-test; ns, not significant; * p<0.05; * *ρ<0.01 ; ***p<0.001. Some of this data is also presented in figures 6-8.

Figure 13 shows that PP7-AIP1S vaccination limits agr function at the site ofS. aureus infection. BALB/c mice were vaccinated twice (i.m.) at 4 week intervals with 10 pg of the indicated VLPs or PBS control. Eight weeks after the second vaccination, mice were challenged by subcutaneous infection with 4 x IO? CPU of L1SA300 LAC. (a) Local RNAf II transcription on day 1 postinfection measured b qPCR (n-^:4 mice per group, Kraskal-Wallis ANOVA p^::::0.O⁽}29}, (b Representative immunoblot (showing 3 replicates) and quantification of Hia levels (relative to PBS control) in clarified abscess tissue homogenate on day 6 postinfection (n^~6 mice per group) (Knsskal-Wallis ANOVA p^:"0.0025) with Dunn's post-test: ns, not significant; * p<O.0S; **p<-HO.0L

Figure 1 shows an anii-AIPI S antibody mechanism of action of the present invention, based upo the results of experimentation described in the Examples section hereof.

Detailed Descri ption of the invention

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skil l of the t. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual"; Ausubel, ed., 1994, "Current Protocols in Molecular Biology" Volumes MH; Cells, ed., 1994, "Cell Biology: A Laboratory Handbook" Volumes I-III; Coligan, ed,, 1994, "Current Protocols in Immunology" Volumes I-III; Gait ed., 1984, "Oligonucleotide Synthesis"; Barnes & Higgins eds., 1 85, "Nucleic Acid

Hybridization"; Hames & Biggins, eds., 1 84, "Transcription And Translation"; Freshney, ed., 1986, "Animal Cell Culture"; IRL Press, 1986, "Immobilized Cells And Enzymes"; Perbal, 1984, "A Practical Guide To Molecular Cloning."

Where a range of values is provided, it is understood that each intervening value, to t¾e tenth of the unit of the lower limit unless the context clearly dictates otherwise,

between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

Unless defined otherwise, all technical ^'and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be usee! in the practice or testing of the present invention, the preferred methods and materials are now described.

It niust be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set out below.

The term "patient" or "subject" is used throughout the specification within context to describe an animal, generally a mammal and preferably a human, to whom treatment including prophylactic treatment (prophylaxis), with the immunogenic compositions and/or vaccines according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, in most instances, the patient or subject of the present invention is a human patient of ei ther or both genders.

The term "effective" is. used herein, unless otherwise indicated, to describe a number of VLP's or an amount of a VLP-eontaining composition which, in context is used to produce or effect an intended result, whether that result relates to the prophylaxis and or therapy of an SA-mduced or SA-related disorder or disease state, including an SA infection or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms (including the term "therapeuticall effective^5*) which are otherwise described or used in the present application.

As used herein, the te : "po lynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynacieoiides, and includes both double- and single- stranded D and NA. A polynucleotide .may include nucleotide sequences having different functions, such as coding regions, and non-coding regions such as regulatory sequences (e.g., promoters or transcriptional terminators), A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques, A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" aiso includes molecules whicli contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that ate joined together, eovalently or noncovalently, as raultimers (e g., diroets, tettaraers). Thus, die terras peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that, these terms do not connote a specific length of polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using

recombinant, techniques, chemical or enzymatic synthesis, or is naturally occurring.

The term "single-chain dimer" refers to a normally dimeric protein whose two sub nits of coat polypeptide of a. RNA bacteriophage ha ve been genetically (chemically, through covaient bonds) fused into a single polypeptide chain. Specifically, in the present invention single-chain dimer versions of PP7 coat proteins were constructed. Bach of these proteins is naturally a dimer of identical polypeptide chains. In the PP? coat protein dirners the N-tenninus of one subu t lies in close physical proximity to the C -terminus of the companion subunit. Single-chain coat protein dimers were produced using recombinant DNA methods by duplicating the DMA coding sequence of the coat proteins and then fusing them to one another in tail to head fashion. The result is a single polypeptide chain in which the coat protein amino acid appears twice, with the C -terminus of the upstream copy

covaleatly fused to the -terminus of the downstream copy, ^'Normally (wild-type) the two subunits are associated only through aoncovalent interactions between the two chains. In the single-chain dimer these noncovalent interactions are maintained, but the two subunits have additionally been eovalently tethered to one another. This greatly stabilizes the folded structure of the protein and confers to it its high tolerance of peptide insertions as described above.

This application makes frequent reference to coat protein's "AB-loop" The RNA phage coat, proteins possess a conserved tertiary structure. The PP7 coat proteins, for example, possess a structure wherein eac of the polypeptide chains is folded into of a number of β-strands. The & -strands A and B form a hairpin with a three-araino acid loop connecting the two strands at the top of the hairpin, where it is exposed on the sur face of the VLP. As evidenced in the present application, peptides inserted into the AB-loop are exposed on the surface of the VLP and are strongly immunogenic. The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional is retained by the polypeptide. N¾ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the earbox terminus of a polypeptide.

The term "valency" is used to describe the density of the SA epitopic peptide

(preferabl a heterologous AIP thio lactone peptide such as AIP 1 or AIPS) displayed on VLPs according to the present invention. Valenc in the present invention may range from low valency to high valency, from less than 1 to more than about i SO, preferably 90 to i SO.

immunogenic compositions according to the present invention comprise VLPs which are preferably high valency and comprise VLPs which display at least 50-60 up to about ISO or more SA epitopic peptides, preferabiy an AIP, more preferabiy ΑΣΡ1 or AIPS.

The term "coding sequence" is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally detemiined by a obosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5'- end of the mRNA and a transcription terminator sequence located j ust downstream of the open reading frame at the 3'~ end of the mRNA. A coding sequence can include, but is not limited to, DMA, cDHA, and recombinant nucleic acid sequences.

A "heterologous" region of a recombinant ceil is an identifiable segment of nucleic acid within a larger nucleic acid molecule that is not found in association with the larger molecule in nature.

An "origin of replication" refers to those DMA sequences that participate in D A synthesis.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream ($' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Profcaryotic promoters contain SMne-Dalgamo sequences in addition to the -10 and -35 consensus sequences.

In bacteria, transcription normally terminates at specific transcription termination sequences, which typically are categorized as rbo-dependent and rho-independent (or intrinsic) terminators, depending on whether they require the action of the bacterial rho-faetor for their activity. These terminators specify the sites at which RNA polymerase is caused to stop its transcription activity, and thus they largely define the 3 '-ends of the E As, although sometimes subsequent action of ribonuc leases further trims the RN A.

An "expression control sequence" is a DMA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control:" of transcriptional and trartslational control sequences in a cell when RN A

polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. Transcriptional and translations! control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylatkm signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell

An "antibiotic resistance gene" refers to a gene that encodes a protein that renders a bacterium resistant to a given antibiotic. For example, the kanamycin resistance gene directs the synthesis of a phosphotransferase thai modifies and inacti vates the drag. The presence on plasmids of a kanamycin resistance gene provides mechanism to select for the presence of the pi asm id within transformed bacteria. Similarly, the chloramphenicol resistance gene allows bacteria to grow in the presence of the drug by producing an acetyitransferase enzyme that inactivates the antibiotic through acety!ation.

The term "PCR" refers to the polymerase chain reaction, a technique used for the amplification of specific DNA sequences in vitro. The term "PGR primer" refers to DNA sequences (usually synthetic oligonucleotides) able to anneal to a target DNA. thus allowing a DNA polymerase (e.g. Taq DNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are used in the polymerase chain reaction to initiate UNA synthesis on each of the two strands of a DMA and to thus amplify the DMA segment between two primers.

Representative PCR primers which used in the present invention are those which are presented in the examples section hereof. Additional PCR primers may be obtained for the various S.A epitopk peptides which are presented herein.

Examples of primers used for PCR described above and otherwise in the presen inventio are presented in the examples section (Methods). In addition to those primers, the following primer E3.2: 5' CGG GCT TTG TTA GCA GCC GG 3' - (SEQ ID No. 39) may serve-as the 3^s (reverse)-primer in PCR reactions to amplify coat protein. Primers useful in the present invention, among others, are otherwise set forth in the examples (Methods) section of the present application.

A cell has been "transformed" by exogenous or heterologous D A when such DMA has been introduced inside the celt. The transforming DNA may or may mi be integrated (covalently linked) into chromosomal DN A making up the genome of the cell In

prokaryotes, yeast, and mammalian cells for example, the transforming DMA may be maintained on an episoraal element suc as a plasmid, which normally replicate

independently of the bacterial chromosome by virtue of the presence on the plasmid of a replication origin. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that i t is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic ceil to establish cell tines or clones comprised of a population of daughter cells containing the transforming DNA,

A "signal sequence" ca be included before the coding sequence. This sequence encodes a signal peptide, N-tenninal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell, Signal sequences can be found associated with a. vari ety of proteins nati ve to prokaryotes and eukaryotes. It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the poK eptide(s) of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which are degenerate to the nucleic acids disclosed herein. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the polypeptide(s) of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which are degenerate to the nucleic acids disclosed herein. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 4 such amino acids, and more often, consists of at least 5-10 such amino acids . Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2 -dimensional nuclear magnetic resonance.

As used herein, the term "coat protem(s}" refers to the protein(s) of a bacteriophage or a RNA -phage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA-phage. These include, but ar not limited to PP7, MS2, AP205, Q¾ R17, SP, PP7, GA, Ml 1, MXl , f4, CbS, CM2r, Cb23r, 7s and £2 RNA bacteriophages. Preferred coat proteins which are used in the present invention include coat proteins from bacteriophages include PP7, MS2, AP205, Q$. Preferably, PP7 or MS2 coat polypeptides are used to create VLPs according to the present invention.

As used herein, a "coat polypeptide" as defined herein is a polypeptide fragment of the coat protein that possesses coat protein function and additionally encompasses the full length coat protein as well or single-chain variants thereof.

