Precision immunoscore for staphylococcus aureus

Abstract

Provided herein are methods and systems for use in determining risk of a Staphylococcus infection, predicting efficacy of a Staphylococcus vaccine, and prognosing clinical outcomes on a subject exposed to Staphylococcus. Methods of treating subject identified as having or at risk of a Staphylococcus infection are further provided.

Description

PRECISION IMMUNOSCORE FOR STAPHYLOCOCCUS AUREUS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made in part with Government support under Grant NumberAI159677 awarded by National Institutes of Health. The Government has certain rights in this invention. FIELD OF THE INVENTION

[0002] The present invention generally relates to methods and systems for determining risk of aStaphylococcus infection, predicting efficacy of a Staphylococcus vaccine, and prognosing clinical outcomes on a subject exposed to a Staphylococcus and applications thereof in prophylactic or therapeutic treatment of a Staphylococcus infection. BACKGROUND OF THE INVENTION

[0003] At the start of this decade, the world again witnessed the remarkable ability ofvaccination to mitigate an infectious disease threat. SARS-CoV-2 spread rapidly within the human population wherein pre-existing immunity was negligible. On this backdrop, targeting a single viral antigen through mRNA-based vaccine technology engendered population-level immunity at an unprecedented speed. In stark contrast, more than 20 years have elapsed since the initial Phase III clinical trial of a vaccine to prevent Staphylococcus aureus infection. Since that time, understanding of the molecular pathogenesis of S. aureus disease has become more sophisticated, kindling additional vaccine trials. These efforts, however, have not yet culminated in the development of a successful vaccine, compelling a much-needed focus on investigation of the human immune response to S. aureus.

[0004] Staphylococcus aureus is a leading bacterial cause of morbidity and mortality in thehuman population. The remarkable pathogenic potential of this organism has been demonstrated over the past 15 years, with the rapid spread of highly virulent S. aureus strains worldwide. S. aureus commonly causes bacteremia and sepsis, pneumonia, infection of the skin and soft tissues, osteomyelitis, and septic arthritis in addition to a host of toxin mediated entities including staphylococcal toxic shock syndrome, enterotoxin-induced gastrointestinal disease, andlife-threatening desquamation caused by a family of epidermolytic toxins. The impact of S. aureus infection is well-documented: epidemiologic studies indicate a population-based incidence of >3.6 / 100,000 person-years for S. aureus bacteremia and >122 / 100,000 person-years for skin and soft tissue infections. Mortality rates for invasive S. aureus disease consistently exceed 20% and in some populations approach 50%. Among hospitalized patients, infection was associated with a 3-fold increase in both cost and length of stay, and a 5-fold increase in risk of in-hospital death. The annual US economic burden ascribed to S. aureus infection exceeds $14 billion. This pathogen strikes individuals of all ages; it remains unclear if any humans are fully immune to disease. Current epidemic strains harbor genes encoding for resistance to methicillin (MRSA), rendering the once highly potent class of β-lactam antimicrobials obsolete as therapeutic agents. Greater than 80,000 cases of invasive MRSA occurred in the US in 2011, resulting in over 11,000 deaths and leading to the CDC designation of this pathogen as a serious threat.

[0005] Observations on S. aureus disease readily illustrate the complexity of the interactionbetween this microbe and its human host. In 1928, twenty-one children in Australia that received von Behring's diphtheria toxin-antitoxin serum were unknowingly injected with the so-called 'Bundaberg staphylococcus' as a contaminant of the vaccine lot. Among these, twelve died from fulminant infection, six became ill but recovered, and three did not experience systemic disease but developed local abscess lesions at the immunization site. In the official report on this incident, it was noted that “a very large number of organisms was directly introduced into the subcutaneous tissues of extremely susceptible, partially susceptible, and almost insusceptible children,” highlighting variation in clinical outcome despite identical inoculation. Nearly a century later, the understanding of human anti-staphylococcal immunity is insufficient to predict susceptibility to infection or to provide prognostic insight on clinical outcomes. This fact underscores the fundamental gap in knowledge that hinders vaccine development.

[0006] Therefore, there is an unmet need for developing methods and systems that objectivelyquantifies one or more attributes of the immune response to Staphylococcus aureus. SUMMARY OF THE INVENTION

[0007] Disclosure encompasses a method of determining risk of a Staphylococcus infection in asubject. The method comprises providing data obtained from immunological tests from abiological sample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies and generating a score for the subject based on the data. A score above a predetermined threshold value is indicative of the subject being at risk of the Staphylococcus infection.

[0008] Further provided is a method of vaccinating a subject with a Staphylococcus vaccine. Themethod comprises providing data obtained from immunological tests from a biological sample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies, generating a score for the subject based on the data, wherein a score above a predetermined threshold value is indicative of the subject being at risk of the Staphylococcus infection, and administering a Staphylococcus vaccine to the subject determined to be at risk of the Staphylococcus infection.

[0009] A method of predicting efficacy of a Staphylococcus vaccine in a subject is furtherprovided. The method comprises providing data obtained from immunological tests from a biological sample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies, and generating a score for the subject based on the data. A score above a predetermined threshold value is indicative of the vaccine being efficacious for the subject.

[0010] Further provided is a method of determining risk of recurrent Staphylococcus infection ina subject. The method comprises providing data obtained from immunological tests from a biological sample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies, and generating a score for the subject based on the data. A score above a predetermined threshold value is indicative of the subject being at risk of recurrent Staphylococcus infection. A score below threshold value may indicate that the subject is free of infection for about 3 months to 15 months.

[0011] The Staphylococcus may be Staphylococcus aureus. The Staphylococcus aureus may beMethicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin- resistant Staphylococcus aureus (VRSA). The Staphylococcus infection may be selected from an invasive infection, a skin and soft tissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea,dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister.

[0012] The biological sample may be a blood sample. The blood sample may be selected fromwhole blood, serum, and plasma.

[0013] The total Hla Ig titer and the Hla neutralizing antibodies may be measured, respectively,as a log of the total amount of Hla Ig titer and the amount of Hla neutralizing antibodies.

[0014] The score may be a combined score determined by weighting the data obtained from eachof the immunological tests with a predefined coefficient to obtain a weighted value for each immunological test and adding the weighted values to obtain the combined score.

[0015] The Staphylococcus infection may be a SSTI, the immunological tests comprise a log ofthe total Hla Ig titer and the amount of Hla neutralizing antibodies; their respective weights are 0.036 and 0.266; and the predetermined threshold value is 0.473. The Staphylococcus infection may be an invasive infection, the immunological tests comprise a log of the total Hla Ig titer and the amount of Hla neutralizing antibodies; their respective weights are 1.335 and -0.323; and the predetermined threshold value is 0.277.

[0016] The method may further comprise providing a biological sex of the subject, an age of thesubject, and a history of S. aureus colonization of the subject. The score is a combined score determined by weighting the log of the total Hla Ig titer, the amount of Hla neutralizing antibodies, the biological sex of the subject, the age of the subject, and the history of S. aureus colonization of the subject. The Staphylococcus infection may be a SSTI; the weight for male sex is -1.17; the weight for age is -0.178; weight for S. aureus colonization is -0.482; the weight for no S. aureus colonization is -2.16; the weight of the amount of Hla neutralizing antibodies is 0.179; the weight of the log of the total Hla Ig titer is 0.399; and the predetermined threshold value is 0.458. The Staphylococcus infection may an invasive infection; the weight for male sex is 0.660; the weight for age is 0.055; the weight for S. aureus colonization is -0.658; the weight for no S. aureus colonization is -1.65; the weight of the amount of Hla neutralizing antibodies is 1.21; the weight of the log of the total Hla Ig titer is -0.348; and the predetermined threshold value is 0.517.

[0017] The method may further comprises providing a biological sex of the subject, an age of thesubject, a history of S. aureus colonization of the subject, and status of SSTI in the subject’shousehold. The score may be a combined score determined by weighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the age of the subject, the history of S. aureus colonization of the subject, and status of SSTI in the subject’s household. The Staphylococcus infection may be a SSTI; the weight for male sex is 1.07; the weight for age is -0.168; the weight for S. aureus colonization of the subject is -0.467; the weight for no S. aureus colonization is -2.20; the weight of the amount of Hla neutralizing antibodies is 0.080; the weight of the log of the total Hla Ig titer is 0.407; the weight for SSTI in the subject’s household is 14.5; the weight for no SSTI in the subject’s household is 13.6; and the predetermined threshold value is 0.520. The Staphylococcus infection may be an invasive infection; and the weight for male sex is 0.625; the weight for age is 0.054; the weight for no S. aureus colonization is -1.75; the weight of the amount of Hla neutralizing antibodies is 1.26; the weight of the log of the total Hla Ig titer is -0.332; the weight for SSTI in the subject’s household is -1.42; the weight for no SSTI in the subject’s household is -0.857; and the predetermined threshold value is 0.480.

[0018] The immunological tests may further comprise a level of SpA neutralization titer, aresponse of T follicular helper cells to Staphylococcus(Tfh), a response of T regulatory cells to Staphylococcus (Treg), a ratio of Tfh over Treg (Tfh:Treg ratio), and a cytokine response profile of T cells. The score may be a combined score determined by weighting the data obtained from each of the immunological tests with a predefined coefficient and adding the weighted data to obtain the combined score. The predefined coefficient may be obtained from a control data set, wherein each of the immunological tests in the control dataset is ranked for the ability to predict risk of the Staphylococcus infection, predict efficacy of a Staphylococcus vaccine, determining risk of recurrent Staphylococcus infection, or predict whether the subject is at risk of SSTI or invasive Staphylococcus infection.

[0019] The score may be generated by a processor using a machine learning model. The machinelearning model may comprise logistic regression.

[0020] The methods may further comprise administering to the subject a vaccine or a treatmentfor Staphylococcus. The vaccine may be StaphVax. The vaccine may comprise an antigen selected from CP5, CP8, ClfA(SA3Ag), CP5-CRM197, CP8-CRM197, MntC, ClfA(SA4Ag), Alpha-toxin,Panton-Valentine Leukocidin, Leukocidin AB, HlgAB, HlgBC, Surface protein A (SpA), enterotoxin B, and a live or attenuated S. aureus.

[0021] The method may further comprise determining a prognosis of the subject, wherein a scoreabove the predetermined threshold indicates a positive outcome selected from positive survival outcome, a positive response to a Staphylococcus treatment, reduced recurrent Staphylococcus infection.

[0022] The method may further comprises administering a treatment to the subject. Thetreatment may comprise providing an antibiotic to the subject. The antibiotic may be selected from cefazolin, nafcillin, oxacillin, vancomycin, daptomycin, and linezolid.

[0023] A score below or equal to the predetermined threshold value may be indicative of thesubject being at low risk of the Staphylococcus infection or recurrent Staphylococcus infection.

[0024] A score below or equal to the predetermined threshold value is indicative of the vaccinebeing unlikely to be efficacious for the subject.

[0025] Disclosed further is a system comprising a processor in communication with a memory,the memory including instructions, which, when executed, cause the processor to execute the methods.

[0026] Provided herein is a kit comprising means for performing one or more immunologicaltests comprising at least amount of Hla neutralizing antibodies and the total Hla Ig titer and instructions for: determining risk of a Staphylococcus infection, vaccinating a subject with a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, or determining risk of recurrent Staphylococcus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a schematic of the conceptual framework for use of an immunoscore to informS. aureus vaccine development. Humans exhibit heterogeneity in the host immune response to S. aureus. Positioning the field to develop and implement functional assessment of the protective immune response will enable stratification of the population that can be utilized to guide targeted vaccine­based interventions.

[0028] FIG. 2 shows acute anti-Hla NAb titer comparison across groups. Acute anti-Hla NAbtiters in S. aureus colonized healthy controls (HC), non-colonized healthy controls (NC), children with SSTI, or invasive infections (INV). NAb titer determined by RBC lysis assay. Data expressed as log NAb titer, comparisons by ANOVA with Tukey’s post-test if significant or paired t-test. **p<0.005, ***p<0.0005, ****p<0.0001.

[0029] FIG. 3A – 3B show comparison of Acute and Convalescent Log Anti-Hla NAb Titers inSSTI and INV. FIG.3A shows acute and convalescent log anti-Hla NAb titers in children with SSTI or invasive infections. FIG.3B shows paired log anti-Hla NAb acute (green circle) and convalescent (blue circle) titers from individuals depicted on the right. Red lines indicate individuals with an increased convalescent to acute titers, black lines depict individuals without an increase in convalescent to acute titer. NAb titer determined by RBC lysis assay. Data expressed as log NAb titer, comparisons by ANOVA with Tukey’s posttest if significant or paired t-test. ***p<0.0005, ****p<0.0001.

[0030] FIG. 4A – 4B show acute and convalescent log Anti-Hla NAb titers in SSTI (FIG. 4A)and invasive (FIG.4B) groups with recurrent infection or no recurrent infection. Anti-Hla acute and convalescent NAb titers in children with SSTI (FIG.4A) or invasive infections (FIG.4B) stratified by those with or without recurrent infections during 12 months follow-up. NAb titer determined by RBC lysis assay. Data expressed as log NAb titer, comparisons by paired t-test. **p<0.005.

[0031] FIG. 5A – 5C show acute anti-Hla NAb titers stratified by age groups. Acute anti-HlaNAb titer in children without a history of S. aureus infections (FIG.5A), SSTI (FIG.5B), or invasive infection (FIG.5C) stratified by age group. NAb titer determined by RBC lysis assay. Data expressed as log NAb titer, comparisons by ANOVA. *p<0.05.

[0032] FIG. 6A – 6B show correlation between fold induction (Ratio Convalescent:Acute loganti-Hla) titer and risk of recurrent infection. Fold induction titer was calculated as a ratio between the convalescent and acute anti-Hla NAb titer. Boxplots of fold induction log anti-Hla NAb ratio depicted for individuals with invasive and SSTI infections (FIG.6A) with recurrent infection (blue) or without recurrent infection (red) during the 12 month follow-up. Simple logistic regression model of fold induction and recurrent infection in the SSTI group (FIG.6B) indicating the odds of recurrent infection decreases as the fold induction titer increases (OR 0.543, 95% CI: 0.308, 0.0960), p=0.036.