As used herein, the terra ".immune response" refers to a humoral immune response and/or cellular immune response leading to the activation or proliferation of B- and/or T- lymphocytes and/or antigen presenting cells. In some instances, however, the immune responses may be of iow intensity and become detectable only when using at least one substance in accordance with the invention. 'Immunogenic" refers to an agent used to stimulate the imimrae system of a living organism, so that one or more functions

of the immune system are increased and directed towards the immunogenic agent. An

"immunogenic polypeptide" is a polypeptide that elicits a cellular and/or humoral

immune response as described above, whether alone or linked to a carrier in the presence or absence of an adjuvant. Preferably, antigen presenting cell may be activated.

As used herein, the terra Vaccine" refers to a formulation -which contains the composition of the present in ven tion and which is in a form that is capable of being administered to an animal, often a human patient or subject.

As used herein, the term -"virus-like particle of a bacteriophage" refers to a virus-like panicle (VLP) resembling the structure of a bacteriophage, being non-replicative and noninfectious, and lacking at least the gene or genes encoding for the replication machinery of the bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.

This definition should, however, also encompass vims-like particles of

bacteriophages, in which the aforementioned gene or genes ar still present but inactive, and, therefore, also leading to non-replicative and noninfectious virus-like particles of a bacteriophage.

VLP of EN A bacteriophage coat protein: The capsid structure formed from the self- assembly of one or more stibonits ofRNA bacteriophage coat protein and optionally containing host. RNA is referred to as a "VLP of RNA bacteriophage coat protein". In a particular embodiment, the capsid structure is formed from the self assembly of 90 coat protein single-chain diniers or 180 coat protein monomers.

A nucleic acid molecule is "operatively linked" to. or "operably associated with", an expression control sequence when the expression control sequence controls and regulates the transcription and translation of nucleic acid sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATGj in front of the nucleic acid sequence to be expressed an maintaining the correct reading frame to permit expression of the nucleic-acid sequence under the control of the expression control sequence and production of the desired product encoded by the nucleic acid sequence, if a gene that one desires to insert into a recombinant DMA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene,

SA-Indnced Disorders, Immunogesicity, and Prophylactic Efficacy

"SA-induced disorders" or "SA-reiated disorders" include, but are not limited to, the disorders identified in this application which are caused by & aureus infections, including the infection itself, which may be a niefhieilim sensitive Staphylococcus aureus SSA) infection or a meihieillin resistant -. Staphylococcus aureus- (MRS A) infection,

immimogenicity and prophylactic efficacy (e.g. whether a composition is prophylactic for SA-induced disorders) may be evaluated either by the techniques and standards mentioned in this section, or through other methodologies that are well-known to those of ordinary skill in the art.

To assess imniunogenicity (e.g. whether a composition has induced a high titer antibody responses against SA), an anti-SA geometric mean titer (GMT) can he

Measured by ELISA, e.g. after a few weeks of treatment (e.g. 3 or 4 weeks) and after administration of a few dosages (e.g. 3 or 4). The percentage of subjects who seroconverted for SA after few weeks of treatment (e.g. 3 or 4 weeks) and after administration of a few- dosages (e.g. 3 or 4) can also be determined to assess immunogenicity.

To determine prophylactic efficacy, an immunogenicity analysis can be conducted on subjects who remain SA seronegative and PCR-negative to SA infection (swab and biopsy) at various endpoints after challenge.

Staphylococcus aureus

"SA epitopic peptide" as used herein includes the S. aureus epitopic peptides of all autoinducing peptides (AIPs), which regulate quorum-sensing dependent virulence in this pathogen, or epitopes from SA toxins and leukocidins. These epitopic peptides include the following, which can be inserted into VLPs in the A-B loop (upstream or downstream. preferably in the downstream Ά-Β loop) or in the amino or carboxyl terminus of a

bacteriophage protein coat dimer.

Production of Virus-Like Particles

The present invention is directed to vims-like phage particles as well as methods, for producing these particles in viva as well as in vitro. As used herein, producing virions "in vitro" refers to producing virions outside of a cell, for instance, in a cell-free system, while producing virions "in viva" refers to producing virions inside a ceil, for instance, an

Escherichia colt or Pseudomonas aeruginosa cell.

.Bacteriophages

The YLPs described here consist of assemblies of the coat proteins of single-strand RNA bacteriophage [RNA Bacteriophages, in The Bacteriophages, Calendar, RL, ed. Oxford University Press. 2005]. The known viruses of this group attack bacteria as diverse as E. coii, Pseudomonas and Acinetobacter. Each possesses a highly similar genome

organization, replication strategy, and virion structure. I particular, the bacteriophages contain a single-stranded (-f )-sense RNA genome, contain maturase, coat and replicase genes, and have small (<300 angstrom) ieosaheclral capskls. These include but are not Limited to PP7, MS2, AP205, Qp\ RI7, SP, PP7, GA_f Ml L MX1, P4, CM, Cbl2r, Cb23t, 7s and i2 RNA bacteriophages.

The information required for assembly of the ieosahedral eapsid shell of this family of bacteriophage is contained entirely within coat protein itself For example, purified coat protein can form eapsids in vitro in a. process stimulated by the presence of RNA [Beckett ei a!., 1988, I, Mol Biol 204: 939-47]. Moreover, coat protein expressed in ceils from a plasmid assembles into a vims-like particle in vivo [Peabody, D.S., 1990, J Biol Chem 265: 5684- 5689],

Examples of PP7 coat polypeptides include but are not limited to the various chains of PP7 Coat Protein Dimer in Complex With Rna Hairpin (e.g. Genbank Accession Nos, 2QUXR; 2QDXO; 2QUXJL; 2QOKJ; 2QUXJ; and 2QUX_C). See aim Example 1 herein and Peabody, et a!., RNA recognition site of PP7 coat protein. Nucleic Acids Research., 2002, Vol. 30, No. 1 4138-4144.

RNA Bacteriophage Coat Polypeptide

The coat polypeptides useful in the present invention, also include those having similarity with one or more of the coat polypeptide sequences disclosed above. The

similarity is referred to as structural similarity. Structural similarity may be determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate amino acid sequence can be isolated from a single stranded RNA virus, or can be produced using recombinant techniques, or chemically or enzymatica!!y synthesized. Preferably; two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wl), or the B!astp program of the BLAST 2 search algorithm, as described by Tatusova, et at (FEMS Microbial Lett 1 99, 174:247-250), and available at

ht^://w^w.ncbi.nlc^mh.gov blast bl2seo bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including matrix -BLOSUM62; open gap penalty - 1 1 , extension ga penalty— 1, gap xdropoff ^~ 50, expect ~ 10, wordsize - 3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "ide ities." Preferably, a coat polypeptide also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, or a least 95% amino acid identit to one or more of the amino acid sequences disclosed above. Preferably, a coat polypeptide is active. Whether a coat polypeptide is active can be determined by evaluating the ability of the polypeptide to form a capsid and package a single stranded RNA molecule. Such an evaluation can be done using a in vivo or in vitro system, and such methods are known in the art and routine. Alternatively, a polypeptide may be considered to be structurally similar if it has similar three-dimensional structure as the recited coat polypeptide and/or functional activity.

Hi SA Epitopic Peptide As described herein, the SA epitopic peptide sequence may be present in the A-B loop, at the N-termmus or the carboxy terminus of a coat polypeptide, but preferably in the A-B loop in the downstream. Preferably, the SA epitopic peptide sequence is expressed on the outer surface of the capsid.

The S A epitopic peptide sequence includes but is not limited to ammo acid sequences derived from the autoinducing peptides (AIPs), which regulate quorum-sensing dependent virulence in this pathogen, or epitopes from SA toxins and Jeukocidins.

In preferred embodiments, the present invention is directed to A-B loop, N-terramal or C-terrninal presentation of SA AIPl -4 wild-type and C4S mutants epitopic SA peptides on VLPs including PP7, MS2, AP205 and Qp. These VLP-AiPs can be used singly or as a combination vaccine. The inventors have generated preliminary data showin protection against infection using a vaccine consisting of a peptide from the SA toxin alpha-hemol sin (Hla) presented on AP205. Sequence alignment of Hla with SA bicomponent leukotoxins (including LukSF components of gamma, hemolysin, Panton- Valentine leukocidin (PVL), Luk ED and LukGH) show similar peptide epitopes which are predicted to also induce neutralizing immunity, with the greatest protection provided by vaccination with a combined YLP-leukocidin cocktail.

Epitopic peptides which are used in the present invention include the following: In. each instance of a peptide, a t least four (4) contiguous amino acids are used a the epitopic peptide and anywhere from 4-9 contiguous amino acids {depending on the epitopic peptide employed).

AiPl YSTCDFIM (SEQ ID NO: 1) or AIP1 S YSTSDFIM fSEQ ID NO: 2)

ΑΪΡ2 GVNACSSLF (SEQ D NO: 3) or AIP2S GVNASSSLF (SEQ ID NO: 4)

AIP3 INCDFLL (SEQ ID NO: 5) or AIP3S I SDFLL (SEQ ID NO: 6)

AIP4 Y^'STCYFJM (SEQ IS NO: 7) or AIP4S YSTSYFJM (SEQ ID NO: 8)

Or truncation^'s of any of the above (i.e. any 4, 5, 6, 7, 8 or 9 contiguous amino acids found within the sequences described above). Additional SA. epitepie peptides (or truncations as described herein below) include the following:

PVL (Panton- Valentine leukocidm

From LakS-PV Q2FGU9

1) INYLPKNKIDSVNVSQTLGYNiGG F SGPSTGGNGSFNYS TTiSY!Si

OQNYIS (SEQ ID O:9)

2} KWGVTQNI (SEQ ID NO: 10) From LukF-PV Q2FGV0

1) VDYAPKNQNEEFQV{^TVGYSYGGD.i lS GLSGGGNGSKSFSET[NYQ ESY T (SEQ ID HO: 11)

2) L ISQIL (SEQ ID NO: 12)

From Hia~ alpha hemolysin P09616

1) STLTYGFNGNV GDDTG iGGL!GANVSiGHTLK (SEQ ID NO; 13)

2) E GMHKKV (SEQ ID NO: 14)

3) SDY YPRNSID E Y (SEQ ID NO: 15)

4) KYVQPDF T (SEQ ID NO: 16)

F!lg ··· gaiiima-liemolysin

From A subi it P0A074

1 ) INYLP KIDSA DVSQ LG YN1GGNFQSAPSIGGSGSF YSKTISYNQK NYVT (SEQ ID NO: 17)