[0033] FIG. 7A – 7C show Receiver Operating Characteristic (ROC) curves for the logisticregression models predicting risk for skin and soft tissue infection. Each curve plots sensitivity (true positive rate) against 1 - specificity (false positive rate) across various classification thresholds. FIG.7A shows the area under the ROC curve (AUROC) representing a 5 feature model with a discriminative abilities at 0.81 for model including the following features: age, sex,history of S. aureus colonization, and level of total Hla serum Ig titer together with Hla- neutralizing antibodies. FIG.7B shows the ROC curve representing a 2 feature model with a discriminative abilities at 0.64 for model including only the following features: Hla serum Ig titer and Hla-neutralizing antibodies. FIG.7C shows the ROC curve representing a 6 feature model including the following features: sex, age, household ssti, colonization at enrollment, lognab_acute, and logigg_acute.

[0034] FIG. 8A – 8C show Receiver Operating Characteristic (ROC) curves for the logisticregression models predicting risk for invasive infection. Each curve plots sensitivity (true positive rate) against 1 - specificity (false positive rate) across various classification thresholds. FIG.8A shows the area under the ROC curve (AUROC) which represents a 5 feature model with a discriminative abilities at 0.82 for model including the following features: age, sex, history of S. aureus colonization, and level of total Hla serum Ig titer together with Hla- neutralizing antibodies. FIG.8B shows the ROC curve which represents a 2 feature model with a discriminative abilities at 0.79 for model including only the following features: Hla serum Ig titer and Hla-neutralizing antibodies. FIG.8C shows the ROC curve representing a 6 feature model with the following features sex, age, household ssti, colonization at enrollment, lognab_acute, and logigg_acute.

[0035] FIG. 9A – 9G show T cell function is altered by infection. FIG. 9A is a schematic of S.aureus infection model and immunologic analysis. FIG.9B shows skin lesion area in mice following infection with either wild-type S. aureus (WT) or its isogenic ^hla variant. FIG.9C shows T cell immunophenotyping gating strategy to identify S. aureus antigen-activated Ki67+ cells, which are then evaluated for proportion of T effector (TEF, CD44+ CD62-) and T memory (TMEM, CD44+ CD62+) populations. The TEF population is then examined to assess number of FoxP3- (non-T regulatory cells) and FoxP3+ T regulatory cells (TREG). Non-TREG cells are then evaluated for percent T follicular helper (TFH) cells. FIG.9D shows TEF and TMEMCD4+ cells generated 10 days post-primary infection with S. aureus or S. aureus ^hla. FIG. 9Eshows analysis of FoxP3- (left), FoxP3+ TREG (center) and TFH populations following infection as in FIG.9D. FIG.9F shows analysis of intracellular cytokine staining of CD4+ T cells harvested from infected mice, quantifying the percent of FoxP3- cells expressing IL-17 (left), IFNγ(center), or IL21 (right). FIG.9G shows FoxP3+ cells expressing IL10.

[0036] FIG. 10A – FIG. 10K show T cells are exposed to Hla during skin infection. FIG. 10Ais a schematic showing timeline of skin infection with S. aureus and harvest of skin draining lymph node (dLN) to quantify active Hla present in the nodal tissue by ELISA. FIG.10B shows ELISA showing quantification of Hla present in skin dLN from mice infected with S. aureus WT or S. aureus Dhla, n=5 per group. FIG.10C is a schematic depicting timeline for evaluation of immune cell-associated AF647-labelled recombinant active Hla following skin injection. FIG. 10D shows Gating strategy for identification of AF647-Hla+cells. Quantification of Hla+CD4 (FIG.10E), CXCR5+Bcl6+(FIG.10F), CD19+(FIG.10G), and CD11b+MHCIIhicells (FIG. 10H) in the skin dLN at 0, 1, and 6 hours post-injection of AF647-Hla. FIG.10I shows confocal microscopy images of the intact dLN as in (FIG.10C- FIG.10H) stained to evaluate CD4+ T cells (green), CD11b+MHCIIhidendritic cells (red), and AF647-Hla (magenta). Images were subjected to 3D Imaris reconstruction (right panels) to show features of cells and localization of Hla. Quantification of T cell volume (FIG.10J) and T cell sphericity (FIG.10K) in the absence or presence of exposure to recombinant Hla.

[0037] FIG. 11A – FIG. 11J show Hla blunts T cell Ca2+ signaling. FIG. 11A is a schematic ofapproach to cell harvest from mouse lymph node (LN) to purify CD4+T cells for analysis of cellular signaling. FIG.11B shows relative T cell membrane potential measured by fluorescence of the Ca2+indicator dye DIBAC in response to 0.03mM (purple), 0.06mM (magenta), or 0.09mM (pink) recombinant active Hla. FIG.11C shows quantification of findings in FIG.11B from n=3 independent mice per group. FIG.11D shows Ca2+influx after isolated addition of Hla to CD4+T cells, showing that toxin alone does not result in influx of Ca2+. FIG.11E shows Ca2+influx after isolated addition of Hla to CD4+T cells, showing that toxin alone does not result in influx of Ca2+. FIG.11F shows Ca2+influx kinetics after addition of ionomycin to untreated CD4+T cells (black) or following addition of 0.03-0.09 mM Hla in addition to ionomycin. FIG. 11G shows quantitative comparison of data in FIG.11F showing dose response impairment of Ca2+influx. FIG.11H is a schematic diagram of Hla pre-pore locked mutant (HlaPPL) that is incapable of forming the membrane-perforating pore until DTT is added to cells. FIG.11I shows relative T cell membrane potential measured by fluorescence of the Ca2+indicator dye DIBAC in response to DTT alone (black), recombinant HlaPPL 0.06mM (purple), or recombinant HlaPPL (0.06mM (pink)) in the presence of DTT. FIG.11J shows quantification of findings in FIG.11I from n=6 independent mice per group.

[0038] FIG. 12A – FIG. 12G show Hla dampens TCR signal strength. FIG. 12A is a schematicof S. aureus infection model with delivery of CD4+OT-II T cells for antigen-specific T cell response analysis. FIG.12B shows FACS-based analysis of CFSE dilution in OT-II T cells 10 days following primary skin infection with WT S. aureus and its Dhla variant or S. aureus and the Dhla variant harboring a plasmid that enables expression of ovalbumin in the context of infection (S. aureusOVA and S. aureus DhlaOVA). FIG.12C shows quantification of total proliferating OT-II T cells as in FIG.12B. Analysis of OT-II T cells with limited CFSE dilution (Divisions 0-1, (FIG.12D)) and maximal observed CFSE dilution (Division 8 (FIG.12E)) from mice as in FIG.12B. FIG.12F shows analysis of OT-II T cell activation by wild type OVA peptide (OVAWT, circles) or the weak T cell receptor binding altered peptide ligand OVAH331R(triangles), measured by CD69 expression on cells exposed to either active Hla (black) or inactive HlaH35L (gray). FIG.12G shows quantification of relative T cell receptor signal reduction upon exposure to OVAH331R compared to OVAWT based on area under the curve (AUC) analysis from data in FIG.12F.

[0039] FIG. 13A – FIG. 13I show HlaHRE vaccine restores T cell function. FIG. 13A is aschematic of Hla vaccine model showing timing of vaccine administration, delivery of CFSE- labelled OT-II cells, infection, and immune cell analysis. FIG.13B shows skin lesion area in mice following infection with either wild-type S. aureus (S. aureusWT) or its the OVA-expressing variant (S. aureusOVA). FIG.13C shows FACS-based analysis of CFSE dilution in OT-II T cells 7 days following primary skin infection as in FIG.13B. FIG.13D shows quantification of total proliferating OT-II T cells as in FIG.13C. FIG.13E shows analysis of CD4+FoxP3+T cells present in the draining lymph node of mice that were vaccinated with either Alum or HlaHRE vaccines, then subjected to primary infection with S. aureus on the right flank and secondary infection on the left flank. FIG.13F shows analysis of CD4+FoxP3+T cells in the draining lymph nodes of mice subjected to infection with S. aureus wild-type (WT), the isogenic Dhla mutant, or the Dhla variant complemented with plasmid-encoded Hla (Dhla::phla). FIG.13G shows analysis of CD4+TFHcells present in the draining lymph node of mice as in FIG.13E. FIG.13H shows analysis of CD4+TFH cells in the draining lymph nodes of mice as in FIG.13F. FIG.13I shows analysis of sialylation of IsdB antibody in serum from mice as in FIG.13F.

[0040] FIG. 14A – FIG. 14B show 7B8-HIgG1 standard curve (FIG. 14A) and quantitation ofthree distinct human sera for Hla neutralizing antibody (FIG.14B).DETAILED DESCRIPTION

[0041] The current disclosure is based partly on developing a group of fundamental attributes ofthe host immune response to S. aureus that may be harnessed to predict susceptibility to infection and to inform potential benefit of vaccination at the level of an individual patient. It has been recognized that failed vaccine trials in the field are attributable at least in part to pre-existing immune responses within the population. To overcome this challenge, the inventors have developed a reliable method to risk stratify patients based on potential vaccination outcome. Referred to herein as Predictive Immunoscore for S. aureus (PISA), it is capable of stratifying immunologic risk in the setting of heterogeneous pre-existing anti-staphylococcal immune responses to not only inform vaccine design, delivery, and outcomes prediction, but stratify riskfor infection. To this end, the inventors have leveraged study of the pediatric immune response toS. aureus to examine this hypothesis. It was demonstrated that the serologic response to S. aureus a-toxin (a-hemolysin, Hla), a major virulence factor of this pathogen, is a correlate of human protective immunity. Relying on a pre-existing and well-characterized population of pediatric subjects with S. aureus infection and matched controls, the ability of a predictive algorithm to utilize individual-specific clinical data together with serum anti-Hla titers and Hla- neutralizing titers were evaluated to identify risk for infection. Data showing that a limited number of features enables the development of a clinically interpretable supervised predictive model that successfully classifies individuals with S. aureus infection from uninfected controls is presented. By leveraging easily obtainable patient characteristics alongside serum biomarkers, this approach has the potential to provide real-world applicable tools to predict infection risk and guide early interventions, specifically targeted vaccine administration.1. Definitions

[0042] The terminology used herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0043] For recitation of numeric ranges herein, each intervening number therebetween with thesame degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[0044] The use of a singular term, such as, “a” is not intended as limiting of the number of items.Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0045] Further, as the present disclosure is susceptible to aspects of many different forms, it isintended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0046] Any term of degree such as, but not limited to, “substantially” as used in the descriptionand the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than orequal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0047] The terms “comprising,” “including” and “having” are used interchangeably in thisdisclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

[0048] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive ormeaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0049] The term “about”, as used herein, may refer to variation of a numerical quantity that canoccur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%, 2%,1%, etc.

[0050] As used herein Staphylococcus, is a major human pathogen, producing a multitude ofvirulence factors making it able to cause several types of infection, from superficial lesions to toxinoses and life-threatening systemic conditions such as endocarditis, osteomyelitis, pneumonia, meningitis and sepsis. Staphylococcus may include Staphylococcus aureus, Staphylococcus epidermidis, and strains such as Staphylococcus carnosus. In one exemplary aspect, Staphylococcus is Staphylococcus aureus.

[0051] As used herein, Staphylococcus infection can range from minor skin problems to life-threatening illness. Signs and symptoms of Staphylococcus infections vary widely, depending on the location and severity of the infection. Skin infections by Staphylococcus may include boils, impetigo, cellulitis, and Staphylococcal scalded skin syndrome. Staphylococcus infection maycause food poisoning, symptoms of which can include nausea, vomiting, diarrhea, dehydration,and low blood pressure. Bacteremia can also occur with Staphylococcus infection and may cause meningitis, endocarditis, pneumonia, infections of bones, muscles, surgically implanted devices, such as artificial joints or cardiac pacemakers. Staphylococcus can also cause toxic shock syndrome, symptoms of which can include high fever, nausea and vomiting, rash, confusion, muscle aches, diarrhea, and pain. Staphylococcus infection may also cause septic arthritis, including joint swelling, pain in the joints, and fever.

[0052] As used herein, skin and soft tissue infections (SSTIs) may refer to a skin or soft tissueinfection (e.g. cellulitis, soft tissue abscess, dermonecrosis, myositis, impetigo, ecthyma, cutaneous abscesses, furuncles, carbuncles, and inflamed epidermoid cysts, erysipelas, faciitis, pyomysitis, gangrene, myonecrosis, or other infections) resulting from Staphylococcus entering the body at a site where a cut, scrape, bite, or other wound has broken the skin. In some instances, S. aureus SSSI is the result of Staphylococcus living on the body, and may occur spontaneously in the absence of a visible site of skin injury or wound. Such infections may affect the layers of the skin or deeper tissues, such as muscle and connective tissue (the interlacing framework of tissue that forms ligaments, tendons, and other supporting structures of the body). Skin abscesses may also occur in areas of the skin where the body has been fighting a Staphylococcus infection. Exemplary strains of S. aureus responsible for skin or soft tissue infections include antibiotic-resistant Staphylococcus known as methicillin- resistant Staphylococcus aureus (MRSA); vancomycin-resistant and daptomycin-resistant strains of S. aureus may also cause SSTI. MRSA is resistant to commonplace antibiotics. SSTIs may also be caused by methicillin sensitive Staphylococcus aureus (MSSA).

[0053] As used herein, invasive infection may refer to a Staphylococcus infection that occurswhen bacteria get inside the body and multiply. Invasive infection may cause bloodstreaminfection, septicaemia, sepsis, toxic shock syndrome, osteomyelitis, septic arthritis, endocarditis, pneumonia, or mortality.

[0054] As used herein, status of household SSTI may refer to the occurrence of SSTIs within ahousehold, where multiple members may be affected or where the infection is likely to spread among residents. These infections often involve bacteria like Staphylococcus aureus, which can be carried by asymptomatic individuals and transmitted within the household.

[0055] Vaccination as used herein will reduce the severity, delay, or prevent the development ofsymptoms. Subjects are at elevated risk of infection if they are hospitalized or living in aninstitutionalized community, antibiotic treated, or immunosuppressed including children having HIV / AIDS or other diseases that compromise immune function, individuals having frequent contact with the healthcare system, having a chronic illness such as diabetes, cancer, HIV / AIDS, being very young or very old, frequent use of antibiotics, having an open wound, dermatitis or skin lesions, poor nutrition or poor hygiene. Other subjects at risk include those living in crowded living conditions, military personnel, especially deployed troops, athletes, and prison inmates. Still others at risk of developing a Staphylococcus skin or soft tissue infection are those individuals previously having such infections or individuals scheduled for or having had a surgical or invasive medical procedure.

[0056] As used herein, the term “subject” may refer to a mammal, and in one example, a human.The mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.

[0057] As used herein, the term “healthy subject,” “control subject,” “normal subject” or asample from a “healthy” subject means a subject, or group subjects, who is / are diagnosed by a physician as not suffering from Staphylococcus aureus infection, or a clinical symptom associated with Staphylococcus aureus infection non-limiting examples include an invasive infection, a skin and soft tissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister) based on qualitative or quantitative test results. A “normal” subject is usually about the same age as the individual to be evaluated, including, but not limited, subjects of the same age and subjects within a range of 5 to 100 years.

[0058] The term “treatment” or “therapy” means to cure, heal, alleviate, alleviate, ameliorate,treat, ameliorate, ameliorate, or treat a condition (e.g., a disease), one or more symptoms of that condition; to prevent or delay the occurrence of symptoms, complications, biochemical indicators of, or otherwise statistically significantly halt or inhibit the further development of, the disease, symptoms or disease. Treatment may mean reducing one or more symptoms of a disease or condition.

[0059] A “threshold value” as used herein is meant to be relative to a number or value derivedfrom population studies, including without limitation, such subjects having similar age range, disease status (e.g., stage), subjects in the same or similar ethnic group. Such reference values can be derived from statistical analyses and / or risk prediction data of populations obtained from mathematical algorithms and computed indices of Staphylococcus aureus infection. Reference indices can also be constructed and used using algorithms and other methods of statistical and structural classification.2. Methods of evaluating infection risk and vaccine status

[0060] Provided herein are methods of determining risk of a Staphylococcus infection for asubject, determining a subject as being in need of a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine for a subject, and determining risk of recurrent Staphylococcus infection in a subject. The methods may comprise providing data obtained from immunological tests obtained from a biological sample from the subject, and may further comprise performing the immunological tests. The methods may further comprise generating a score for the subject based on the data. A score above a predetermined threshold value may be indicative of the subject being at risk of the infection, the subject being in need of a Staphylococcus vaccine, the vaccine being effective for the subject, or that the subject is at risk of recurrent Staphylococcus infection. The score may also comprise additional traits of the subject, which may include one or more of biological sex of the subject, the subject’s age, the subject’s history of S. aureus colonization, and the status of SSTI in the subject’s household. a. Immunological tests

[0061] Disclosed herein is one or more immunological tests.(1) Total α-hemolysin (Hla) Immunoglobulin G titer and Hla neutralizingantibodies The immunological tests may comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies. The total amount of Hla Ig titer and the amount of Hla neutralizing antibodies may be determined using any known method in the art. In some instances, the amount of Hla neutralizing antibodies may be determined using solid phase antibody detection assays such as enzyme-linked immunosorbent assay (ELISA) and a LUMINEX®Bead based assay. The amount of Hla neutralizing antibodies may also be measured using a Hla neutralizing antibody titer method, as described herein, such as in the methodsection. The method may use an Hla antibody, such as a 7B8-hIgG1 antibody. Briefly, a sample may be incubated in serial wells with a fixed quantity of recombinant active Hla. These samples may then be added to a fixed quantity of rabbit red blood cells. Lysis of the rabbit red blood cells may be quantified by an absorbance-based assay (OD450), where lysis may be inversely proportional to Hla neutralization by the antibody. IC50 titer may be used based on the absorbance to determine log of the amount of Hla neutralization antibodies or standard absorbance curves may be used to determine amount of Hla neutralization antibodies. An exemplary standard curve of 7B8-hlgG1 is shown in FIG.14A.

[0062] To determine total amount of Hla Ig, any established method in the art may be used,including but not limited to ELISA like assay, as described in the method section, below, and a LUMINEX®Bead based assay. Briefly, recombinant Hla may be utilized to coat the ELISA plate, after which a dilution range of human serum is incubated in serial wells to enable anti-Hla binding to recombinant Hla. Specific IgG to Hla may be detected using standard methods with an HRP-conjugated antibody to human IgG. Curves may be generated representing the absorbance per well across the dilution series for each serum sample. The half maximal titer may be defined based on absorbance and log transformed to determine the log of total amount of Hla Ig titer.

[0063] The total amount of Hla Ig titer and the amount of Hla neutralizing antibodies may bedetermined at any stage of the disease, such as an acute stage or a convalescent stage. In such instances, the total amount of Hla Ig titer and the amount of Hla neutralizing antibodies comprises an acute titer, a convalescent titer, or a log-fold induction titer.

[0064] The total amount of Hla Ig titer and the amount of Hla neutralizing antibodies obtainedmay be a conversion of a log of the total Hla Ig titer and a log of the amount of Hla neutralizing antibodies, respectively. In a specific instance, the log of total Hla Ig titer and the amount of Hla neutralization antibodies may be used in the method. (2) SpA neutralization titer

[0065] The immunological tests may further comprise an amount of antibodies that canneutralize the activity of Staphylococcus aureus protein A (SpA). SpA neutralization titer can be determined using a neutralization assay, i.e., by determining the presence and / or level of neutralizing antibodies. Methods for determining the presence and / or level of neutralizing antibodies of SpA are known in the art, and any of the available method may be used herein. Non-limiting examples of methods include ELISA, lateral flow tests, microneutralization assay,bead-based assay, and phagocytosis assay. Neutralization titer may be determined by identifying the highest dilution of the sample that shows a significant neutralizing effect and shows a certain percentage of alive cells. (3) Response of T follicular helper cell to Staphylococcus

[0066] The immunological tests may further comprise a response of T follicular helper (Tfh)cells in a sample obtained from the subject to Staphylococcus. Tfh facilitate B cells to produce antibodies that opsonize Staphylococcus cells and / or recruit neutrophils and macrophages for phagocytosis of Staphylococcus cells, resulting in clearance of Staphylococcus cells. Response of Tfh to Staphylococcus may be determined using one or more methods established in the field, examples of which include flow cytometry, intracellular cytokine staining, PCR, Cytokine and chemokine array analysis, sequencing, co-culture assays with for example B cells, ELISpot assays, expression analysis of genes associated with Tfh function such as Bcl-6, CXCR5, ICOS, IL-21, IL-4, CD40L, and in vitro differentiation assays. (4) Response of T regulatory cell to Staphylococcus

[0067] The one or more immunological test may further comprise a response of T regulatory(Treg) cells to Staphylococcus. Treg cells are involved in regulating immune responses in a subject to a Staphylococcus infection, inflammation caused by Staphylococcus infection, and disease severity. Response of Treg to Staphylococcus may be determined using one or more methods established in the field, examples of which include flow cytometry, intracellular cytokine staining, PCR, Cytokine and chemokine array analysis, sequencing, co-culture assays, ELISpot assays, expression analysis of genes associated with Treg, neutralization assay, histology, transcription factor staining, suppression assay, and in vitro differentiation assays. (5) Ratio of Tfh to Treg

[0068] The immunological tests may further comprise a ratio of amounts or frequencies of Tfhover the amount of Treg. An increase in ratio may be positively correlated with a favorable immunologic response. The ratio of Tfh over Treg may be obtained by determining the level or frequence of Tfh cells and Treg cells in a sample obtained from the subject. Non-limiting examples of such methods include, flow cytometry, staining using cell specific biomarkers, ELISA, PCR, sequencing, Cytokine and chemokine array analysis, ELISpot assays, expression analysis of genes associated with Tfh or Treg, neutralization assay, histology, transcription factor staining, suppression assay, and in vitro differentiation assays.(6) Cytokine response profile of T cell

[0069] The immunological tests may include a cytokine profile of T cells. The cytokine profilemay include level of expression of one or more cytokines including IFNγ, IL1, IL2, IL10, IL4, IL13, IL-16, IL17, IL21, TNF, GM-CSF, MIP1in the T cells obtained from the subject. Determination of the cytokine response profile of T cells from the subject may be undertaken by methods established in the art, including but not limited to flow cytometry, PCR, ELISA, cytokine arrays, intracellular cytokine staining, ELISpot assay, and bead based assays. b. Other traits(1) Subject characteristics

[0070] The method may comprise providing one or more of the subject characteristics (for e.g.,sex, age, weight, etc.), demographic information, and other personal health information. Such information may be collected by a medical professional or by using a questionnaire. In a specific instance, one or more, or both, of the biological sex and the age of the subject may be used in the method. (2) Colonization

[0071] The method may further comprise providing the Staphylococcus colonization status ofthe subject. The Staphylococcus colonization status may be determined by detecting Staphylococcus in a subject’s body including but not limited to nose and skin. Colonization status then may be categorized as subject as having Staphylococcus colonization or subject as not having Staphylococcus colonization. Colonization status of Staphylococcus may be determined using any established method. For example, a cotton swab may be used to collect a sample from the subject from skin or nose. Alternatively, a sample of blood, urine, sputum, or pus from an abscess may be collected. The collected sample may then be cultured in a laboratory, and colonies of the bacteria may be identified for the presence of Staphylococcus. Non-limiting examples of tests for identifying Staphylococcus include biotyping, tests for clumping factor, coagulase, hemolysins and thermostable deoxyribonuclease, molecular tests such as Polymerase Chain Reaction (PCR), sequencing, and identification of genetic markers or other methods such as Matrix-assisted laser desorption / ionization-time of flight. Other tests include determining the colonization status using wound culture, examination of biopsy, or imaging using CT scans or MRI. A subject may present with one or more symptoms of a Staphylococcus infection including but not limited to an invasive infection, a skin and soft tissue infections (SSTI) infection, skinboil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister. In such instances, the colonization status may be determined using physical examination and symptom assessment. Alternatively, the subject may not present any symptoms of a Staphylococcus infection. (3) Household SSTI

[0072] The method may further comprise providing the status of SSTI in the subject’s householdof the subject. The status of Staphylococcus infection may comprise household or non-household transmission of Staphylococcus infection, i.e., the source and setting of the subject’s Staphylococcus infection. Household SSTI may be acquired by a subject through skin to skin contact or touching contaminated surfaces within a household. Non-household SSTI may be acquired though contact with contaminated surfaces or items in a public space or a communal setting. The status of household SSTI of the subject may be determined using a questionnaire with questions related to status of Staphylococcus infection or SSTI of the members of the subject’s family, or using one or more diagnostic tests described above conducted on person(s) living with the subject or who have come in contact with the subject.

[0073] The subject or a member of the subject’s household may present with one or moresymptoms of a Staphylococcus infection including but not limited to an invasive infection, a skin and soft tissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded skin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister. In such instances, the status of SSTI in the subject’s household may be determined using physical examination and symptom assessment of the subject or one or more members of the subject’s household. Alternatively, the subject or a member of the subject’s household may not present any symptoms of a Staphylococcus infection. In such instance, one or more diagnostic tests may be performed to determine the status of SSTI in the subject’s household. c. Staphylococcus

[0074] A Staphylococcus infection disclosed herein may be caused in one instance byStaphylococcus aureus. Staphylococcus aureus may be an antibiotic sensitive strain or anantibiotic insensitive strain. In one instance the Staphylococcus aureus may be selected from Methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin- resistant Staphylococcus aureus (VRSA). Other strains of Staphylococcus aureus may include USA300, Newman, N315, GEM, USA200, USA400, ST5, ST8, CC30, CC1, NCTC8325, COL, JH1, JH9, MSSA476, FRI1169, FPR3757, CA-MRSA MW2, CA-MRSA c99529, MSSA Levy, CA-MRSA MNWH, MSSA CDC587, MSSA MN8, and MSSA MNPE.

[0075] The Staphylococcus aureus infection may be any Staphylococcus aureus of a subject,including, for example, a Staphylococcus aureus biofilm infection, a Staphylococcus aureus osteomyelitis infection, a biofilm-associated Staphylococcus aureus osteomyelitis infection, a Staphylococcus aureus indwelling medical device infection, a Staphylococcus aureus endocarditis infection, a Staphylococcus aureus diabetic wound or ulcer infection, a Staphylococcus aureus chronic rhinosinusitis infection, a Staphylococcus aureus ventilator associated pneumonia infection, a Staphylococcus aureus intravenous catheter associated infection, a Staphylococcus aureus skin infection, a Staphylococcus aureus nectrotizing fasciitis, a Staphylococcus aureus keratitis, a Staphylococcus aureus endophthlamitis, a Staphylococcus aureus pyopneumothorax, a Staphylococcus aureus empyema, and a Staphylococcus aureus septicemia. d. Subject

[0076] The methods as described herein may be performed on a subject, or performed with abiological sample obtained from a subject. As used herein, the term “subject” may refer to a mammal. The mammals may include, but are not limited to, humans, primates, livestock, rodents, or a pet mammal. The subject may be a laboratory animal, optionally wherein the laboratory animal is genetically engineered.

[0077] The subject may be a healthy subject. The subject may have no clinical signs orsymptoms of a Staphylococcus infection. The subject may be undiagnosed with aStaphylococcus infection. The subject may be negative for Staphylococcus infection. In anotherinstance, the subject may be positive for Staphylococcus infection.

[0078] The Staphylococcus infection may be selected from an invasive infection, a skin and softtissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure,bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister. In one instance, the Staphylococcus infection may be an invasive infection or a skin and soft tissue infections (SSTI) infection.

[0079] The subject may or may not be symptomatic. An “asymptomatic subject,” as usedherein, may refer to a subject that does not show any signs or symptoms of Staphylococcus infection. One or more symptoms may be include skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister.

[0080] In the methods disclosed herein, the subject may present with one or more symptoms of aStaphylococcus infection including but not limited to an invasive infection, a skin and soft tissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia, toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister. Alternatively, the subject may not present any symptoms of a Staphylococcus infection.