2) RLAITQNI (SEQ ID NO: 18)

From B submit P0A077

1) VDYAPKNQNEEFQVQNTLGYTFGGDISIS GLSGGLNGNTAFSETINY Q

ESYRT (SEQ ID NO: 19)

2) FKISQIL (SEQ ID NO:20)

From C subunit Q07227 1 ) 1NYLPKMKIESTN VSQTLG Y IGG FQSAPSLGG GSFH YS SIS YTQQ

NYVS (SEQ ID N0:21)

2) KWGVTQNi (SEQIDNO:22)

LukED -

From L¾k E Q2FXB0

1) m P N mTTDYGQTLGYTSilGGNFFQSAPSlGGHGSFNYSKlTISYTQK

SYVS (SEQ ID NO:23)

2} .WGVTQ V (SEQ ID O:24)

FromLukD 054082

1) VDYAPKHQHEEFQVQQTLGYSYGGDlHISNGLSGGmGSKSFSET Y QESY RT (SEQ ID NO-25)

2) LNIFQIL (SEQ ID NO:26) LukAB (also called LukGH)

From LukA/G sequence taken from SAUSA300 1974

1 } TDFAP NQDESREVK YTYGY TGGDFSl RGGLTGNITKESNYSETiSYQQPS Y^'RT (SEQ ID NO:27)

2) KNITQSL (SEQ ID NO:28)

Frora iikBH sequence taken from SAUSA3G0 1 75 i) LDQLPKNKIS1A \^?DSTFSYSSGGKFDST GIGRTSS SYSKTISYNQQ

NYDT (SEQ ID NO:29>

2} KTNILQNL (SEQ ID MO:30)

LukF, P3171S

LukS,P3l716

Or epitopic truncations of any of the above sequences (i.e. any 4, 5, 6, 7, 8, 9, 10, 11, 1:2, 13, 14, 15, !6, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 35 contiguous epitopic amino acids, where relevant or any e iiopte amino acid sequence .from- ny of the above am no acid sequences thereof comprising at least 4 contiguous amino acids).

In order to determine a corresponding position in a structurally similar coat polypeptide, the amino acid sequence of this structurally similar coat polypeptide is aligned wit , the sequence of the mimed coat polypeptide as specified above.

In a particular embodiment, the coat polypeptide is a single-chain dimer containing an upstream and downstream subtmit Each subunit contains a functional coat polypeptide sequence. The SA epitopk peptide sequence may be^■inserted in the upstream and/or downstream subtmit at the sites mentioned herein above, e.g., the A-B loop, the N-terminus or a earboxyi terminus. n a particular embodiment, the coat polypeptide is a single chain dimer of a PP7 or MS2 coat polypeptide, preferably a PP7 coat polypeptide, although a number of bacteriophage coat polypeptides may be used.

Preparation of Transcription Unit

The transcription unit of the present invention comprises an expression regulatory region,, (e.g., a promoter), a sequence encoding a coat polypeptide and transcription terminator. The RN A polynucleotide may optionally include a coat recognition site (also referred to a "packaging signal", "translations! operator sequence", "coat recognition site"). Alternatively, the transcription unit may be free of the tmnsiationai operator sequence.

The promoter, coding region, transcription terminator, and, when present, the coat recognition site, are generally operably linked. ' perably linked" or ''operably associated with" refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to, or "operably associated with^'5, a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. The coat recognition site, when present, may be at any location within the RNA. polynucleotide provided it functions in the intended manner.

The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. The promoter used in the invention can be a constitutive or an inducible promoter. Preferred promoters are able to drive high levels of RNA encoded by me coding region encoding the coat polypeptide Examples of such promoters are known in the art an inc de, for instance, the lac promoter, T7, T3, and SP6 promoters.

The nucleotide sequences of the coding regions encoding coat polypeptides described herein are readily determined. These classes of nucleotide sequences are large but finite, and die nucleotide sequence of each m mbe of the class can be readil determined by one skilled in the art by reference to the standard genetic code. Furthermore, the coding sequence of an NA bacteriophage single chain coat polypeptide comprises a site for insertion of SA epitopic peptide-encoding sequences . In a particular embodiment, the site for insertion of the SA epitopic peptide-encoding sequence is a restriction enzyme site. In another embodiment, the A epitopic peptide-encoding. se ence is- inserted using polymerase chain reaction

(PCR) using standard techniques. in a particular embodiment, the coding region encodes a single-chain dime of the coat polypeptide. In a most particular embodiment, the coding region encodes a modified single chain coat polypeptide dimer, where the modification comprises an insertion of a coding sequence at least four amino acids at the insertion site, which four amino acids represent an epitopic SA peptide as otherwise described herein. The transcription unit may contain a bacterial promoter, such as a lac promoter or it may contain a bacteriophage promoter, such as a T7 promoter. S ^■ynthesis

The VLPs of the present invention may be produced in vivo by introducing

transcription units into bacteria, especially if transcription units contain a bacterial promoter. Alternatively, it ma be synthesized m vitro in a coupled cell-free transcri tion/translation system.

Assembly f FLPs Encapsidaiig Heterologous Substances

As noted above, the VLPs of the present invention encapsidate a A epitopic peptide- encoding sequence. These VLPs may be also be assembled in combination with another substance, such as an adjuvant. Specifically, purified coat protein subunits are obtained from VLPs that have been disaggregated with a denaturant (usually acetic acid). The adjuvant is mixed with coat protein, which is then reassembled in its presence. In a particular embodiment, the substance has some affinity for the i terior of the VLP and is preferably negativel charged.

In another embodiment, the adjuvant is passively diffused into the VLP through pores that naturally exist in the VLP surface. In a particular embodiment, the substance is small enough to pass through these pores and has a hig affinity for the interior of the VLP.

The following experiments were conducted to determine the potential for providing immunogenic VLPs which incorporate heterologous peptides from Staphylococcus .aureus as potential compositions for inducing protection against Staphylococcus aureus infection, especially including MRSA infection.

The following facts formed the basis of the rationale for the present invention:

• Antibiotic resist ance of Staphylococcus aureus emphasizes the importance of

al ternati ve approaches;

• The S. aureus accessory gene regulator (agr) pathway utilizes secreted autoinducing peptides (AfPs) for virulence regulation. See figure 9, which shows the agr pathway.

• AIPs (7-9 amino acids) are too small: to invoke natural immune response, but might be effective targets if presented on Virus-1 ike-particles ( V LPs) which are natural adjuvants and can serve as effective vaccines.

Thus, the hypothesis emerged that, vaccination with VLP-AIP will induce antibodies wit structural recognition of nati ve AIP, and thereby will confer immune protection from

Staphylococcus aureus SSTI via agr disruption. The following examples test that hypothesis.

Examples

In certain preferred embodiments, the present invention is directed to A-B loop, N- terminal or C-terminal (preferably A-B loop) presentation of SA AlPI-4 wild-type and C4S mutant epitopic SA peptides on VLPs racluding PP7, MS2, AP205 and ζ>β. These VLP-AlPs can be used singly or as a combination vaccine. The inventors have generated data showing protection against mfection using a vaccine consisting of a peptide from the SA toxin alpha- hentetyshi (Hla) presented on AP205. Sequence alignment of Hla with SA bicomponent leukoioxins (including LukSF components of gamma hemolysin, Panton- Valentine

lenkocidin (PVL), Lok ED and LukGH) show similar peptide epitopes which the inventors have predicted would also induce neutralizing immunity, with the greatest protection provided by vaccination with a combined VLP-leukocidin cocktail With the expectation of immunogenic activity, further testing will concentrate on thes embodiments, as well as others.

Additional Examples

The production of virulence factors required for S. attrem SSIl is largely regulated by the accessory gene regulator operon ( gry'' through a bacterial communication system known as quorum sensing. Induction of agr signaling depends upon the accumulation of small, secreted autoinducing peptides (AIPs) to activate a receptor histidme kinase, AgrC, in the bacterial cell membrane ^{9 J,>}. AgrC activation dri ves downstream production of the effector molecule, RNAJH, which in turn regulates expression of over 200 virulence genes contributing to invasive infection'^'. S. aureus isolates express one of four agr alleles (agr~l to agr~W), with each secreting a unique AIP (ΑΪΡ1-ΑΙΡ4) and expressing a corresponding AgrC. Previously, both an anti-AIP4 monoclonal antibody (mAb)^{, U2} and an AIP4

immunologic mimotope vaccine " showed protection against infection caused by agr type IV isolates. However, antibody or vaccine targeting of signaling by agr type I isolates, which are most associated with invasive \^' aureus infection^54-15, has not been reported.

8. aureus AIP I is an eight amino acid peptide (YSTCDF1M, SEQ IDNO:i) cyclized by a thiolactone bond between the Cys4 side-chain and the carboxyl group of the C-terminal residue (Met8) (Fig. 10a). Given that cye!ization is essential for function, immune

recognition of the cyclic form of AIPI may be necessar for antibody-mediated

neutralization. However, the small size of these peptides makes them innately non~ immunogenic and, together with the labile nature of the thiolactone, increases the difficulty of vaccine development ^>l*^>lf>. The inventors sought to overcome these challenges using a bacteriophage virus-like particle (VLP) vaccine platform. These VLPs self-assemble from recombinantiy expressed bacteriophage coat proteins which can be genetically altered for surface presentation of practically any epitope in a multivalent format that virtually guarantees strong tramunogeni city resulting in high titer, high affinity and long-lasting antibodies' ^"'. Specifically, the inventors hypothesized that a vaccine produced by co¾f n«aiionaS|y~resiricted presentation of the AO? i -amino acid sequence on the surface of bacteriophage VLPs would elicit antibodies against native AIPl and induce immune control of agr type Lregulated virulence, despite the absence of a thiolactone in the heterologous epitopic peptide incorporated in to the VLP.