[0081] The subject may be waiting for medical care or treatment, maybe under medical care ortreatment, or may have received medical care or treatment. The subject may be alive, or may be deceased (e.g., as in the case of performing a method as described herein on or with a biological sample).

[0082] As used herein, the term “control”, “normal”, or “healthy” subject or population ofsubjects may refer to that subject or population not having any clinical signs or symptoms of a Staphylococcus infection. For instance, a control subject or population would not have an invasive infection or a skin and soft tissue infections (SSTI) infection, or a clinical symptom associated with Staphylococcus infection, which may optionally be confirmed by qualitative or quantitative test results. e. Sample

[0083] Suitable biological samples for one or more immunological tests or in the methodsdescribed herein may be any biological tissue, fluid, or cell from the subject. The sample can be solid or fluid. The sample can be a heterogeneous cell population. Non-limiting examples ofsuitable biological samples include sputum, serum, blood, blood cells (e.g., white cells), a biopsy, urine, peritoneal fluid, pleural fluid, nasal swab, skin swab, or cells derived therefrom. The biopsy can be a fine needle aspirate biopsy, a core needle biopsy, a vacuum assisted biopsy, an open surgical biopsy, a shave biopsy, a punch biopsy, an incisional biopsy, a curettage biopsy, or a deep shave biopsy. Biological samples may also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological purposes. Methods of collecting a biological sample from a subject are well known in the art.

[0084] The biological sample used in a specific immunological test may be any suitable samplefor the type of test performed. For example, the biological sample from the subject that may be used for determination of the total amount of Hla Ig titer and the amount of Hla neutralizing antibodies may be a blood sample. The blood sample may be derived from blood, preferably peripheral or circulating blood. The blood sample may be whole blood, plasma, or serum. For example, the blood sample may be serum.

[0085] For the colonization test or for determining the status of SSTI in the subject’s household,a sample from a swab may be used. For example, a cotton swab may be used to collect a sample from the subject from skin or nose. Alternatively, a sample of blood, urine, sputum, or pus from an abscess may be used. Further, the sample for determining SpA neutralization titer may be any biological sample obtained from the subject adequate for testing SpA neutralization titer such as a blood sample. The blood sample may be derived from blood, preferably peripheral or circulating blood. The blood sample may be whole blood, plasma, or serum. In one instance, the blood sample may be serum.

[0086] Determination of response of Tfh to Staphylococcus, Treg response to Staphylococcus,and ratio of amount or frequency of Tfh over Treg cells may be from any biological sample obtained from the subject adequate for testing or determining subsets of T cells. For example, the sample may be a blood sample or a sample form lymph node such as drainage from lymph node. The blood sample may be derived from blood, preferably peripheral or circulating blood. The blood sample may be whole blood, plasma, or serum. In one instance, the sample may be sample from lymph node or blood serum.

[0087] Any biological sample suitable for determining the cytokine profile of T cells may beused. For example, the sample may be a blood sample or a sample from lymph node such as drainage from lymph node. The blood sample may be derived from blood, preferably peripheralor circulating blood. The blood sample may be whole blood, plasma, or serum. In one instance, the sample may be sample from lymph node or a peripheral blood sample.

[0088] The sample may be large enough to allow the measurement required for the one or moreimmunological tests. For example, a blood sample may be from about 0.5 ml to about 10 ml. More than one sample may be pooled for a particular time point. The sample may be collected directly as part of the method. Alternatively, a previously-obtained sample may be used. Methods of collecting a sample are well known in the art. For example, venipuncture, with or without a catheter, may be used to collect a blood sample. In another example, a finger stick, or the equivalent, may be used to collect a blood sample. Additives may or may not be added to the collected sample prior to analysis. Suitable additives include citrate, heparin, EDTA, Tween, and protease inhibitors.

[0089] The methods as described herein may be performed by using or analyzing a biologicalsample obtained from a subject at a timepoint that is prior to, or is concurrent with, the timepoint that the method is performed. The timepoint of the obtainment of the biological sample from the subject may be the same day that the method is performed; or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15-30 days inclusive, 31-45 days inclusive, 46-60 days inclusive, 61-90 days inclusive, 90-365 days inclusive, or greater than one year prior to the timepoint that the method is performed.

[0090] In one instance, a first sample may be obtained from the subject, followed by obtaining asecond sample from the subject. The second sample may be obtained at a later time from obtaining the first sample. For example, the second sample may be obtained from the subject at 1 hour, 12 hours, 24 hours, 2 days, 3 days or more after obtaining the first sample. f. Score

[0091] The methods may comprise generating a score for the subject. The score may be acombined score determined by weighting the data obtained from the immunological tests, and optionally data related to one or more of the additional traits of the subject, with a predefined coefficient to obtain a weighted value for each immunological test and each additional trait. The weighted values may then be added to obtain the combined score. The predefined weight coefficient for each immunological test and trait may be calculated using any suitable method including but not limited to generalized models (e.g. logistic regression, generalized additive models), multivariate analysis (e.g. discriminant analysis, principal components analysis, factoranalysis), and time-to-event “survival” analysis. In one example of determining appropriate weighting coefficients, logistic regression models may be performed using the data from the immunological tests and the other traits and model performance may be further analyzed. Diagnostic utility of the models may be analyzed by ROC curve and AUC analysis using the R packages pROC and ROCR. The one or more immunological tests may then be examined in control samples from healthy subjects or samples from subjects previously determined to be at a specific stage or intensity of Staphyloccous infection, such as SSTI or invasive infection. Prediction models may be performed with multiple iterations of undersampling to balance the sample sizes of the cases and controls per phenotype. In an instance, the data from one or more immunological tests not included or unavailable may be eliminated from the model and the weighting coefficients for each data from one or more immunological tests remaining in the model may be further determined or adjusted.

[0092] In one instance, the predefined weight coefficient may be selected from Table 3,Table 5, Table 7, Table 10, Table 12, and Table 14. The range of the predefined weight coefficient for each of the immunological tests may be about ±10% of the weight coefficient disclosed in one or more of the Table 3, Table 5, Table 7, Table 10, Table 12, and Table 14.

[0093] The score, in addition to including weighted expression level obtained from each of theimmunological tests, may comprise weighted values of one or more of the sex of the subject and the age of the subject. Such weighted values may be obtained by weighting the sex of the subject and the age of the subject. The weighted values may then be added to obtain a combined score. The predefined coefficient for the sex of the subject and the age of the subject may be determined using the methods described above. For example, a logistic regression model may include the covariates of sex, and age at sample draw. The predefined weight coefficient or the range of the predefined weight coefficient for each of the sex of the subject and the age of thesubject are provided in Table 3, Table 5, Table 7, Table 10, Table 12, and Table 14. The rangeof the predefined weight coefficient for each of the sex of the subject and the age of the subject may be about ±10% of the weight coefficient disclosed in one or more of the Table 3, Table 5, Table 7, Table 10, Table 12, and Table 14.

[0094] In determining the risk of a Staphylococcus infection, vaccinating a subject with aStaphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, determining risk of recurrent Staphylococcus infection, or prognosis of a subject determined to have Staphylococcusinfection, the score generated for the subject may be compared to a predetermined threshold value. Suitable threshold levels may be determined for certain combinations of immunological tests and features such as sex and the age of the subject. The threshold value may be determined using, for example a ROC curve. ROC curve comprises a plot of the true positive rate (sensitivity) against the false positive rate (specificity) for a binary classifier system as its discrimination threshold is varied. A ROC curve can be represented equivalently by plotting the fraction of true positives out of the positives (TPR=true positive rate) versus the fraction of false positives out of the negatives (FPR=false positive rate). Each point on the ROC curve represents a sensitivity / specificity pair corresponding to a particular decision threshold value. The predetermined threshold values used in the methods described herein may be the valuesdisclosed in Table 2, Table 4, Table 6, Table 9, Table 11, and Table 13, which may be basedon the immunological tests and co-variates and their respective weight coefficients in Table 3, Table 5, Table 7, Table 10, Table 12, and Table 14.

[0095] The score may be generated by a processor using a machine learning model. The machinelearning model may comprise logistic regression, Support Vector Machines (SVM), naïve bayes, decision trees, linear regression, k nearest neighbors (kNN), random forest, boosting algorithm, K-means, or hierarchical clustering. In one example, the machine learning model may comprise logistic regression. g. Risk of infection

[0096] In one instance, a score above a predetermined threshold value may indicate that thesubject is at risk (which may be high) of a Staphylococcus infection. In such instances, the method may comprise generating a score based on the data obtained from the immunological tests from a biological sample from the subject. A score above a predetermined threshold value may be indicative of the subject as being at risk of the Staphylococcus infection. A score below the predetermined threshold value may be indicative of the subject as being at low risk of the Staphylococcus infection

[0097] In another instance, the method comprises determining risk of a recurrent Staphylococcusinfection in a subject. The method may comprise generating a score based on the data obtained from the immunological tests from a biological sample from the subject. A score above a predetermined threshold value is indicative of the subject as being at risk of recurrent Staphylococcus infection. A score below the predetermined threshold value may be indicative ofthe subject as being at low risk of the recurrent Staphylococcus infection. In such instances, the subject may be free of Staphylococcus infection for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, or more. (1) SSTI

[0098] The Staphylococcus infection may be a SSTI. The immunological tests may comprise alog of the total Hla Ig titer and the amount of Hla neutralizing antibodies. In one example, the corresponding weights of the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 0.036 ±10% and 0.266 ±10%, respectively. In another instance the corresponding weights of the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 0.036 and 0.266, respectively. The predetermined threshold value may be 0.473.

[0099] In some instances, the score may include additional traits such as the biological sex of thesubject, the age of the subject, and the subject’s history of S. aureus colonization. The combinedscore may be determined by weighting the log of the total Hla Ig titer, the amount of Hlaneutralizing antibodies, the biological sex of the subject, the age of the subject, and the history of S. aureus colonization and adding the weighted values. In one example, the weight for male sex may be -1.17±10%, the weight for age may be -0.178±10%, weight for S. aureus colonization may be -0.482±10%, the weight for no S. aureus colonization may be -2.16±10%, the weight of the amount of Hla neutralizing antibodies may be 0.179±10%, and the weight of log of the total Hla Ig titer may be 0.399±10%. Alternatively, the weight for male sex may be -1.17, the weight for age may be -0.178, weight for S. aureus colonization may be -0.482, the weight for no S. aureus colonization may be -2.16, the weight of the amount of Hla neutralizing antibodies may be 0.179, and the weight of log of the total Hla Ig titer may be 0.399. The predetermined threshold value may be 0.458.

[0100] In yet another instance, the score may further include status of SSTI in thesubject’s household. The combined score may be determined by weighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the ageof the subject, the history of S. aureus colonization, and status of SSTI in the subject’s household and adding the weighted values. The weight for male sex may be 1.07±10%, the weight for age may be -0.168±10%, weight for S. aureus colonization may be -0.467±10%, the weight for no S. aureus colonization may be -2.20±10%, the weight of the amount of Hla neutralizing antibodies may be 0.080±10%, the weight of log of the total Hla Ig titer may be 0.407±10%, the weight for SSTI in the subject’s household may be 14.5±10%, and the weight for no SSTI in the subject’s household may be 13.6±10%. In one instance, the weight for male sex may be 1.07, the weight for age may be -0.168, weight for S. aureus colonization may be -0.467, the weight for no S. aureus colonization may be -2.20, the weight of amount of Hla neutralizing antibodies may be 0.080, the weight of log of the total Hla Ig titer may be 0.407, the weight for SSTI in the subject’s household may be 14.5, and the weight for no SSTI in the subject’s household may be 13.6. The predetermined threshold value may be 0.520. (2) Invasive infection

[0101] The Staphylococcus infection may be an invasive infection. The immunologicaltests may comprise the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies. In one example, the corresponding weights of the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 1.335 ±10% and -0.323±10%, respectively. In another instance the corresponding weights of the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 1.335 and -0.323, respectively. The predetermined threshold value may be 0.277.

[0102] In some instances, the score may include additional traits such as the biologicalsex of the subject, the age of the subject, and history of S. aureus colonization. The combinedscore may be determined by weighting the log of the total Hla Ig titer, the amount of Hlaneutralizing antibodies, the biological sex of the subject, the age of the subject, and the history of S. aureus colonization and adding the weighted value. In one example, the weight for male sex may be 0.660±10%, the weight for age may be 0.055±10%, weight for S. aureus colonization may be -1.65±10%, the weight for no S. aureus colonization may be -2.16±10%, the weight of the amount of Hla neutralizing antibodies may be 1.21±10%, and the weight of log of the total Hla Ig titer may be -0.348±10%. Alternatively, the weight for male sex may be 0.660, the weight for age may be 0.055, weight for S. aureus colonization may be -0.658, the weight for no S. aureus colonization may be -1.65, the weight of the amount of Hla neutralizing antibodies maybe 1.21, and the weight of log of the total Hla Ig titer may be -0.348. The predetermined threshold value may be 0.517.

[0103] In yet another instance, the score may further include status of SSTI in thesubject’s household. The combined score may be determined by weighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the age of the subject, the history of S. aureus colonization, and status of SSTI in the subject’s household and adding the weighted value. The weight for male sex may be 0.625±10%, the weight for age may be 0.054±10%, the weight for no S. aureus colonization may be -1.75±10%, the weight of amount of Hla neutralizing antibodies may be 1.26±10%, the weight of log of the total Hla Ig titer may be -0.332±10%, the weight for SSTI in the subject’s household may be 1.42±10%, and the weight for no SSTI in the subject’s household may be -0.857±10%. In one instance, the weight for male sex may be 0.625, the weight for age may be 0.054±10%, the weight for no S. aureus colonization may be -1.75, the weight of amount of Hla neutralizing antibodies may be 1.26, the weight of log of the total Hla Ig titer may be -0.332, the weight for SSTI in the subject’s household may be 1.42, and the weight for no SSTI in the subject’s household may be -0.857. The predetermined threshold value may be 0.520.3. Methods of treatment

[0104] Provided herein is a method of vaccinating a subject using a Staphylococcusvaccine or treating a subject determined to be at risk of Staphylococcus infection. The method identifies a subset of subjects that are at risk of Staphylococcus infection and therefore are inneed of a vaccine or a treatment. The method may comprise administering the Staphylococcusvaccine or the treatment to the subject determined to be at risk of the Staphylococcus infection by virtue of a score above the predetermined threshold value as described herein. A score below the predetermined threshold value may be indicative of the subject as being at low risk of the Staphylococcus infection.