To test this, the inventors produced a VLP-base agr type I vaccine by cloning a modified AIPl amino acid sequence (YSTSDFIM, SEQ ID O:2) into an immuno-prormnent surface loop (the AB-loop) of the Pseudomonas aeruginosa RNA bacteri ophage PP7 coat protein¹*^*21. As expected, the resulting vaccine (PP7-AIP S) elicited antibodies which recognized AIPl in vitro and was efficacious in a murine SSTI model upon challenge with a highly virulent MRSA agr type I isolate. Compared to controls, PP7-AIP1S vaccination resulted in reduced agr function and agr-regulated virulence factor production at the site of infection. Importantly, PP7-AI IS vaccination significantly reduced $. aureus pathogenesis, based on dermonecrosis and weight loss, and increased bacterial clearance, findings consistent with enhanced host innate defense in the absence of agr function ²²"*. Together, these results demonstrate the protective benefits of vaccine-induced immune control of agr type I-regulated virulence. Given that several important pathogens utilize similar structurally constrained peptides for virulence regulation²'', the findings highlight the potential clinical utility of VLP-based vaccines targeting virulence regulators as an alternative or adjunct approach to combat infections caused by other human pathogens.

Results

Presentation of the 8. aureus AIPl sequence on VLPs induces AlPI-recogtiizing antibodies.

The icosahedral capsid. of the Pseudomonas aeruginosa RNA bacteriophage PP7 self- assembles from coat protein monomers, with each monomer presenting a highly constrained β-tura, called the AB-loop, on the surface of the assembled capsid" ^{¾ 2s 2} . In an effort to promote kntnunogenkity and maintain the structural integrity of AIPl presentation to the adaptive immune system, the inventors inserted a modified AI l sequence into the second AB-loop of the previously reported PP7 single-chain coat protein dimer which self-assembles into stable VLPs (Fig. la-c)ⁱ*^"20"* '². To avoid potential interrnolecuiar disulfide bond formation that could negatively impact VLP purification and immune presentation, the inserted AIPl sequence included a cysteine to serine mutation in position 4 (YSTCDP SEQ ID NO: I to YSTSDFIM, SEQ ID NO: 2) (referred to as AIP1S). Recombinant^- expressed PP7-A1P1 S protein dimers self-assemble into soluble VLPs as indicated by a single protein band (Coomassie staining) upon agarose gel electrophoresis, and by co-migration of encapsidated EN A (ethidnim bromide staining) (Fig. Id). The resulting highly purified PP7- Al LS VLPs consist of 90 single-chain coat protein dimers, which therefore display 90 copies of AI l 5 per VLP to be presented for immune stimulation.

The inventors first sought to determine whether vaccination with PP7-AIPI S would induce production of antibodies capable of recognizing S. aureus AIPl. To address this, we vaccinated mice with PP7-AIP1 S (twice with a 4-week interlude) and the measured, the ability of serum antibodies to specifically bind the AIP1S sequence. Serum collected at two-, four- and eight- weeks after the last vaccination with ΡΡ7-ΑΙΡΪ S, but not after PP7 control vaccination, showed dose-dependent binding to the AlPiS sequence present on PP7-AIP1S VLPs (Pig. 2a). Importantly, in competitive dose-response assays, AIP1 S binding by eight- week post-vaccination antiserum (geometric mean titer- 4,550) was inhibited by synthetic cyclic AIPl, but not synthetic ΑΪΡ2 (GVNACSSLF, SEQ ID NO:3) (Fig. 2b), demonstrating specificity and the ability to bind native AIPL These results, showing the production of specific antibodies which recognized sol uble, native AIPl, suggested .maintenance of the conformational integrity of AIP presentation within the P 7 AB-loop.

PF7-AI IS vaccination provides protection In a Murine model vtS. aureus

dermoneerosis. MRSA isolates of the piilsed-field gel electrophoresis type USA300 (agr type [) have long been the cause of most community-associated MRSA (CA-MRSA) infections, and now also cause an increasing number of health-care associated infections¹ In mouse models of USA300 SSTI, infection with an isogenic agr-deletion mutant ( agr) results in significantly decreased pathogenesis and increased bacterial clearance compared to infection with the wild- type agr* strain '^22*25. Therefore, we postulated that vaccination with PP7-AIP1 S would induce immune suppression of gr-signaling in vivo, thus reducing pathogenesis and increasing bacteria! clearance during SSTL To evaluate the efficacy of PP?~ AIP1S vaccination against agr type [-mediated virulence and to avoid potential non-specific effects of VLP administration-*⁴, we challenged mice eight weeks after final vaccination using a well-established mouse model of.?, aureus SSTL* and the highly virulent USA300 isolate LAC^{' 6}. As expected, PP7-A1P1 S vaccinated mice showed reduced abscess formation, dermonecrosis and weight loss (used as a measure of morbidity) over the coarse of a six-day infection compared to controls (Fig. 3a~e). Importantly, bacterial burden on day 6 postinfection was also significantly reduced in the PP7-AIP1S vaccinated group (Fig. 3f consistent with, lower local Ievels of the inflammatory ^'cytokines IL-Ι β and TNFa (Fig. 3g). Given the contributions of #g -signaling to -pathogenesis and inflammation in this infection model, these data demonstrate the efficacy of PP7-AI 1 S vaccination against S. aureus agr type J-regulated pathogenesis during skin infection,

PF7~AiFlS vacclimtion inijibits & aureus ^r-slgnaling in viva. S. aureus agr~^gn^ g induces expression of the effector molecule RNAHl as well as production of alpha-hemolysin (Hla), the causative agent of dermonecrosis³ " ⁱ. The results of our challenge studies, as well as our in vilro studies showing that antibodies from PP7-AIPIS vaccinated mice bind soluble AIP1, suggested that vaccination with PP7-AIP1S results in immune suppression of agr- signaling during S. aureus SSTX if correct, we would expect reduced RNAIII transcription and Hla expression at the site of infec tion (local) in PP7-AIP1 S vaccinated mice compared to contols. To test this, we measured local RNAIII expression and Hla protein levels on days one and six, respecti vely, following subcutaneous infection. As expected. RNAIII expression was reduced at die site of infection in PP7-AI 1 S vaccinated mice compared to controls (Fig. 4a), as were local levels of Hla (Fig. 4b). Together, these data support a mechanism of action whereby vaccination with PP7-AIPIS induces^' immune control of . aureus agr type I signaling and virulence regulation dining SSTX

Diseossion

The ongoing antibiotic resistance crisis highlights the urgent need for non- conventional approaches to combat infectious disease, including approache to inhibit bacterial virulence⁴²'⁴''. In the case of the important human pathogen Staphylococcus aureus, virulence regulation is largely mediated by the agr operon via secretion of A3Ps^{:>, i}. These small, confomiationally-restrained, secreted peptides bind in an autocrine and paracrine fashion to the bacteria! membrane receptor AgrC, which in turn regulate downstream virulence factor expression. Therefore, antibody-mediated sequestration of secreted AlPs could neutralize agr-signa!i«g and virulence factor expression on -a population level. Of the four & aureus agr types, agr type I isolates are most frequently associated with invasive infection ^M'^{T S}. Here we -report that multivalent, confotmarionally-restricted presentation of a modified AIPl amino acid sequence on VLPs elicits immune control of 5. aureus agr type regulated virulence. Specifically; PP7-AIPI S vaccination (1 ) induced the production of anti- AIP! antibodies. (2) limited agr type I-signaling in vivo and (3) demonstrated efficacy

(reduced pathogenesis and increased bacterial clearance) in a mouse model of 8. aureus

SSTI. Give these results and the contr i bution of agr type I isolates to human 5. aureus infection*^4,15, vaccine prevention of agr type I-mediated virulence could h ve a major clinical impact and make a significant contribution to the fight against antibiotic resistance.

The diversity of virulence factors -produced by S. aureus' _t mmy-of which disable innate immune cells ^^*46, and the range of infection types (skin, pneumonia, bacteremia, etc.)^i>4?, suggests that multiple anti-virulence approaches ma be needed to limit human disease. For example, targeting specific- virulence factors, in particular HIa which is a major contributor to pathogenesis**, has shown efficacy in numerous animal models^ ^,9,49" " and a monoclonal antibody targeting Hla (MEDI4893) is currently in human clinical trials³*.

Broader approaches aimed at inhibiting S. aureus virulence regulation have included peptide and small molecule targeting of the agr system^{* 2} "*⁶'* ^"60, as well as development of a monoclonal antibody (mAb) against S. aureus AIP4^ii>6\ However, agr-signaling has been shown to occur early post-infection and disruption of this early signaling correlates with reduced pathogenesis in the host²\ suggesting a possible limit to the window of opportunity tor therapeutic agr-inhib on. Therefore, the development of an efficacious anti~¾r vaccine could expand the impact of 8. aureus virulence regulation strategies to have the broadest potential clinical benefit to patients. In this regard, we previously developed a VLP-based AIP4 mimotope vaccine by screening a VLP-peptide library against an anti*AIP4 mAb, AP4- 24H 1 1 ^{ί2 1Λ}^⁷, shown by passive transfer to be protective in a mouse model of agr type IV SSTI. Here we advance this work by demonstrating the efficacy of PP7-A1P1S vaccination against 5. aureus agr type I-regulated virulence. Our findings suggest that this VLP-based approach may be utilized to produce a combined vaccine against virulence regulation by each of the agr types, thus serving as a valuable component of an overall anti-virulence strategy.

In addition to Staphylococcal species other human pathogens using agr-\ike quorum sensing systems and secreted peptides to coordinate virulence factor expression"" could be targeted by VLP-based vaccination. For example, the food-bome pathogen Listeria monocytogenes uses a variety of communication systems to regulate v fuie.nce^f>fi,i'⁹, including an agt locus and recently identified secreted AIP^''°^" in 1. monocytogenes _t the agr system regulates over 650 genes contributing to virulence including ones involved in biofilm formation and host ceil invasion'¹. Similarly, Enterococeus faecali?_y an important cause of drug resistant infections ' uses the agr-likefir gene locus and the secreted, cyclic peptide gelatin se biosyo hesis-aetivati g pherornone (GBAP) ^'" ^' to regulate expression of virulence factors important for hiofihn formation and pathogenesis^''^ importantly, St has also recently been shown that a agr locus regulates production of toxins A and B by the multidrug resistant pathogen Clostridium difficile*⁴' *. These C difficile toxins are directly responsible for disease manifestation^8*6 which, in severe cases, can result in sepsis and death^¾?, suggesting that interference with «gr-signaling b this pathogen could significaii ly limit disease. Therefore, a VLP -vaccine platform could provide a straight-forward approach to elicit immune inhibition of agr- and agrAike virulence signaling by these and other important human pathogens.

Virus-like particles have proven to be a flexible and highly imnnmogenic platform for vaccine design, and are currently used in FDA-approved vaccines⁸*, including Hepatitis B vaccines⁸* and the current nonavaient HP V vaccine (GardasiS 9) designed to induce protection against nine HPV types ⁰. Although non-replicating, the dense, repetitive array of coat proteins comprising VLPs is largely unique to microbial antigens and this multivalency triggers a robust immune response in mammals. Therefore, VLPs can dramatically increase the immunogenicity of otherwise poorly immunogenic peptides' ⁹ⁱ even, including self- antigens^¾i'^¾\ This property, along with the potential for presentation of conformation- dependent antigens, has resulted in investigation of VLP-based vaccines against numerous pathogenic viruses, allergies, cancer, autoimmune disease, Alzheimer's disease and chronic diseases such as hypertension^57'94"9' . However, reports of the use of VLP-based vaccines to elicit adaptiv immunity against specific bacterial pathogens or proteins have com mainly from our own work and from research targeting Streptococcal species^13,98"10", suggesting that the flexibility of VLP-based vaccine approaches to address bacterial diseases remains largely untapped. Given the FDA approval and success of VLP vaccines against viral pathogens, the use of VLP-based vaccines to prevent infections by the many important human bacterial pathogens warrants further investigation, in this era of diminishing antibiotic efficacy, a multi-pronged approach, including novel antibiotics, host-targeted therapeutics, vaccines, anti-virulence strategies and combined therapies will .likely be crucial for combating disease caused by antibiotic resistant pathogens⁵ . Here we present a novel approach to achieve vaccine induced immune control of S aureus agi-regiilated virulence. This work highlights the potential clinical utility of VLP-based vaccines as part of an overall strategy to combat infections caused by MRSA and other important antibiotic resistant human pathogens ^'utilizing secreted peptides for virulence regulation^{"5 ,} .

Summary

The inventors can summarize the successful results of the experimentation described herein as follows.

* VLPs can be constructed which present AIP epitopes (AIPl , AIP1S_., among others) on their surface.

• Vaccination with PP7-A1P1S induces antibodies that recognize native A!P l .

PP7~AiPIS vaccination limits pathogenesis (abscess, dermonecrosis) and promotes bacterial clearance during X aureus SST!.

* PP7-AIP1S vaccination limits the local pro-inflammatory cytokine response dining S. aureus SSTL

• ΡΡ7-ΑΪΡ1 S vaccination disrupts ¾gr-signalrag and gr-mediated virulence. Methods

Ethics statement Animal studies described herein were approved by the institutional Animal Care and Use Committee (IACUC) of the University of New Mexico Health Sciences Center (Animal Welfare Assurance number D 16-00228) and conducted in strict accordance to recommendations in the Guide for the Care and Use of laboratory A mab , the Animal Welfare Act, and U.S. federal law.

Bacterial strains and growth conditions. The CA-MRSA USA3G0 isolate LAC³⁶

(generously provided by Dr. Frank DeLeo, Rock Mountain National Laboratories, National institutes of Health, Hamilton, MT) was used for infection studies. Early exponential-phase bacteria were prepared as previously described^{" i>5} and stored at -80°C for no more than two weeks prior to use. For infection studies, bacteria were diluted in USP~grade saline (B. Braun. Medical, Irvine, CA) to yield 4 l ^' CPU per 50 iiL. The number of CFU was verified by platteg ten-fold serial dilutions onto Trypticase soy agar containing 5% sheep blood (Becton, Dickinson and Company; Franklin Lakes, NJ).

VLP cloning, expression and purification. The pET2P?K32 plasmid²¹', encoding the PP7 single-chain diroer nder the T? promoter and transcription terminator, was used for synthesis of PP7-AIPIS VLPs in £ colt With pBT2P7 32 as a template, PC was used to produce an insert fragment encoding a Kpnl restriction site, the modified ΑΪΡ1 sequence (YSTSDFIM, SEQ ID NO:2), and a downstream BamHI site (forward primer 5^*-GGC GGT ACC TAC ACT ACC TCT GAC TTC ATC ATG GAG GCT ACT CGC ACT CTG ACT GAG-3' (SEQ ID MO;31); reverse primer S'-CGG GCT TTG TTA GCA GCC GG-3 ' (SEQ ID NO:32). The PCR fragment was cloned into the pET2P7K32 at the Kpnl and BamHI restriction sites and insertion was verified by sequence analysis.

E. coU C41 cells (Lncigen, Middle-ton, WI) transformed with pET2P?K32 or the

pET2P7K32-AIP 1 S expression plasmid were grown at 37°C to an OD< of 0.8. Expression was induced with 1 mM EPTG, cells cultured for an additional 3 hours, and harvested by eenaifugation. Cell pellets were lysed and VLPs purified essentially as described

previously²⁰ but. with size exclusion purification using a 16/60 Sephacryl S-400 HR column (GE Healthcare, Pittsburgh, PA). VLP purity was verified by agarose ge! electrophoresis pins ethidium bromide and Coomassie staining, VLPs were concentrated using Amicon Ultra Centrifugal filter units (10 MWCO) (EMD iliipore. Biileriea, MA), and concentrations determined by SDS-PAGE comparison to hen egg lysoso ie concentration standards (Sigma- Aldrieh, St. Louis, MO) VLP aliquots were stored at ~20°C until use.

Mouse immunizations. Four wee old, female BALB/eJ mice (Jackson Laboratories, Bar Harbor, ME, USA) were immunized by injection into the caudal thigh muscle with 50 L of PBS alone or containing 10 fig of either PP7-AIP 1 S or PP7. Mice received an identical injection four weeks after the initial dose. Serum for ELISA analysis was coilected by cardiac puncture at two. four or eight weeks after the second vaccination, with challenge experiments performed at the eight week time-point.

ELISA, ELiSA plates to measure semm antibody binding to Al l S were prepared by coating Ultra Cruz ELISA High Binding plates (Santa Cruz Biotechnology, Santa Cruz, CA) with 125 ng per well of recombinant PP7 or PP7-AI I S in 50 uL PBS and incubating 20 hours at room temperature (RT) with shaking. Alter removing excess liquid, plates were blocked for 2 hours with PBS containing 0.05% Tween-20 and 1% casein. To reduce PP7- and potential E. coii-hm^' dmg antibodies (depleted serum) mouse serum was treated as follows: Serum was diluted 1 :50 in PBS and incubated for one hour at RT with end-over-end rotation together with recombinant PP7 (10 « per 300 uL diluted serum) and FBS~washe C41. cells (the E. coli strain used for VLP-expression) (-9 x 10 CPUs). The mixture was centrifuged (5 mia at 11 ,600 x g) to remove antibody bound to C41 cells, and the intermediate depleted serum processed through an Amkon Ultra Centrifugal filter unit (I00K MWCO) to remove antibody bound to PP7, The final depleted serum was serially diluted onto PP7- or PP7- AlPl -coated BU S A plates and incubated for I hour at R . Murine antibodies bound to VLPs were detected using goat anti-mouse poiy-HRP secondary antibody (ThermoFisher Scientific, WaSthani, MA) and developed using 1-Step™ Ultra TMB-ELISA according to manufacturer's directions (ThermoFisher Scientific). For each serum sample and dilution, ΑΪΡ1 S specific binding (ΔΑ 30) was equal to the A 50 for ΡΡ7-ΑΪΡ1 S binding minus the A 50 for PP7 binding. For competition ELISAs, depleted serum was incubated for 1 hour at 37°C with the indicated concentrations of AiPl or AIF2 (BioPeptide Co., Inc., San Diego, OA) before addition to VLP-coated EL IS A plates.

Mouse skin infection .model. The mouse model of derrrtoneerosis was implemented essentially as previousl described'³. One to three days before infection (eight weeks after the second vaccination). Naif™ was used to depilate the right flank of the mice (site of infection). On the da of infection, mice were anesthetized b isotlurane inhalation and infected by subcutaneous injection of 50 μL of saline containing 4 lO' CFU of LAC. Mice were weighed the day of injection and daily thereafter until sacrifice. Injection sites were photographed daily and abscess and dermoiiecrosis areas determined by analysis with

ImageJ ^* . Six days after infection, mice were sacrificed by C⁽¾ asphyxiation and a 2.25-cm" section of skin surrounding the abscess was excised for mechanical disruption. Abscess homogenate was serially diluted and plated on sheep blood agar to determine infection site bacterial burden. The remaining homogenate was clarified by centrifngation and the clarified fraction stored at -80°C until cytokine analysis.

Cytokine anal sis by multiplex assay. Clarified abscess tissue homogenates were quick thawed at 37°C and concentrations of the indicated cytokines determined us ng a BioPlex 200 system and BioPJex manager software (Bio-Rad, Ifcreules, CA) together with a custom- designed mouse multiplex assay (HMD Millipore, Billeriea, MA) according to

manufacturer's directions.

RNA isolation from tissue aad quantitative PCR analysis. For analysis of day one postinfection bacterial gene transcription, 2, 25 -cm^* sections of skin surrounding the. infection si te were harvested, minced, and stored in RMA a/er (Qiagen, Valencia, CA) at ~20°C. SNA was isolated using QIAzol (Qiagen) and purified using RNeasy kits (Qiagen) according to manufacturer's directions. cDNA conversion from RNA was performed with a High Capacity c NA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and specific primers for S. aureus 16$ (reverse, 5'-TTC OCT CGA CTT GCA TGT A-3 \ SEQ ID NO:33) or RNAIII (reverse, 5^*-GATGTTGTTTACGATAGCTTACATGC-3', SEQ ID NO:34)

(Integrated DNA Technologies, Coralvilie, ΪΑ). Quantitative PCR (qPCR) was performed using a V'iiA-7 RT-PCR system (Applied Biosystems), the specific primers and probes"" for 16S (forward primer, 5 '~TGA TCC TGG CTC AGO ATG A-3\ SEQ ID NO:35; reverse primer above and probe S'-CGC TGG CGG CGT GCC TA-3\ SEQ ID NO:36) and RNAIII (forward primer, 5'~AAT TAG CAA GTG ACT AAC ATT TGC TAG T~3\SBQ ID NO:37 ; reverse primer above and probe 5 '-ACT TAG TTT CCT TGG ACT CAG TGC TAT GTA TTT TTC TT-3\ SEQ ID NO:38) (Integrated DNA Technologies) and TaqMan Gene Expression Master Mix according to the manufacturer's protocol (Applied Biosystems), Data are shown as the bid expression of^'RNAHI versus 16S and relative to the PBS control.