[0105] Further provided is a method predicting efficacy of a Staphylococcus vaccine in asubject. The method may comprise administering a Staphylococcus vaccine to a subject with ascore above the predetermined threshold value as described herein. A score below the predetermined threshold value is indicative of the vaccine as not being efficacious for the subject.

[0106] The method may also comprise providing a prognosis to the subject. The methodmay comprise generating a score for the subject as described herein, with a score above a predetermined threshold value being indicative of a positive outcome. The positive outcome may be selected from positive survival outcome, a positive response to a Staphylococcus treatment, and reduced recurrent Staphylococcus infection.

[0107] The method disclosed herein may further determine whether a subject isresponsive to any of the therapeutic agents disclosed herein. In such instances a score above apredetermined threshold value may be indicative of a subject as responsive to a treatment. Inother instances, the subject may be identified as likely to be responsive or as not responsive or not likely to be responsive to treatment based on the score. a. SSTI

[0108] The Staphylococcus infection may be a SSTI. The immunological tests maycomprise a log of the total Hla Ig titer and the amount of Hla neutralizing antibodies. In one example, the corresponding weights of log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 0.036 ±10% and 0.266 ±10%, respectively. In another instance the corresponding weights of the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 0.036 and 0.266, respectively. The predetermined threshold value may be 0.473.

[0109] In some instances, the score may include additional covariates such as biologicalsex of the subject, an age of the subject, and history of S. aureus colonization. The combinedscore may be determined by weighting the log of the total Hla Ig titer, the amount of Hlaneutralizing antibodies, the biological sex of the subject, the age of the subject, and the history of S. aureus colonization and adding the weighted value. In one example, the weight for male sex may be -1.17±10%, the weight for age may be -0.178±10%, weight for S. aureus colonization may be -0.482±10%, the weight for no S. aureus colonization may be -2.16±10%, the weight of the amount of Hla neutralizing antibodies may be 0.179±10%, and the weight of log of the total Hla Ig titer may be 0.399±10%. Alternatively, the weight for male sex may be -1.17, the weight for age may be -0.178, weight for S. aureus colonization may be -0.482, the weight for no S. aureus colonization may be -2.16, the weight of the amount of Hla neutralizing antibodies may be 0.179, and the weight of log of the total Hla Ig titer may be 0.399. The predetermined threshold value may be 0.458.

[0110] In yet another instance, the score may further include status of SSTI in thesubject’s household. The combined score may be determined by weighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the age of the subject, the history of S. aureus colonization, and status of SSTI in the subject’s household and adding the weighted values. The weight for male sex may be 1.07±10%, the weight for age may be -0.168±10%, weight for S. aureus colonization may be -0.467±10%, the weight for no S. aureus colonization may be -2.20±10%, the weight of amount of Hla neutralizing antibodies may be 0.080±10%, the weight of log of the total Hla Ig titer may be 0.407±10%, the weight for SSTI in the subject’s household may be 14.5±10%, and the weight for no SSTI in the subject’s household may be 13.6±10%. In one instance, the weight for male sex may be 1.07, the weight for age may be -0.168, weight for S. aureus colonization may be -0.467, the weight for no S. aureus colonization may be -2.20, the weight of amount of Hla neutralizing antibodies may be 0.080, the weight of log of the total Hla Ig titer may be 0.407, the weight for SSTI in the subject’s household may be 14.5, and the weight for no SSTI in the subject’s household may be 13.6. The predetermined threshold value may be 0.520. b. Invasive infection

[0111] The Staphylococcus infection may be an invasive infection. The immunologicaltests may comprise the log of the total Hla Ig titer and the amount of Hla neutralizing antibodies. In one example, the corresponding weights of log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 1.335 ±10% and -0.323±10%, respectively. In another instance the corresponding weights of log of the total Hla Ig titer and the amount of Hla neutralizing antibodies may be 1.335 and -0.323, respectively. The predetermined threshold value may be 0.277.

[0112] In some instances, the score may include additional traits such as biological sex ofthe subject, an age of the subject, and history of S. aureus colonization. The combined score maybe determined by weighting the log of the total Hla Ig titer, the amount of Hla neutralizingantibodies, the biological sex of the subject, the age of the subject, and the history of S. aureus colonization and adding the weighted values. In one example, the weight for male sex may be 0.660±10%, the weight for age may be 0.055±10%, weight for S. aureus colonization may be - 1.65±10%, the weight for no S. aureus colonization may be -2.16±10%, the weight of the amount of Hla neutralizing antibodies may be 1.21±10%, and the weight of log of the total HlaIg titer may be -0.348±10%. Alternatively, the weight for male sex may be 0.660, the weight for age may be 0.055, weight for S. aureus colonization may be -0.658, the weight for no S. aureus colonization may be -1.65, the weight of the amount of Hla neutralizing antibodies may be 1.21, and the weight of log of the total Hla Ig titer may be -0.348. The predetermined threshold value may be 0.517.

[0113] In yet another instance, the score may further include status of SSTI in thesubject’s household. The combined score may be determined by weighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the age of the subject, the history of S. aureus colonization, and status of SSTI in the subject’s household and adding the weighted values. The weight for male sex may be 0.625±10%, the weight for age may be 0.054±10%, the weight for no S. aureus colonization may be -1.75±10%, the weight of amount of Hla neutralizing antibodies may be 1.26±10%, the weight of log of the total Hla Ig titer may be -0.332±10%, the weight for SSTI in the subject’s household may be 1.42±10%, and the weight for no SSTI in the subject’s household may be -0.857±10%. In one instance, the weight for male sex may be 0.625, the weight for age may be 0.054±10%, the weight for no S. aureus colonization may be -1.75, the weight of amount of Hla neutralizing antibodies may be 1.26, the weight of log of the total Hla Ig titer may be -0.332, the weight for SSTI in the subject’s household may be 1.42, and the weight for no SSTI in the subject’s household may be -0.857. The predetermined threshold value may be 0.520.

[0114] The vaccine may comprise an antigen selected from CP5, CP8, ClfA(SA3Ag),CP5-CRM197, CP8-CRM197, MntC, ClfA(SA4Ag), Alpha-toxin,Panton-Valentine Leukocidin, Leukocidin AB, HlgAB, HlgBC, Surface protein A (SpA), enterotoxin B, or a live or attenuated S. aureus.

[0115] The vaccine may be formulated to comprise different amounts of the particularpeptides and polypeptides from which they are prepared. Further, the total amount of protein in the formulations will vary based on the particular use to which the formulations are put (for e.g., administration to the subject pre- or post-exposure to Staphylococcus aureus), the age and size of the subject, and the general health of the subject, to name only a few factors to be considered. The vaccine formulations may comprise sufficient Staphylococcus aureus protein to induce an immune response in a subject in need thereof, to the components of the vaccine. For example, the vaccines formulations may contain between about 1 to about 1000 µg of total Staphylococcusaureus protein per kg of body weight of the subject to which the dose of the vaccine formulation will be administered, more preferably between about 10 to about 200 µg, even more preferably between about 15 to about 100 µg.

[0116] The vaccine may also be used in methods of inhibiting a Staphylococcus infectionin a subject. Such methods comprise administering a therapeutically effective amount of a vaccine to a subject at risk of developing a Staphylococcus aureus infection, thereby inhibiting a Staphylococcus infection in a subject. The method may further comprise administering an antimicrobial agent to the subject at risk of developing a Staphylococcus aureus infection in conjunction with the administration of the vaccine formulation.

[0117] Providing a treatment may comprise administering an antibiotic or anantimicrobial agent to the subject determined to be at risk of Staphylococcus infection, or determined to be at high risk of recurrent Staphylococcus infection.

[0118] In some instances, when the subject is determined to be at risk of Staphylococcusinfection or at risk of recurrent Staphylococcus infection, the method may comprise modifying a treatment.

[0119] The antibiotic may be selected from cefazolin, nafcillin, oxacillin, vancomycin,daptomycin and linezolid. An antimicrobial agent may be selected from the group that includes, but is not limited to, an Aminoglycoside, such as Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin or Paromomycin; a Carbacephem, such as Loracarbef; a Carbapenem, such as Ertapenem, Doripenem, Imipenem / Cilastatin or Meropenem; a Cephalosporin, such as Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime or Ceftobiprole; a Glycopeptide, such as Teicoplanin or vancomycin; a Macrolide, such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Erythroped, Roxithromycin, Troleandomycin, Telithromycin or Spectinomycin; a Monobactam, such as Aztreonam; a Penicillin, such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Meticillin, Nafcillin, Oxacillin, Penicillin, Piperacillin or Ticarcillin; a Polypeptide, such as Bacitracin, Colistin or Polymyxin B; a Quinolone, such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin or Trovafloxacin; a Sulfonamide, such as Mafenide, Prontosil (archaic), Sulfacetamide,Sulfamethizole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim or Trimethoprim-Sulfamethoxazole (Cotrimoxazole) (TMP-SMX); a Tetracycline, such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline or Tetracycline; as well as Chloramphenicol, Clindamycin, Lincomycin, Fusidic acid, Furazolidone, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin, Macrobid, Platensimycin, Quinupristin / Dalfopristin, Rifampin or Rifampicin.

[0120] When an antimicrobial agent is included in the methods of the present inventionthe antimicrobial agent may be administered prior to, concurrent with or after the vaccine formulation is administered to the subject. Where the antimicrobial agent is administered prior to or after the vaccine formulation, the period of time between when the antimicrobial agent and the vaccine formulation are administered may be a period of hours (such as 6, 12, 18 or 24 hours), days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6 or more months).

[0121] The vaccine or anti-microbial agent may be administered to a subject usingdifferent dosing schedules, depending on the particular use to which the formulations are put (e.g., administration to the subject pre- or post-exposure to Staphylococcus), the age and size of the subject, and the general health of the subject, to name only a few factors to be considered. In general, the vaccine formulations may be administered once, or twice, three times, four times, five times, six times or more, over a dosing schedule. The timing between each dose in a dosing schedule may range between a few hours, six, 12, or 18 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days. The same quantity of protein in the formulation may be administered in each dose of the dosing schedule, or the amounts in each dose may vary. The identity of the particular peptides and polypeptides in the formulation may also vary or remain the same in each dose in a dosing schedule.

[0122] Appropriate doses and dosing schedules can readily be determined by techniqueswell known to those of ordinary skill in the art without undue experimentation. Such a determination will be based, in part, on the tolerability and efficacy of a particular dose.

[0123] Administration of the vaccine or anti-microbial agents may be via any of themeans commonly known in the art of vaccine delivery. Such routes include intravenous, intraperitoneal, intramuscular, subcutaneous and intradermal routes of administration, as well asnasal application, by inhalation, ophthalmically, orally, rectally, vaginally, or by any other mode that results in the vaccine formulation contacting mucosal tissues.

[0124] In one instance, the subject may be determined to have an SSTI. A suitabletreatment for SSTI may be provided based on the type of the symptom developed by the subject, the treatment. Incision and drainage for purulent infections (like abscesses) and antibiotics for both purulent and non-purulent infections, may be recommended with specific choices depending on severity and presence of MRSA. For severe or complicated SSTIs, broader spectrum antibiotics and potential hospitalization may be needed.

[0125] For example, impetigo may be treated with mupirocin or retapamulin. Oraltherapy for ecthyma or impetigo should be a 7-day regimen with an agent active against S. aureus unless cultures yield streptococci alone (when oral penicillin is the recommended agent) (strong, high). Because S. aureus isolates from impetigo and ecthyma are usually methicillin susceptible, dicloxacillin or cephalexin is recommended. When MRSA is suspected or confirmed, doxycycline, clindamycin, or sulfamethoxazole-trimethoprim (SMX-TMP). Ecthyma or impetigo may be treated an agent active against S. aureus unless cultures such as oral penicillin. Because S. aureus isolates from impetigo and ecthyma are usually methicillin susceptible, dicloxacillin or cephalexin may be recommended. When MRSA is suspected or confirmed, doxycycline, clindamycin, or sulfamethoxazole-trimethoprim (SMX-TMP) may be recommended. An antibiotic active against MRSA may be recommended for patients with carbuncles or abscesses who have failed initial antibiotic treatment or have markedly impairedhost defenses or in patients with SIRS and hypotension. For cellulitis with systemic signs ofinfection, systemic antibiotics may be indicated. For subjects whose cellulitis is associated with penetrating trauma, evidence of MRSA infection elsewhere, nasal colonization with MRSA, injection drug use, or SIRS, vancomycin or another antimicrobial effective against both MRSA and streptococci may be recommended. In severely compromised subjects, broad-spectrum antimicrobial coverage may be considered . Vancomycin plus either piperacillin-tazobactam or imipenem / meropenem may be recommended as a reasonable empiric regimen for severeinfections. Hospitalization may be recommended if there a deeper or necrotizing infection, forsubjects with poor adherence to therapy, for infection in a severely immunocompromised subject, or if outpatient treatment is failing. Further, systemic corticosteroids (for e.g., prednisone 40 mg daily for 7 days) could be considered in nondiabetic adult subjects with cellulitis. A briefcourse of systemic antimicrobial therapy may be recommended in subjects with surgical site infections following clean operations on the trunk, head and neck, or extremities that also have systemic signs of infection. Further, a first-generation cephalosporin or an antistaphylococcal penicillin for MSSA, or vancomycin, linezolid, daptomycin, telavancin, or ceftaroline where risk factors for MRSA are high (nasal colonization, prior MRSA infection, recent hospitalization, recent antibiotics), may be recommended. Cephalosporin or fluoroquinolone in combination with metronidazole, may be recommended for infections following operations on the axilla, gastrointestinal tract, perineum, or female genital tract. For fasciitis, ancomycin or linezolid plus piperacillin-tazobactam or a carbapenem; or plus ceftriaxone and metronidazole may be recommended. Penicillin plus clindamycin may be recommended for treatment of documentedgroup A streptococcal necrotizing fasciitis. Cefazolin or antistaphylococcal penicillin (eg,nafcillin or oxacillin) may be recommended for treatment of pyomyositis caused by MSSA. Antibiotics may be administered intravenously initially, but once the subject is clinically improved, oral antibiotics may be appropriate for subject in whom bacteremia cleared promptly and there is no evidence of endocarditis or metastatic abscess.