Tissue Hta quantification by Western blot. For Western blot analysis of Hla levels in clarified abscess homogenate, frozen samples were quick thawed and equal amounts of total protein (based on A ) were electrophoresed on 16% Tris-giyeine SDS-PAGE gels (Life Technologies, Grand Island, NY).

membranes were blocked overnight, at 4°C with TBST (20 raM Tris, pH 7.5, 150 mM NaCI, 0.1% Tween 20) with 5% nonfat dry milk. Hla was detected using sheep anti-Hla primary antibody (ab 15948, Abeam, Cambridge, MA) and alkaline phosphaiase-conjiigated rabbit, polyclonal anti-sheep secondary. Membranes were developed with mtroblue tetrazolium ( BT)/5-biOmo-4-chloro-3-indolyl-phosphate (BOP) (Thermo Scientific). Band intensity relative to recombinant Hla control was measured on FluorChem R system using

AlphaView software (ProteinSimple, San Jose, CA). S atistica analysis. GraphPad Prism version 5.04 (GraphPad Software, San Diego

CaJifoffiia) was used for all statistical evaluations. One-way ANOVA parameters followed Barilett's test for equal variances and were used with Bonferronfs (ANOVA) or Dumfs (Kruskal- Wallis test, non-pararaetrics) post-hoc .multiple comparison analyses. Results were considered statistically significant at /X0.05.

References

1 Lee, G. C. et al incidence and Cost of Skin and soft Tissue Infections la the united States. Value.. Health X doi : 10,10 Wj d .2015.03,.1424 (2015).

2 oran, G . I. et al. Methicillin-resistant S. aureus infections among patients in the emergency department N. Engl, J, Me 355, 666-674, doi:10.1056/N£jMoa05S356 (2006).

3 Tahm, D. A. et-al. Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008. -.Clin, infect. Dis. 53, 144449, doi:10J093/cid/cir308 (2011).

4 Labreche, M , J. et al. Treatment failure and costs in patients with nietrsicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections: a South Texas Ambulatory Research Network (STAR.Net) study . J. Am. Board Fain. Med. 26. SOS- SI?, doi: 10.3122 jabfni.2013.05.120247 (2013).

5 Montgomery, C. P.. David, M. Z. & Daum, R. S. Host factors that contribute to

recurrent staphylococcal skin infection. C r. Opin. infect. Dis. 28, 253-258, doi: 10;i097/QCO.0000000000000156 (2015).

6 Fowler, V. G._t Jr. & Proctor, R. A, Where does a Staphylococcus aureus vaccine stand? Clin. Microbial infect. 28 Sttppi 5, 66-75, doi: J 0.11 11/1469-0691.12570 (2014).

7 Cheung, G. Y., Wang, R., Khan, B. A., Sturdevant, D. E. & Otto, M. Role of the accessory gene regulator agr in community-associated methicillin-resistani

Staphylococcus aureus pathogenesis. Infect. Immun. 79, 1927-1935,

doi: 10.1 128/1 AL 00046-11 (2011).

8 Montgomery, C P.. Boyle- Vavra, S. & Damn, R. S. Importance of the global

regulators Agr and SaeRS in the pathogenesis of CA-MRSA USA300 infection. PLo One 5, el 5177, doi: 10.1371/joumal.pone.OOl 5177 (2010). Novick, R. P. & Geisinger, E. Quorum sensing in staphylococci. Annu. Rev. Genet 41, 541 -564, dot : 10.1 46/anfiof cv.genet.42.1 10807.091640 (2008).

Thoendel, M., Kavanaugh, J . S., Flack, C. E. & Horswill, A. R. Peptide signaling in the staphylococci. Chem. Rev, 111 , 117-151, doi: 10. 021 cr 100370η (201 1).

Kauimanrs, G. F._s Park, J. & Janda, . D. Bacterial quorum sensing: new target for anti-infective immunotherapy. Experf Opm. Biol Ther. 8, 719-724,

doi: 10 J 517/14712598.8.6.719 (2008).

Park, J . el al Infection control by antibody disruption of bacterial quorum sensing signaling. Chem. Biol Ϊ4, 11 19-1 127, doirl0.1016/j.die-¾hio].2007.08.013.(2007). O^' Rourke, J. Ψ. et. al. Development of a mtmotope vaccine targeting the

Staphylococcus aureus quorum sensing pathway. PLoS One 9, el 1 11 8,

doi: 10.1371/jouraaI.pone.Ol 1 i 1 8 (2014).

larraitd, S. et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (Alkies), and human disease. Infect, Immun. 70, 631- 641, doi:Doi 10.1 128/lai 70.2.631-641.2002 (2002).

Traber, K. E. et al agr function in. clinical Staphylococcus aureus isolates.

Microbiology 154, 2265-2274, doi: 10.1099/mic.0.2007/01 1874-0 (2008).

Kau&uino, G. F., Park, J., Mayorov, A. V., ubitz, D. M. & Janda, K. D. Generation of quorum quenching antibodies. Method's Moi. Biol. 692, 299-3 1, doi: 10.1007/978- i-60761 -971-0 22 (201 1).

C¾acfcerian, B. Virus-like particles: flex ible platforms for vaccine development Expert review of vaccines 6, 381-390 (2007).

Caldeira, .). C. & Peabody, D. S. Thermal stability o RNA phage virus-like particles displaying foreign peptides. Journal of riatwbiotecknology 9, 22-3155-3159-3122 (201 1),

Caldeira, J. C, & Peabody, D, S. Stability and assembly in vitro of bacteriophage ΡΡ7 virus-like particles. J Manobioiechnology 5, 10, doi 10.1186/1477-3155-5-10 (2007). Caldeira Mo, C. el al Immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus L2 epitope, on virus-like particles of the RNA bacteriophage PP7. Vaccine 28, 4384-4393,

doi: 10.1016/j.vaccine.20I0.04.049 (2010).

Chao, . A.. Patskovsky, Y., Almo, S. C. & Singer, R. H. Structural basis for the coevoliition of a viral RNA-protein complex. Nat. Struct. Moi. Biol 15, 1 3-105, doi: 10.1038/nsmb!327 (2008). Wright, J, 3rd, Jin, R. & Movick, R. P. Transient interference with staphylococcal quorum sensing blocks abscess formation. Proc, Nai , Acad, Sci. U. S. A. 102, 1691 - 1696, doi: 10.1073.· 'pnas.0407661 102 (2005).

Peterson. ML M. et al. Apolipoprotein B Is an innate barrier against invasive

Staphylococcus aureus infection. Cell Host Microbe 4, 555-566.

doi; 10.1016/j. c om.2008.10.001 (2008).

Sully, E. . el al. Selective chemical inhibition afagr quorum sensing in

Staphylococcus aureus promotes host defense with minimal impact on resistance.

Plo Paihog 10, ©1004174, doi : 10 J 371/journal.ppa 1004174 (2014).

Hall, P. R. et al. ox2 modification of LDL is essentia! for optimal apolipoprotein B- mediated control of gr type 111 Staphylococcus aureus quorum -sensing, PLoS

Pafhog. 9, 61003166, doi:10. ! 37L(joumalppatJ003166 (2013).

Daly, S. M. et al omega- Hydroxyeniodm limits Staphylococcia aureus quorum serising-niediated pathogenesis and inflammation. Anfimkrob. Agentf Chemother. 59,

2223-2235, doi: 10.1 128/ A AC.04564- 14 (2015).

Gray, B., Hall, P. & Gresham, H. Targeting agr- and agr- Like quorum sensing systems for development of common therapeutics to treat multiple gram-positive bacteria! infections. Se ors 13, 5130-5166, doi: 10.3390/s 130405130 (2013).

Tars. ., Fridborg, K., Bundule, M. & Liljas, L. The three-dimensional structure of bacteriophage PP7 from Psendomonas aeruginosa at 3,7-A resolution. Virology 272, 331 -337, dot: 10.1 06/viro.2000.0373 (20001.

Tars, K.., Pridborg, K.., Bundiile, M. & Liljas, L. Structure determination of bacteriophage PP7 from Psendomonas aeruginosa: from poor data to a good map. Acta Crystallogr. D Biol. CrysiaHogr. 56, 398-405 (2000).

Tumban, E., Peabody, J., Peabody D. S. & Chackerian, B. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the il V minor capsid protein, L2. PLoS One 6 (201 1 ).

Ko, X, Park, H., Heo, L. & Seok, C. Galaxy WEB server for protein structure

prediction and refinement. Nucleic Acids Res. 40. W294-297, doi : 1.0.10 3/nar/gks493

(2012).

Park, H._} Lee, G. R.. Heo, L. & Seok, C. Protein loop modelmg using a new hybrid energy function and its application to modeling in inaccurate structural environments. PLoS One 9, e i 1381 1 , doi: 10.1371/joumaLpone.Ol 13811 (2014). Carrel, ^'M, Perenceviek £. N. & David, M. Z. USA3GQ Metbict lin-Resistant Staphylococcus aureus, United States, 2000-2013. Emerg. Infect, Dis, 21, 1973-1980, doi:103201/eid21.11.150452 (2 15).

Rynda- Apple, A. e( al. Virus-like particle-induced protection against MRSA pneumonia is dependent o IL-13 mid enhancement of phagocyte' function. The Amen-eanjourrmt of pathology 181, 196-2 0 (2012).

Malachowa, N., obayashi, S. D., Braughton, . R. & DeLeo, F. R. Mouse model of tap lococ us aureus skin infection. Methods Mol Biol, 1031, 109-1 16,

doi: 10, 1007/978-1 -62703-481 -4 14 (2013).

Public health dispatch; outbreaks of co niunity-associated nietbicillin-resistant Staphylococcus aureus skin infections— Los Angeles Comity, California, 2002-2003, Can. Comm n. Dis. Rep 29, 1 10-1 2 (2003).

oshinia, N., Wang, Y . & Bubeck Wardenbitrg, J . Genetic requirement for ADAM 10 in severe Staphylococcus aureus skin infection. J. Invest. Dermatol 132, 1513-1516, doi; 10.1038/jid.201 1.462 (2012).

Kennedy, A. D. et at. Targeting of alpha-hemolysia by active or passive

immunization decreases se ver ity of USA300 skin infection in a mouse model. J Infect Dis 202, 1050-1058, dot: 10.1086/656043 (2010).

Sampedro, G. . et al. Targeting Staphylococcus aureus alpha-toxin as a novel approach to reduce severity of recurrent skin and soft-tissue infections, J Infect Dis 210, 1012-1018, doi;i©.1093/m s/}t«223 (2014).

Kobayashi, S. D. et al. Comparative analysis of USA3O0 virulence determinants in a rabbit model of skin and soft tissue infection. J infect Dis 204, 37-941,

doi; 10. i 93/mfo¾/?ir44 i (20 i 1 ).

Berabe, B. 3. & Bubeck Wardenburg, 3. Staphylococcus aureus alpha-toxin; nearly a century of intrigue. Toxins (Basel) 5, 1140-1 166 (2013).

NIAID Antimicrobial Resistance Program: Current States and Future Directions 2014 - ARstrategieplan20t4.pdf, <bttp://www.ncbi,nIm.aih.gov ¾?ubaied > (2015). Spellberg, B.„ Bartlett, J . G. & Gilbert. D. N . The future of antibiotics and resistance. N. Engl. J. Med. 368, 299-302, doi : i0.1056 NEJMp 1215093 (2013).

DeLeo, F. R., Diep, B. A. & Otto, M. Host defense and pathogenesis in

Staphylococcus aureus infections, Infect. Dis. Clin. North Am, 23, 17-34 (2009). Cheung, G. Y. & Otto, M. The potential use of toxin antibodies as a strateg for controlling acute Staphylococcus aureus infections. Expert Opin, Ther, Targeis 16, 601-612 (2012).

Tkaczyk, C. et ai Staphylococcus aureus alpha toxin suppresses effective innate and adaptive immnne responses in a murine dexmonecrosis model,. PLoS One 8, e75103, doLl 0.137 l/joumakpoflfi.0075 03 (2013).

Proctor, . A. Recent developments for Siaphyloc<iccu$ aureus vaccines: clinical and basic science challenges. European cells &. materials 30, 315-326 (2015).

Berube, SB, J. &. Warderiburg, J, B, Staphylococcus aureus alpha-Τοχίιι; Nearly a Century of Intrigue. Toxins. (Basel} 5, 1 140-1 1,66, doi:DOI. i ,3390/toxi«s506i l40 (2013),

Bubeck. Wardenburg, J. & Schneewind, 0. Vaccine protection against Staphyiococcm aureus pneumonia, J. Exp. Med 205, 287-294, doi: f0.IQ84/jem.200?220S (2008). Adhikari, R. P. et al. Novel, structurally designed vaccine for S. aureus alpha* hemolysin; protection against bacteremia and pneumonia. PLoS One 7, e38567, doi: i0.1.371, journal.pone.0038567 (20.12).

Oscherwitz, J., Munoz-Planillo, R., Yu, F., Nunez, G. & Cease, . B. In vivo mapping of a protecti ve linear neutralizing epitope at. the N-terminus of alpha hemolysin from Staphylococcus aureus. Mol Immunol, 60, 62-71 (201.4).

Oscherwitz, j. & Cease, K, B. identification and validation of a linear protective neutralizing epitope in the beta-pore domain of alpha toxin. PLoS One 10, eOl 6882, doi:10J37i/joumal.pone.0116882 (2015).

Yu, X. Q. et ai Safety, Tolerability, and Pharmacokinetics o M£DI4893, an

Investigational, ExtendedHalf-Life, Α^ηύ-Staphylococcus aureus Alpha-Toxin Human Monoclonal Antibody, in Healthy Adults. Antimicrah. Agents Chemother. , dot: 10. ! 128/aae.O102O-I6 (2016).

Figueroa, M. et al. Polyhydroxyanihraqmnones as Quorum Sensing Inhibitors from the Giittates of Peniciliiiim restriction and Their Analysis by Desorption E!ectrospray Ionization Mass Spectrometry. J. Nat. Prod. 77, 1 . 1 -1358, doi; Doi

10.1021 Np50OO7O4 (2014).

hodaverdian, V. et al. Discovery of Antivirulenee Agents against MethicilHn- Resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 57, 3645-3652 (2013). Kuo, IX et at Novel qoor m-quenc iag agents promote meihicillin-resistant

Staphylococcus aureus (MRSA) wound healing and sensitize MRSA to beta-Iactam antibiotics. Antimicroh Agents Chemother, 59, 1512-1518, doi: 10.1128 AAC.04767- 14 (2015).

Yu, CL, Kuo, D., Shoham, M. & Yiswanataan, , Combinatorial Synthesis and m vitro evaluation of a biaryl hydroxyketone library as antiv ralenee agents against MRSA. ACS combinatorial science 16, 85-91, doi:10.102I/co400142t (2014).

Cech, N. B., Junto, H. A., Ackemtann, L. W._s Kavanaugh, J. S. & Horswill, A. R. Quorum quenching and antimicrobial activity of goldenseal (Hydrastis canadensis) against methicUtra-resistant Staphylococcus aureus (MRSA). Planta Med. 78, 1556- 1561 , doi:10,1055/s-0032-1315042 (2012),

Quave, C. L. et ai. Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oieanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance. PLoS One 10, eOt 36486,

doi; 10.1371 /journal .pone. 136486 (2015).

Vermote, A. et ai. Eamametitannin Analogues thai Modulate Quorum Sensing as Potentiators of Antibiotics against Staphylococcus aureus. Angew. Chem, Int. Ed. EngL 55, 6551-6555, doi:10.1 02/anie.201 0i973 (2016).

Nakayama, J. et ai, Ambuie acid inhibits the biosynthesis of cyclic peptide quormones in gram-positive bacteria. Antimicrob. Agents Chemother, 53, 580-586,

doi: 10.112S/AAC.O0995-08 (2009).

Tal-Gaa, Y., Stacy, D. M., Foegen, M. JL, oeaig, D. W. &. Blackwell, H. B. Highly potent inhibitors of quorum sensing in Staphylococcus aureus revealed through a systematic synthetic study of the group-Ill autoinducing peptide. J. Am. Chem, Soc. 135, 7869-7882, doi:10.102I./ja31121 15 (2013).

Tal-Gaa., Y„ Ivaneic, M., Coraikseu, G., Yang, T. & Blackwell, H. B. Highly Stable, Amide-Bridged Automducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aurem. Angew. Chem, Int. Ed. EngL 55, 891 -8 17, doi : 10.1002/aoie.201602974 (2016) .

Gordon, C. P.. Williams, P. & Chan, W. C. Attenuating Staphylococcus aureus virulence gene regulation: a. medicinal chemistry perspective. J, Med. Chem, 56, 1389-1404 (2013). Murray, E. L et ai Targeting Staphylococcus aureus quorum sensing with

nonpeptidie small molecule inhibitors, J. Med. Ckem, 57, 2813-2819,

doi:10.l021/jm500215s (2014).

Chan, W. C, Coyle, B. J. & Williams, P. Virulence regulation and quorum sensmg in staphylococcal infections competitive AgrC antagonists as quorum sensing inhibitors. J Med. Chem. 47, 4633-4641, doi:lQ.l02l/jm04 0754 (2004).

irchdoerfer, R. N. ei at. Structural basis for ligand recognition and discrimination of a quorum-quenching antibody. J Biol Chem 286, 17351-17358,

doi: 10, 1074/jbc.M 1 11.231258 (201 1 ).

Freitag, . E, Port, G. C. & Miner, M. D. Listeria monocytogenes - from saprophyte to intracellular pathogen, Nat. Rev. Microtia!. 7, 623-628, doi: 10.1038/nrmkro2171 (2009).

Gamiyn, D., Gal, L.„ Lemaitre, 1, P., Hartmann, A. & Pivetean, P. Communication and autoinduction in the species Listeria monocytogenes: A central role for the agr system. Comm n. Integr. Biol. 2, 371-374 (2009).

Atttret, ., Raynaud, C, Dubail, I., Berche, P. & Charbit, A. Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence. Infect Immun. 71, 4463- 4471 (2003).

Pviede!, C. U. et ai AgrD-dependent quorum sensing affects biofilm formation, invasion, virulence and global gene expression profiles in Listeria monocytogenes. MM. Microbiol 71, 1177-1 ] 89, doi: 10.111 1365-2958.2008.06589.X (2009).

Rieu, A., Weidrnann, S._s Garmyn, D., Piveteau, P. & Guzm, J. Agr system otlisieria monocytogenes EGD-e: role in adherence and differential expression pattern. Appl Environ, Microbiol 73, 6325-6133, doi: 10.1128/aem.00608-07 (2007).

Rieu, A. et al Listeria monocytogenes EGD-e hiofilnis: no mushrooms but a network of knitted chains. AppL Environ, Microbiol 74, 4491-4497, doi: 10.1128/aera.00255- 08 (2008).

Zetzmann, M., Sanchez-Kopper, A., Waidmann, M. S.. Blombacii, 8. & iedel, C. U. Identification of the agr Peptide of Listeria monocytogenes. Front Microbiol. 7, 989, doi: 103389/&aicb.2016.00989 (2016).

Gilmore, M. S., Clewell, D. B.. Ike, Y. & Shankar, N. Enierococci. (Massachusetts Eye and Ear Infirmary, 2014). 76 Makayama, J. et at Ge!aiinase hiosynihesis-acti vatiag pheromone: a peptide lactone thai mediates a quorum sensing in Enterococcus faecalis, Mol Microbiol 41, 1 5- 154 (2001).

77 Nakayama, J. et ai Revised model for Enterococcus faecalis far quorum-sensing system: the small open reading feme fsrD encodes h gelatinase biosynthesis- activating pheromone propeptide corresponding to staphylococcal agrd. J, B ctertoi 188, 8321-8326, doklO.l 128/jb.00865-06 (2006).

78 Cook, L. C. & Federle, M 1 Peptide pheromone signaling in Streptococcus and

Enterococcus. F EMS Microbiol. Rev, 38, 473-492, doUOJ l l 1/1574-6976, 12046 (2014).