[0126] In another instance, the Staphylococcus infection may be an invasive infection.MRSA bacteremia (invasive MRSA) is a systemic MRSA infection that is defined as the presence of MRSA in typically sterile sites, including the bloodstream, cerebrospinal fluid, joint fluid, bone, lower respiratory tract, and other body fluids. MRSA bacteremia has a far worse prognosis compared to MRSA infections confined to the skin, with 20% of cases resulting in death. The difference in prognosis, location of the infection, and clinical symptoms of the condition make it clinically distinct from skin and soft tissue infection MRSA infections. MRSA bacteremia causes multiple complications not seen in skin and soft tissue infections, including infective endocarditis, septic arthritis, and osteomyelitis. For invasive MRSA, daptomycin and vancomycin may be recommended for treatment. The difference in clinical symptoms as well as treatment methods for invasive infection provides clear evidence for invasive infection as a clinically distinct condition from SSTI.

[0127] For recurrent Staphylococcus infection, intranasal mupirocin, daily chlorhexidinewashes, and daily decontamination of personal items such as towels, sheets, and clothes may berecommended. Administration of prophylactic antibiotics, such as oral penicillin or erythromycinfor 4–52 weeks, or intramuscular benzathine penicillin every 2–4 weeks, may be recommendedin subjects who have 3–4 episodes of cellulitis per year despite attempts to treat or control predisposing factors.

[0128] The methods may further comprise adjusting the treatment administered to thesubject based on the one or more immunological tests or a score based on the one or more immunological tests. Adjusting the treatment includes, but are not limited to, changing the dose and / or administration of the therapeutic agent used in the current treatment, switching the current medication to a different therapeutic agent, or applying a new therapy to the subject, which can be either in combination with the current therapy or replacing the current therapy.4. System

[0129] Further provided herein is a system. The system may include a a processor incommunication with a memory, the memory including instructions, which, when executed, cause the processor to determine a risk of Staphylococcus infection, determine whether a subject is in need of a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, or determine risk of recurrent Staphylococcus infection.

[0130] The system may comprise a device for executing one or more of the methodsdescribed herein. The device may comprise one or more network interfaces (e.g., wired, wireless, PLC, etc.), at least one processor, and a memory interconnected by a system bus, as well as a power supply (e.g., battery, plug-in, etc.).

[0131] Network interface(s) may include the mechanical, electrical, and signalingcircuitry for communicating data over the communication links coupled to a communication network. Network interfaces may be configured to transmit and / or receive data using a variety of different communication protocols.

[0132] Processor comprises hardware elements or logic adapted to execute the softwareprograms (e.g., instructions) and manipulate data structures. An operating system, portions of which are typically resident in memory and executed by the processor, functionally organizes device 200 by, inter alia, invoking operations in support of software processes and / or services executing on the device. These software processes and / or services may include AD determination processes / services, which can include aspects of the methods and / or implementations of various modules described herein.

[0133] Memory may include a plurality of storage locations that are addressable byprocessor and network interfaces for storing software programs and data structures associatedwith the embodiments described herein. In one instance, device may have limited memory or no memory (e.g., no memory for storage other than for programs / processes operating on the device and associated caches). Memory can include instructions executable by the processor that, when executed by the processor, cause the processor to implement aspects of the system and associated methods outlined herein.

[0134] It will be apparent to those skilled in the art that other processor and memorytypes, including various computer-readable media, may be used to store and execute program instructions pertaining to the techniques described herein. Also, while the description illustrates various processes, it is expressly contemplated that various processes may be embodied as modules or engines configured to operate in accordance with the techniques herein (e.g., according to the functionality of a similar process). In this context, the term module and engine may be interchangeable. In general, the term module or engine refers to model or an organization of interrelated software components / functions.

[0135] The methods described herein can be carried in conjunction with an automatedsystem or computer. For instance, the methods can be performed using an automated system, in which a subject’s blood sample is analyzed to make the determination or determinations of immunological tests, generation of a score using one or more of immunological tests, the sex of the subject, the age of the subject, colonization status, and status of SSTI in the subject’s household, and the comparison of the score with threshold value may be carried out automatically using appropriate software. Computer software, or computer-readable media for use in the methods may include: a computer readable medium comprising: (a) code for receiving data corresponding to one or more immunological tests; (b) code for generating a score using one or more of immunological tests, the sex of the subject, the age of the subject, colonization status, and status of SSTI in the subject’s household, and for retrieving a threshold level; and (c) code for comparing the score with threshold value to make a determination on risk of Staphylococcus infection, determine whether a subject is in need of a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, or determine risk of recurrent Staphylococcus infection.

[0136] In one instance, the one or more predefined coefficient or predefined coefficientranges of the immunological tests, the sex of the subject, the age of the subject, colonization status, and status of SSTI in the subject’s household, and threshold values may be stored in a memory associated with a digital computer. After data corresponding to one or more of one ormore of immunological tests, the sex of the subject, the age of the subject, colonization status, and status of SSTI in the subject’s household is obtained (e.g., from an appropriate analytical instrument, questionnaire), the digital computer may generate a score and compare the score with one or more appropriate threshold values. After the comparisons, the digital computer may automatically calculate if the score is indicative of Staphylococcus infection, determine whether a subject is in need of a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, or determine risk of recurrent Staphylococcus infection.5. Kits

[0137] Also provided in this disclosure are kits. Such kits may include means fordetermining for performing one or more immunological tests and, optionally instructions for performing the method as described herein, such as determining risk of a Staphylococcus infection, vaccinating a subject with a Staphylococcus vaccine, predicting efficacy of a Staphylococcus vaccine, or determining risk of recurrent Staphylococcus infection. Such kits may facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to systems, assays, binding agent (e.g., antibodies for neutralization titer), reagents, internal standards, or software. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components. In one instance, the kit may include one or more therapeutic agents.

[0138] Kits may also include reagents in separate containers such as, for example, sterilewater or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such asaluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0139] Kits may be supplied with instructional materials. Instructions may be printed onpaper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit. Said instructions may be derived from any of the methods as described herein.

[0140] The present invention has multiple aspects, illustrated by the following non-limiting examples. Example 1 Immunoscore for Staphylococcus aureus

[0141] Humans serve as the primary ecologic niche for S. aureus. While the precisedefinition of a 'niche' has been the subject of debate in ecology, it is reasonable to consider the S. aureus niche as that environment within its human host that provides both the physical and biological context necessary for survival. Niche acquisition depends upon competitive fitness within the human microbiome, but also requires protection from sterilizing host immunity. Initial population of the S. aureus niche occurs in the first weeks to months of human life, documented both by assessment of colonization frequency and detection of host immune responses specific to this microbe. While persistent colonization with S. aureus is limited to ~20-30% of the adult population, carriage in infancy can approach 70%. Infants and children under the age of 4 exhibit an increased burden of S. aureus disease, likely reflecting the fact that colonization is a risk factor for infection. Together, these observations highlight early life as a unique developmental period in which adaptation of S. aureus to its human niche occurs.

[0142] The importance of canonical microbial ‘virulence’ factors in bacterial nicheestablishment has only recently been recognized. Two broad classes of well-studied S. aureus virulence factors include surface-exposed proteins and secreted molecules. While surface proteins are instrumental for S. aureus to interact with host tissue and acquire nutrients, manysecreted exoproteins and toxins facilitate evasion of innate and adaptive immunity. Through an ecological lens, these two classes of virulence factors may cooperate in homeostatic niche acquisition and maintenance. Interestingly, it was recently revealed that discrete, non-protective immune responses are raised against an array of S. aureus surface proteins in response to natural infection. In contrast, the elicited immune response to several toxins exhibit neutralizing capacity, consistent with prior observations in the field. It is interesting to speculate that perturbation of the adaptive immune response by S. aureus may specifically bias the host toward the generation of non-productive responses to staphylococcal surface proteins, thereby safeguarding the molecular interactions vital to niche maintenance. Multiple human-adapted bacterial species including M. tuberculosis, K. pneumoniae, E. coli, C. trachomatis, C. difficile, and P. aeruginosa exhibit a dynamic interface between niche ecology and host immunity. Like S. aureus, these are the very pathogens for which vaccine development has proven most challenging.

[0143] For each of these microbes, immunologic adaptations that favor niche acquisitionmust be durable in order to ensure resilience of the niche. By virtue of antigen specificity and memory, the T and B cell compartments underlie durability of the immune response. Whether in the setting of microbial exposure or vaccination, the initial elicited T and B cell response will be amplified upon re-encounter of the stimulating antigen(s). This knowledge has led to an application of the doctrine of 'original antigenic sin' (also referred to as antigenic or immunologic imprinting) to explain the failures of vaccine-elicited immunity against S. aureus. In the case of S. aureus, however, it may be more instructive to consider perturbation of the adaptive immune response as a microbe-induced immunologic adaptation that ensures potential for long-term maintenance of the ecologic niche. It is proposed that, this type of adaptation is a 'conditioning' response predicated on the existence of specific molecular interactions between a microbe and its host that preserve niche homeostasis.

[0144] Analysis of the S. aureus pan-genome reveals the preservation of multipleconserved core genome-encoded immunomodulatory proteins across human host-adapted S. aureus isolates. Specifically relevant to the adaptive immune response, a growing body of data points to a critical role for α-toxin (Hla) and staphylococcal protein A (SpA) in modulation of the T and B cell response to S. aureus, respectively. While the difference between ‘imprinting’ and microbe-driven 'conditioning' may appear little more than semantics, considering modification ofthe adaptive immune response as a product of co-evolution of S. aureus and its human host has important implications for vaccine development.

[0145] Recognition of the intrinsic link between niche acquisition and staphylococcalantigen exposure compels the development of a pre-exposure vaccine specifically designed to protect against S. aureus-induced immunologic adaptation early in life. Neonates and infants likely represent the only population in which a truly preventive S. aureus vaccine may be implemented.

[0146] Beyond the expected regulatory challenges that must be embraced in vaccinedevelopment for this population, several important biologic considerations must also be acknowledged. Targeting staphylococcal factors that modulate immunity to facilitate niche homeostasis may engender changes in S. aureus ecology. Moreover, development and implementation of a S. aureus vaccine for infants cannot be expected to confer immediate widespread protection against disease. The field must commit to the ‘long game’, recognizing that vaccine-elicited immunity in this population has the unique potential to confer lifetime protection for each population-based vaccine cohort.

[0147] Development of a S. aureus vaccine for pre-exposed populations of children andadults has proven to be a formidable task. Among individuals who have already been exposed to S. aureus, two broad populations are likely to exist: 1) those who exhibit a favorable immune profile sufficient to confer protection against disease, i.e., a population that has developed some degree of natural immunity, and 2) those whose functional immunologic profile suggests a non- protective response. In the former group, vaccine delivery may not confer benefit, and harbors risk of degrading the native immune response. In the latter group, vaccine design will need to either augment specific antigenic responses that are partially protective or elicit a novel response, avoiding amplification of non-protective responses. Rather than focus on development of a single vaccine expected to elicit protective immunity across all populations (the so-called ‘universal vaccine’), it will be prudent to stratify the pre-exposed population into biologically distinct groups based on immunologic profiling. The tumor-associated immune signatures to discriminate heterogeneous responses among individuals is very useful. The application of ‘immunoscoring’ guides personalized clinical care and prognostication based on quantifiable immune response traits. Adaptation of this concept within the S. aureus field through the development of a Precision Immunoscore for Staphylococcus aureus (PISA) may enablestratification of immunologic risk in the setting of anti-staphylococcal immune response heterogeneity to inform vaccine delivery and outcomes prediction (FIG.1). The foundation for such an immunoscore must rely on functional analysis of human immunity to S. aureus.

[0148] To this end, and moreover the greater goal of vaccine development, the field willneed to seek a detailed understanding of the humoral and cellular responses present in humans that exhibit protection against infection while being absent or diminished in susceptible individuals. Candidate vaccine targets must be defined based on quantifiable, functional immunologic outcomes, distinguishing immunogenicity of a vaccine antigen(s) from its ability to elicit protective immunity. Rigorous mechanistic evaluation of both the targeted antigen(s) and vaccine formulation will be essential, necessitating complementary studies in human populations and reductionist model systems to support vaccine design and clinical trials.

[0149] These considerations do not offer assurance that a S. aureus vaccine for pre-exposed individuals is close at hand. Now is the time to accelerate translational research within the field through the development of diverse teams of investigators whose expertise spans microbiology, immunology, and investigation of human clinical disease. Much as the mounting morbidity and mortality attributable to SARS-CoV-2 drove innovation in vaccine development and implementation, so must the S. aureus field be driven by the recognition that nearly 400,000 newborns worldwide are beginning to 'adapt' to this microbe each day.

[0150] A precision Immunoscore for Staphylococcus aureus (PISA) was developed toquantify specific attributes of the human immune response to Staphylococcus aureus. PISA enable the following: determination of risk for S. aureus infection; determination of clinical impact of pre-exposure to S. aureus on potential outcome from infection; risk stratification and prediction to enable vaccine-mediated interventions; and prediction of specific vaccine formulation that will benefit an individual.

[0151] At present, the field of infectious disease has no viable strategy to predict risk forS. aureus infection, prognosticate outcomes from S. aureus infection, or define individuals that would benefit from vaccines that are currently in development in the field.

[0152] PISA incorporates specific objective immunologic tests performed on humanblood into a single algorithm that will permit stratified ‘immunoscoring’ to address the above problem in the field.