79 Hancock, L, E, & Perego, M, The Enterococcm faecalis fsr two-component system controls hiofilm development through production of gelatinase, J. Bacteriol. 186, 5629-5639, doi: 10.1128 jb.186.17,5629-5639.2004 (2004).

80 Qi«, X., Singh, K. V., Weinstock, G. M & Murray, B. E. Effects of E terococcm faecalis fir Genes on Production of Gelat inase and a Serine Protease and Virulence, doi: 10.1128/ΪΑΙ.68.5.2579-2586.2000 (2000).

81 Thurlow, L. R. et ai Gelatinase contributes to the pathogenesis of endocarditis caused by Enierococcm faecalis..Infect Immun. 78, 4936-4943, doi.TO.3128/i.aL011 18-09 (2010).

82 Engeiberi, M,, Mylonakis, E., Ausubel, F. M., Calderwood, S. B. & Gilmore, M. S.

Contribution of gelatinase, serine protease, and fsr to the pathogenesis of

En terococcus faecalis endophthalmitis, infect Immtm, 72, 3628-3633,

doi:10. I 12S/iai.72.6.3628-3633.2004 (2004).

83 Shankar, I,, Walker, R. G., Ward, D. & Horsburgh, M. J. in PLoS One Vol. 7

(2012),

84 Darkoh,€., DuPont, H. L., orris, S. J. & Kaplan, H. 8. Toxin synthesis by

Clostridium difficile is regulated through quorum signaling, mliio 6, e02569, doi:10.1128/ Bio.G2569-14 (2015).

85 Darkoh, C,, Odo, C. & DuPont, E. L. Accessory Gene Regulator-} Locus is Essential for Virulence and Pathogenesis of Clostridium difficile, mBio 7,

doi: 10.1128/mBio.01237-16 (2016).

86 Kuehne, S. A. et ai The role of toxin A and toxin B in Clostridium difficile infection.

Nature 467, 711-713, doi: 10.1038/nature09397 (2010). 8? C ben, S. if, et at Clinical practice guidelines for Clostridium difficile infection in adults 2010 update by the society for healthcare epidemiology of America (SH EA) and the infectious diseases society of America (IDSA). Infect. Control Hasp.

Epidemiol 31, 431-455, doi: 10.1086/651706 (2010).

88 Naskalska, A, & Pyre, . Vires like Particles as iinrnnrrogeris and Universal

auoeamers. Pol. J. Microbiol 64, 3-13 (2015).

89 Lacson, E. et al. Antibody response to Engerix~B and ecorobivax-HB hepatitis B vaccination in end-stage renal disease. Hemodialysis international. International Symposium on Home Hemodialysis 9, 367-375, doi: 10.1 11 1 /j .1492- 7535.2005.01155.x (2005).

90 GAJtl.X4SlL4^:M>9 (Human Papillomavirus 9-v leni Vaccine, Recombinant) for Healih Care Professionals, <!iitB : w^

pgii1 JoXimiC yLRSR0EK44lluV0Tn0000rKPQB0Na

sid=¾z3-n BtiBPk:i a zyyb3i5Babwl0BqEolB-> (2016).

91 Chackeriao, B., Dirrfee, M. R. & Schiller, J. T. Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model. Immunol ISO, 5816-5825 (2008).

92 Chackerian, B., Lowy, D. R. & Schiller, J. T. Conjugation of a. self-antigen to

papiHoraavirus-like particles allows for efficient induction of protective

autoantibodies. 1 din. Invest 108, 15-423 (2001 ).

93 Chackerian, B., Lowy, D. R. & Schiller, J. T. Induction of autoantibodies to mouse CCR5 with recombinant papilloraaviras particles. Proc. Natl Acad. Scl U. S, A, 96, 2373-2378 (1999).

94 Frietze, K, M., Peabody, D. S. & Chackerian, B. Engineering virus-like particles as vaccine platforms. Curr. Opin. Viml. 18, 4-49, doi: 10.1016 j.coviro .2016.03.001 (2016),

95 Effio, C, L. & Hubbueh, J. Next generation vaccines and vectors: Designing

downstream processes for recombinant protein-based virus-like particles.

Biotechnology journal 1.0, 715-727, doi :10.1002/biot.201400392 (2015).

96 Bachmann, M. F. & Jennings, G. T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nature reviews. Immunology 10, 787-796 (2010).

97 Jennings, G. T. & Bachmann, M. F. Imraimodrags: therapeutic VLP -based vaccines for chronic diseases. Anna. Rev, Pharmacol, Toxicol 49, 303-326 (2009). 98 Rivera-Heniandez, T. ei ai. Self-adjuvanting modular virus-like particles for mucosal vaccination against grou A streptococcus (GAS). Vaccine 31, 1950-1955, doi : 10.1016/j .vaccine .2013.02.013 (2013).

99 Seth, A. ei ai. Modular virus-like particles for sublingual vaccination against group A streptococcus. Vaccine, doi:!0»! 0165.vaccine.2016J LOOS (2016).

100 Tamborrint, M. et ai. A Synthetic Virus-Like Particle Streptococcal Vaccine

Candidate Using B-Cell Epitopes from the Praline-Rich Region of Pneumococcal Surface Protein A. Vaccines 3, 850-874, doi: 10.3390/vaccines3040850 (2015).

101 Alksne, L. E, & Projan, S, J. Bacterial virulence as a target for antimicrobial

chemotherapy. C rr. Opm. iot.echnoi 11, 625-636, doL'Doi I0.1O16/S 958- 1669(00)00155-5 (2000),

102 in National Research Council (US) Committee for the Update of the Guide for

theCare and Use of Laboratory Animals (National Academies Press (US), 2 11).

103 Rothfork, J. M., Dessus-Babus, S., Watnel, W. J. V., Cheung, A. L. & Gresham, H. D.

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-reguktion. J Immunol 171, 5389-5395 (2003).

104 Schneider, C. A., Rasband, W. S. & Eiiceiri, . W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675 (2012).

Claims

hat is claimed is:

1. A composition comprising: (a) a viras-like particle; and (b) at least one ami gen or antigenic determinant; wherein said antigen or antigenic determinant is displayed on said viras-Hke particle, and wherein said antigen or antigenic determinant comprises an epitopic peptide derived from Staphylococcus aureus autoindueing peptides

(AIPs), SA toxins or leukociditts.

2. The composition of claim 1 , wherein said antigen or antigenic determinant is

displayed at tfce A-B loop, N-terminn or carboxy terminus of a bacteriophage coat protein.

3. The composition of claim 1 or 2, wherein the bacteriophage coat protein is a single- chain, dimer coat protein derived from PP7 bacteriophage.

4. The composi tion of claim 1 or 2, wherein the bacteriophage coat protein Is a single- chain dimer coat protein derived from MS2 bacteriophage.

5. The composition of claim I or 2, wherein the bacteriophage coat protein is a single- chain dimer coat protein derived from the family of single-stranded RNA

bacteriophage, including, but not limited to, PP7, MS2, Qp\ R.I7, SP, GA, M i l, MX i , f4, Cb5, Cbl2r, Cb23r, 7s and £2.

6. The composition of any of claims 1 -5, wherein said SA epitopic peptide is derived from SA autoinducing peptides.

7. The composition according to any of claims 1-5 wherein said SA epitopic peptide is according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO;! I , SEQ ID NO; 1 , SEQ ID NO: 13, SEQ ID NQ:34 SEQ ID HO: 15, SEQ ID NO: 1.6, SEQ ID NO: 17, SEQ ID ^'NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO;2I, SEQ ID NO;22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO;25₅ SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28_} SEQ ID NO:29, SEQ ID NO:30, or an y epitopic amino acid sequence thereof comprising at least 4 continguous amino acids.

8. The composition according to claim 7 wherein said SA epitopic peptide is SEQ ID NO: I , SEQ ID NO:2, SEQ ID NO:3, SEQ I.DNO:4₅ SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO; 7 or SEQ ID NO: 8.

9. The composition according to any of claims 1 -8 wherein said SA epitoptic peptide i SEQ ID NO: 1 or SEQ ID O:2.

10. The composition according to any of claims US, wherein said S A epi topic peptide is derived from SA Toxins,

11. The composition according to any of claims 1.-5, wherei said SA epitopic peptide is derived from SA leukocidins.

12. A composition according to claim 2 or 3 wherein said VLP comprises a

bacteriophage coat protein which is a single-chain dimer coat protein derived from PP7 bacteriophage, said SA epitopic peptide is SEQ ID NG;1 or SEQ ID MO:2 and said epitopic peptide is displayed at the A-B loop of said bacteriophage dinner coat protein.

13. The composition according to claim 12 wherein said SA epitopic peptide is SEQ 3D NO: l.

14. The composition according to claim 12 wherein said SA epitopic peptide is SEQ IDE KO:2.

15. A . population of virus-like particles according to any of claims 1-14.

16. A pharmaceutical composition comprising a population of virus-like particles

according to claim 15 in combination with a pharmaceutically acceptable carrier, additive and/or excipienl

17. A method for enhancing an immune response against an antigen in an animal

comprisin introducing the compositio of any of claims 1 -14 and 16 into said animal, wherein an enhanced immune response against said antigen is produced in said animal.

18. The method of claim 17, wherein the composition is prophylactic for S- aureus

induced disorders.

19. The method of claim 18 wherein said S. aureus induced disorder is a MRSA infection.

20. The method of claim 18 wherein said 8, aureus induced disorder is a MSSA

infection.

21. The method according to any one of claims 17-20, wherein said immune response is an enhanced 8 ceil response and or an enhanced T cell response.

22. The method according to any one of claims 17-21 , wherein said animal is a human.

23. The method according to any one of claims 17-22. wherein said composition is

introduced into said animal subcutaneously, intramuscularly, intravenously, intranasaliy, intravaginally or directly into the lymph node.

24. A vaccine comprising an immanologtealJy effective amount of the composition of aiiy of claims 1 -14 together with a phamiaceotiealiy acceptable diluent, carrier and/or excipient.

25. A method of immunizing or treating an animal comprising administering to said

animal an immunologically effecti ve amoun -of the vaccine of claim 24.

26. The method of claim 1 , wherein said animal is a human.

27. The vaccine of claim 14, wherein said vaccine former comprises an adjuvant.