[0153] Non-limiting components of the PISA for investigation of predictive significanceinclude: Anti-Hla titer; SpA neutralization titer; colonization status; response of T follicular helper cell to S. aureus; response of T regulatory cell to S. aureus; Tfh:Treg ratio, with increase in ratio being predictive of favorable immunologic response; and cytokine response profile of T cells: may include IFNg, IL10, IL4, IL13, IL17.

[0154] Given the emerging evidence for Hla as a potential target for anti-S.aureustherapeutic and vaccine development, the aim of this study was to correlate the anti-Hla neutralization antibody (Nab) response and define the threshold of protection against risk of recurrent S. aureus infection in a cohort of children with asymptomatic S. aureus colonization, culture-confirmed S. aureus skin and soft tissue infections (SSTI), or invasive infection.

[0155] Multiple studies of both the T and B cell response to infection indicate thatconsiderable inter-individual differences exist in the human immune response to S. aureus. Evidence of an antibody-mediated response to S. aureus antigens is nearly ubiquitous in humans even within the first year of life. Investigation of the anti-S. aureus IgG / IgA response in nearly 1000 healthy individuals revealed a high degree of variability in the nature and magnitude of the response to 79 distinct antigens. A similar study conducted by Radke, et al, assessed the host IgG response to over 100 S. aureus proteins in 7 S. aureus-infected patients, demonstrating broad reactivity to S. aureus antigens. As neither of these studies evaluated serologic responses as a correlate of protective immunity, a highly curated population of pediatric patients with S. aureus infection and healthy controls were leveraged to examine whether the serologic response to S. aureus a-toxin (Hla) correlated with protection against recurrent infection. Hla is a pore-forming cytotoxin produced by almost all clinical S. aureus isolates. Hla causes host injury in the contextof pneumonia, primary and recurrent skin infection, and corneal infection, also playing a role in the pathogenesis of sepsis, central nervous system infection, and toxic shock syndrome. Multiple pre-clinical studies have demonstrated that active and passive immunization approaches targeting Hla elicit protection against the major forms of S. aureus infection. The studies from this disclosure support a central functional role for Hla in perturbation of host protective immunity to S. aureus, thus prompting for evaluation of the serum anti-Hla response in the pediatric cohort. It was revealed that the anti-Hla response was associated with protection against S. aureus infection out to 12 months, defining this response as a serologic biomarker of protective immunity. Adhikari, et al, reported that hospitalized adult patients with S. aureus sepsis exhibited a lower anti-Hla serologic titer. The anti-Hla response demonstrates that the serum neutralizing antibody response to Hla develops over the first 10 years of life, being most variable and underdeveloped in the 24 months following birth. These findings indicate that distinct features of the serologic response to S. aureus may be quantified and serve as a ‘fingerprint’ that provides insight on immunity to this pathogen at the level of the individual patient.

[0156] To enable the development and analysis of PISA, an existing study population ofinfants and children with S. aureus infection (and corresponding healthy controls) that have been curated for investigation of the human immune response to S. aureus was leveraged. This study, entitled 'Pediatric Immunity to Staphylococcus aureus' (The 'PISA Study'), initiated in early 2021 to enable detailed immunophenotypic analysis of the development of human immunity to S. aureus infection during childhood. Subject cohorts examined in the PISA study include infants and children who present with primary or recurrent skin and soft tissue infection (SSTI) or invasive S. aureus infection and age-matched healthy controls. At the time of enrollment during acute infection (or upon enrollment for healthy controls), each subject provided a whole blood sample to permit analysis of serologic and cellular markers of immunity as well as a saliva sample for genomic DNA extraction. These biospecimens are reposited and paired with a detailed case report for each enrollee containing personal medical and family history, assessment of risk factors for S. aureus acquisition and disease, and detailed clinical data during the course of care for S. aureus infection. Unique to the PISA study, each subject is followed longitudinally for 1 year to enable immunophenotyping analysis during the convalescent period (6-8 weeks post-infection) and long-term follow up for recurrent infection. This rich clinical research population and existing / ongoing specimen collection will be leveraged for the proposed studies.Example 2 Acute and Convalescent Anti-Hla IgG and NAb Response Following Infection

[0157] Acute and convalescent anti-Hla IgG and NAb responses were compared acrosspatients in the 4 cohorts. Children with invasive infections had the highest acute anti-Hla NAb titers while noncolonized controls had minimal anti-Hla NAb titers (FIG.2). Children with invasive infections had the highest convalescent anti-Hla NAb titers, which was significantly higher than the invasive acute titers and SSTI anti-Hla NAb convalescent titers (FIG.3A-3B). Significant trends were not observed in the anti-Hla IgG titer between children with recurrent and no recurrent infections at 12 months of follow-up. However, children with SSTI and no recurrent infections had higher convalescent to acute anti-Hla NAb titers (FIG.4A-4B). Children with invasive infections had higher convalescent to acute anti-Hla NAb titers, regardless of whether they developed recurrent infections or not. To characterize the development of the anti- Hla NAb titer response as a function of increasing age, we stratified the anti-Hla NAb titer by age groups (FIG.5A-5C). A statistically significant increase in the anti-Hla NAb response was observed with increasing age in the control and SSTI group, but not the invasive group. Example 3 Anti-Hla NAb Fold Induction Titer Associated with Reduced Risk of Recurrent SSTI

[0158] To evaluate the individual immune response to infection, the fold induction titeras a ratio between the convalescent and acute anti-Hla NAb titers for individuals with paired samples available were calculated (FIG.6A-6B). For each unit increase in the log fold induction anti-Hla NAb titer, children with SSTI had a 0.54 (95% CI 0.31-0.96) decreased odds of recurrent infection within 12 months of follow-up. A similar trend in the invasive group was not observed. Example 4 Predictive analytical modeling of S. aureus risk for infection

[0159] To examine whether it was possible to derive a model based on patient-specificdata that would discriminate individuals without S. aureus infection from those with infection, a 207 subject model development cohort with well-defined clinical and demographic characteristics derived from the PISA study detailed above was leveraged. Serum from each ofthese 207 subjects was subjected to anti-Hla serum titer analysis and quantification of the neutralizing anti-Hla titer (Table 1). For model development, a logistic regression model architecture utilizing an initial subset of the available features including patient sex, history of S. aureus colonization at enrollment, in addition to antibody biomarker profiles were used.

[00160] Logistic regression (LR) methodology is a widely used supervised machinelearning technique to predict binary outcomes, such as the presence or absence of infection. This method is particularly useful when the goal is to classify patients based on their clinical and biological characteristics, as it estimates the probability that a patient has an infection versus no infection by learning from a labeled dataset that includes features such as patient demographics (e.g., sex and colonization status) and laboratory biomarkers (e.g., antibody levels). By applying a logistic function to the linear combination of these predictors, the model outputs probabilities that are then converted to binary classification labels based on a threshold typically set at 0.5 (however adjustable based on specific needs). Table 1 Test cohort demographics and S. aureus colonization statusCharacteristics SSTI (n=70) Invasive (n=56) Controls (n=81)Median age in years (IQR) 1.8 (1.3 2.8) 3.4 (2.2 5.7) 2.7 (1.3 4.4)

[0161] Clinical epidemiology and analysis of the host immune response to S. aureusindicates that SSTI is distinct from invasive S. aureus infection. These clinical disease states were analyzed separately in logistic regression modeling. For SSTI, logistic regression modeling yielded good performance metrics with an area under the receiver operator curve (AUROC) of 0.81 for a 5-feature model accounting for age, sex, history of S. aureus colonization, and level of total Hla serum Ig titer together with Hla-neutralizing antibodies (FIG.7A, Table 2 and Table 3). At the point of best cutoff, where sensitivity (recall) and specificity are both optimized, the model had a sensitivity of 0.74 specificity of 0.78 and an F1-score (harmonic mean of precision and recall) of 0.46. This model outperformed a 2-feature model not including the sex, age, or colonization status (AUROC 0.64, FIG.7B, Table 4 and Table 5) highlighting the biologic relevance of these patient-specific characteristics and simultaneously illustrating the sensitivity of logistic regression modeling. FIG.7C shows ROC that includes six features including sex, age, SSTI in the subject’s household, colonization at enrollment, lognab_acute, and logigg_acute. Table 6 shows sensitivity and specificity of the six-feature model and Table 7 shows coefficients of the six feature model. Table 8 shows different model permutations. Table 2 SSTI five feature modelAUROC 0.8095 B 4 4 4 7 8 8Table 3 Coefficients of five feature modelEstimate Std.Error z value Pr(>|z|)Table 4 SSTI two feature modelAUROC 0.6432 Best 0.47369025 7 6 1 Tableature modelEstimate Std.Error z value Pr(>|z|) (Intercept) -1.16744 0.47714 -2.447 0.0144 *Table 6 SSTI six feature modelArea under the curve: 0.817 Best Cutoff: 052080477 5 5Table 7 Coefficient of six feature modelCoefficients: Estimate Std. Error z value Pr(>|z|)5522*Table 8 Model permutations of SSTICombinations AUROClogig 352697097467548396418743438268066478628858025841620672092576household colonization_enr 52 _sstiollment 0.7418605colonization_enr 4495518133934395731708440797743208592507684626226227

[0162] For invasive disease, logistic regression modeling yielded performance metricswith an area under the receiver operator curve (AUROC) of 0.82 for a 5-feature model accounting for age, sex, history of S. aureus colonization, and level of total Hla serum Ig titer together with Hla-neutralizing antibodies (FIG.8A, Table 9 and Table 10). At the point of best cutoff, where sensitivity (recall) and specificity are both optimized, the model had a sensitivity of0.88 specificity of 0.63 and an F1-score (harmonic mean of precision and recall) of 0.52. In the case of invasive infection, the 2-feature model not including the sex, age, or colonization status also exhibited good performance with an AUROC 0.79 (FIG.8B, Table 11 and Table 12). At the point of best cutoff, where sensitivity (recall) and specificity are both optimized, the model had a sensitivity of 0.60 specificity of 0.88 and an F1-score (harmonic mean of precision andrecall) of 0.27. These findings reflect the distinct biological processes of infection, wherein invasive disease does not exhibit similar epidemiologic links to patient factors such as sex, age, and colonization status as observed for SSTI. FIG.8C shows six feature model of invasive infection which includes sex, age, SSTI in the subject’s household, colonization at enrollment, lognab_acute and logigg_acute. Table 13 shows sensitivity and specificity of the six-feature model and Table 14 shows coefficients of the six feature model. Table 15 shows different model permutations. Table 9 Five feature model for invasive infectionArea under 0.816 the curve: 7 2 1 4 Table 10 Five feature coefficientsEstimate Std.Error z value Pr(>|z|)Table 11 Two feature model of invasive infectionAUROC 0.7921 7Sensitivity 0.6049383 Specificity 0.8823529 4 Table 1feature modelEstimate Std.Error z value Pr(>|z|) (Intercept) -1.0687 0.479 -2.231 0.0257 *Area under the curve: 0.8201 Best Cutoff: 0.48097587 2 5abe Coe cents o t e sx eature modelCoefficients: Estimate Std. Error value Pr(>|z|) Sx - m l 062545 045406 1377 016845353Table 15 Model Permutations for invasive infectionCombination AUROC 04 52 07 45 37 47sex colonization_e lognab_acute logigg_acute 0.8073106 nrollment colonizat lognab_acute logigg_acute 0.8036795 91 81 18 11 42 21 06 82 03 05 52 41 65 83 01 42 73 86 33 56 74 05 88 71 77 63 65 95 14 22nrollment 49 age colonization_e 0.6853198 nrollment 75 55 56 04 69 06 75 16 65 88 56 66 78 84 39 16 52 31

[00163] Currently, these immunoscore models predicting the risk of infections are basedon logistic regression, data-driven probabilistic models rather than fixed-weight scoring systems. Features included in the models as variables were selected from a set of immunologic and demographic features. Each model calculates the predicted probability of infection using logistic regression. As such, the coefficients (i.e., weights) for each feature are estimated from the model and vary by outcome. For example, in the invasive infection model, log Nab had a strong positive association (β = 1.22, p < 0.0001), while log IgG had a negative association (β = –0.35, p = 0.026). In contrast, in the skin infection model, log Nab showed a weaker association (β = 0.17, p = 0.47), whereas log IgG was positively associated (β = 0.40, p = 0.009).

[00164] While each coefficient reflects the weight of that feature in predicting theoutcome, not all were statistically significant on their own. However, the models were selectedbased on overall performance, suggesting that some features contribute meaningfully through interactions with others, even if they are not strong independent predictors. Optimal thresholds were identified to maximize the F1-score (which balances sensitivity and precision by optimizing true positives while minimizing false positives). The best model thresholds were 0.52 for the invasive infection model, yielding sensitivity of 0.88 and specificity of 0.63, and 0.458 for the skin infection model, balancing sensitivity (0.74) and specificity (0.78).

[0165] As compared to other model architectures, logistic regressions provideinterpretable metrics that help clinicians understand how different patient characteristics and biomarkers influence the likelihood of disease, bypassing the growing concerns about machine learning techniques which many find to be un-interpretable. One of the main hinderances to the widespread adoption of machine learning predictive algorithms in healthcare, is their opaqueness and lack of explainability, known as the “Black Box” problem of Artificial Intelligence (AI) which often leads to criticism, skepticism, and reluctance to accept these models, even when they show promising results. These concerns emphasize the need to both use the least complex predictive models that maintain optimal predictive performance, in addition to providing explanations of the contribution of the included features in the model performance. Logistic regression models are recognized to have relatively simple structure as compared to more complex machine learning models in addition to a clear understanding of how individual predictors influence the predictive outcomes with easily interpretable coefficients. For example, the model generates coefficients for each feature, allowing for assessment of effect size and significance of specific variables (such as serologic titers). These coefficients can indicate whether higher levels of certain antibodies significantly increase or decrease the odds of infection. Additionally, performance metrics such as sensitivity, specificity, and the area under the receiver operating characteristic curve (AUROC) provide insight on the model’s accuracy and reliability. Logistic regression is thus a valuable tool in clinical decision-making, as it can be used to support diagnostic strategies and inform interventions based on patients’ risk profiles.

[0166] Currently, the immunoscore models predicting the risk of invasive and skininfections are based on logistic regression, data-driven probabilistic models rather than fixed- weight scoring systems. We included five features: sex, age, colonization status, log Nab antibody response, and log IgG antibody response. These variables were selected from a set of immunologic and demographic features. Each model calculates the predicted probability ofinfection using logistic regression. As such, the coefficients (i.e., weights) for each feature are estimated from the model and vary by outcome. For example, in the invasive infection model, log Nab had a strong positive association (β = 1.22, p < 0.0001), while log IgG had a negative association (β = –0.35, p = 0.026). In contrast, in the skin infection model, log Nab showed a weaker association (β = 0.17, p = 0.47), whereas log IgG was positively associated (β = 0.40, p = 0.009).

[0167] While each coefficient reflects the weight of that feature in predicting theoutcome, not all were statistically significant on their own. However, the models were selected based on overall performance, suggesting that some features contribute meaningfully through interactions with others, even if they are not strong independent predictors. Optimal thresholds were identified to maximize the F1-score (which balances sensitivity and precision by optimizing true positives while minimizing false positives). The best model thresholds were 0.518 for the invasive infection model, yielding sensitivity of 0.88 and specificity of 0.63 and 0.458 for the skin infection model, balancing sensitivity (0.74) and specificity (0.78). Example 5 Molecular mechanism of Hla

[0168] Bacterial pathogens utilize secreted toxins to evade human immunity.Staphylococcus aureus is a leading cause of human mortality related to bacterial infection. This pathogen relies on a diverse array of toxins to cause tissue injury and modulate the host immune response. S. aureus a-toxin (Hla) is a conserved pore-forming toxin produced by nearly all clinical isolates. It was found that Hla causes rapid membrane depolarization on activated CD4+T cells, precluding sustained calcium signaling that is required for proliferation and differentiation of the antigen-specific T cell response. By this means, Hla dampens T cell receptor-mediated signal strength, confirmed by the use of an altered peptide ligand.. These studies define a molecular mechanism by which a bacterial toxin directly thwarts the CD4+T cell response by tuning the strength of signaling elicited by antigen receptor engagement.

[0169] Bacterial pore-forming toxins (PFTs) have been well-studied for theircontribution to cell and tissue injury in the context of infection. The identification of the cellular receptors for many PFTs over the past decade has provided unprecedented insight on cell-type and species specificity of toxin function. Staphylococcus aureus is a leading cause of globalmortality from bacterial infections. Vaccine development to prevent S. aureus infection has been hampered by a paucity of knowledge on the cellular mechanisms by which S. aureus impairs the development of protective immunity. Understanding the molecular and cellular pathways by which S. aureus alters host immunity is thus critical for guiding rational vaccine design. In a cohort of pediatric patients, it was observed that primary skin infections were associated with nearly 50% rates of recurrent S. aureus disease, whereas invasive infections typically conferred protection, suggesting that the site of infection influences host immunity. Murine studies revealed that skin infection with S. aureus impairs the development of antigen‑specific CD4⁺ T cell immunity via the action of α‑toxin (Hla). In this study, it was observed that exposure to Hla in the context of primary S. aureus skin infection amplifies differentiation of regulatory T cells, associated with a reduction in T follicular helper cell differentiation (FIG.9A-9E). In the presence of Hla, a uniform decrease in the ability of antigen-specific T cells to generate cytokines in response to infection was observed suggesting that the toxin elicits a significant impairment of T cell responses (FIG.9F-9G). It was found that Hla reaches the skin‑draining lymph node (dLN) following subcutaneous infection (FIG 10A-10D), and that toxin exposure of the CD4+ T cell results in immediate depolarization of the T cell membrane dependent on toxin pore formation (FIG.10E-10K). As a result, induction of store-operated calcium influx is impaired in activated T cells, effectively dampening the strength of signal generated by T cell receptor (TCR) engagement (FIG.11A-11N). Using an ovalbumin-specific CD4+ T cell system, it was demonstrated in vitro and in vivo that Hla decreases the T cell proliferative response to antigenic stimulus. This effect is reversed by an Hla-targeting vaccine known to elicit toxin- neutralizing antibodies (FIG.12A-12G and FIG.13A-13I). These findings, which span both murine and human systems, suggest that Hla acts during infection to broadly disable the host’s ability to mount effective CD4+ T cell–mediated immunity. A deeper understanding of this pathway may provide insight on the evolutionary basis for the interaction of S. aureus with its human host and guide the development of vaccine strategies to protect the T cell compartment. SUMMARY OF EXAMPLES

[0170] This study provides additional evidence that the anti-Hla serologic response is arobust marker of protection against S. aureus infections in children. Specifically, a higher anti- Hla Nab convalescent to acute ratio significantly protected against recurrent SSTI over 12months of follow-up. This suggests that an individual’s ability to elicit a stronger anti-Hla neutralizing antibody response after acute SSTI infection correlates significantly with protection and may be a better marker of protection than measuring quantitative IgG alone. Significant trends in the anti-Hla IgG titer, with individuals with SSTI having higher anti-Hla IgG convalescent titers compared those with invasive infections were observed, a significant difference between anti-Hla IgG titer and recurrent infections in either group was not observed. Children with invasive infections had higher acute and convalescent anti-Hla NAb titers compared to children with SSTI, however a correlation between titer and risk of recurrent infection in the invasive group was not found. This is likely due to the smaller sample size and low event rate of recurrent infections in the invasive infection group, and also supports previous findings indicating that S. aureus elicits tissue-specific immune responses.

[0171] The pediatric cohort of S. aureus study participants is one of the largest to date,given the uncommon incidence of S. aureus invasive infections in children and difficulty retaining participants in longitudinal, clinical studies. Furthermore, our study is the first to correlate functional anti-Hla serologic response with short and long-term outcomes of pediatric S. aureus SSTI and invasive infections, which is essential given that up to 50% of children with an SSTI will have a recurrent infection within a year and children with invasive infections will have a wide spectrum of clinical responses. The potential utility of the anti-Hla NAb response as a biomarker for S. aureus disease severity would enable risk stratification of individuals and lay the foundation for future, interventional studies to reduce the significant morbidity and mortality from S. aureus infections. Another unique attribute of this study is the utilization of a human monoclonal antibody against Hla to standardize the neutralization assay, which will facilitate direct comparison of anti-Hla NAb titers across different populations in future studies.

[0172] Similar to other studies, heterogeneity in the anti-Hla serologic response betweenparticipants was found, which is reflective of the real-world variability seen in clinical responses to S. aureus and likely in part attributable to differences in host genetics. A recent study of pediatric patients with S. aureus infections also found that approximately 40% of children did not have an increase in convalescent anti-Hla, LukE, or LukS-PV IgG titers after acute SSTI or invasive infection. While disorders affecting phagocyte development or function, such as chronic granulomatous disease or leukocyte adhesion deficiency, have traditionally been linked to an increased susceptibility to S. aureus infections, an increased understanding of human inbornerrors of immunity has led to increasing recognition of rare chromosomal abnormalities, such as OTULIN haploinsufficiency, as a genetic etiology of severe staphylococcal infections. In addition, a genome-wide associated study of 50,000 participants with S. aureus infection or matched controls found an association between two single nucleotide polymorphisms in the HLA class II region and increased risk for S. aureus susceptibility. Future studies are needed to better understand host genetic determinants of S. aureus infections, particularly as high-risk individuals could be prioritized for S. aureus vaccination development and implementation.

[0173] While this study focused specifically on the serologic response to S. aureusinfection, protection is multifactorial, involving several facets of the immune system including T-cell and innate immune responses. Importantly, differences elicited by tissue-specific adaptive immune responses can in part explain the higher rate of recurrent infections in children with S. aureus SSTI compared to those with invasive infections seen in our study and previous studies. S. aureus primary skin infection in mice resulted in an impaired CD4+ T-cell response upon reexposure, whereas primary invasive infection elicited antigen-specific memory T-cell responses, which correlated with protection against secondary skin infection. Furthermore, immunization of mice against Hla prior to skin challenge resulted in preservation of the antigen- specific T-cell response and decreased infection severity, suggesting targeting Hla prior to S. aureus exposure in early life may be essential for a vaccine to be effective. Previous studies have also demonstrated differences in the innate immune response to S. aureus skin and invasive infection, with altered IL-1β production resulting in more severe skin infections and protection against lethal pneumonia in Hla-receptor knockout mice. Additional study of the interplay between anti-Hla serologic and T-cell responses will be essential towards understanding S. aureus long-term immunity.

[0174] There are likely other antibody functional properties that could play a role inprotection against S. aureus infection, such as differences in Fc-glycosylation profiles. While it cannot be directly inferred that the anti-Hla neutralizing antibody response from natural infection would be the same as elicited from vaccine-induced protection, there is a high degree of concordance between immune correlates of protection from seroepidemiologic studies of other pathogens such as SARS-CoV-2, respiratory syncytial virus, and group B Streptococcus.

[0175] In conclusion, it was observed that higher anti-Hla NAb fold induction titercorrelated significantly with a decreased risk of recurrent SSTI infection over 12 months offollow-up in a prospective, longitudinal cohort of children and that a threshold of protection can be inferred to guide future S. aureus vaccine studies. This study supports the advancement of Hla as an antigenic target for S. aureus vaccine development. METHODS

[00176] Study Population: Children birth through 21 years of age with:Group 1: Asymptomatic Staphylococcus aureus colonization or non-colonized controls Group 2: Children with S. aureus skin and soft tissue infections (SSTI) Group 3: Children with invasive S. aureus infections (bacteremia, osteomyelitis / septic arthritis, pneumonia, deep abscess)

[00177] Study Design: Prospective, longitudinal cohort study. Children were enrolled attime of infection (for SSTI or invasive), followed up 4-8 weeks later and then every 3 months for 12 months with serial surveys to determine whether they had a recurrent infection or not. Controls (Group 1) have one in-person visit (enrollment) but are also followed longitudinally.

[00178] Specimens: Blood samples were collected at time of enrollment (acute sample)for all study participants and 4-8 weeks later (convalescent sample) for SSTI and invasive infection only (Table 16). Table 16 Blood samples were collected at time of enrollment (acute sample) for all studyparticipants and 4-8 weeks later (convalescent sample) for SSTI and invasive infection only. Healthy Non-invasive Invasive Controls SSTI InfectionEnrollment Visit ^Enrollment interview^ ^ ^healthy noncolonized controls, 23 S. aureus asymptomatic colonized controls) with at least one follow-up visit.126 with both acute and convalescent samples available (71 with SSTI, 55 with invasive infections)

[00180] Study Aim: Determine a threshold of protection for anti-Hla Nab titer againstrecurrent S. aureus infection. Examine different models using: 1) Acute or convalescent time point; 2) Fold induction ratio= convalescent / acute titer; 3) Delta Nab titer= (convalescent- acute) / acute titer.

[00181] A total of 254 participants who completed at least one follow-up visit wereincluded in the overall analyses: 96 with SSTI, 67 with invasive infection, 60 healthy non- colonized controls, and 31 healthy S. aureus-colonized controls. For fold-induction analyses, participants without both acute and convalescent samples available and at least one additional follow-up visit, were excluded leaving 126 participants: 71 with SSTI and 55 with invasive S. aureus infections. Demographic and other characteristics of the participants are shown in Table 1. Participants with invasive infections were more likely to be older (p<0.001), male (p=0.025), and less likely to be colonized with S. aureus at enrollment (p<0.001) or have had a previous SSTI (p<0.001). The majority of invasive infections occurred in the bone / joint (53%) or blood (40%). Participants with SSTI were more likely to have MRSA isolated from sterile culturecompared to those with invasive (p<0.001). Participants with asymptomatic S. aureus colonization, SSTI, or invasive infections were more likely to have an underlying skin disorder such as eczema or psoriasis (p<0.001). Children with SSTI had a 4.77 (95% CI: 2.15 to 10.19) increased odds of having a recurrent infection within the 12 months of follow-up compared to children with invasive infection (Table 17, p<0.001). Table 17 Participant CharacteristicsHealthy Non Healthy SSTI Invasive P-valueaColonized (n=60) Colonized (n=31) (n=93) Infection (n=68) Age 1 5 2 1 1 1 1 1Psoriasis 0 (0%) 1 (3%) 0 (0%) 1 (1%) p<0.001 Acne 0 (0%) 1 (3%) 0 (0%) 0 (0%) p<0.001

[00182] Study Population, Sample Collection, and Longitudinal Follow-up: Previouslycollected serum and plasma samples from two Washington University in St. Louis IRB-approved prospective, observational cohort studies were utilized. The initial study enrolled four cohorts of pediatric patients aged ≥6 months between August 2008 and March 2021 St. Louis Children’s Hospital in St. Louis, Missouri, as previously described. The first cohort comprised of healthy children with asymptomatic MRSA colonization at enrollment or based on previous culture results. The second cohort of patients comprised of children presenting with first time SSTI requiring incision and drainage whose cultures yielded S. aureus. The third cohort included patients with recurrent culture-confirmed S. aureus SSTI undergoing drainage procedure. The fourth cohort included hospitalized patients with invasive S. aureus infections, determined by isolation of S. aureus in culture obtained from a sterile site. For the analysis, participants with primary and recurrent SSTI were combined into a single SSTI group. Additional samples were obtained from an ongoing, subsequent prospective cohort study which started enrollment in June 2021. The study enrolled a similar cohort of pediatric patients with the following characteristics: 1) healthy controls with or without asymptomatic S. aureus colonization, 2) children with culture-confirmed S. aureus SSTI, or 3) children hospitalized with an acute invasive S. aureus infection. Participants were included if they had completed 12 months of follow-up. All participants provided written informed consent for their children and assent was obtained for children older than 7 years of age.

[00183] Patients with traditional risk factors for healthcare-associated MRSA infections(e.g., indwelling catheter or percutaneous medical device, dialysis, post-operative infection, residence in a long term care facility) were excluded from participation. Immunocompromised individuals were excluded, including preterm neonates, children with diabetes, renal disease, oncologic disease, or HIV. Participants where S. aureus was recovered from a surface culture (e.g., decubitus ulcer) or for whom culture was not obtained were excluded. Participants who did not complete any follow-up visits were excluded from analysis. For paired fold induction analyses, participants without both acute and convalescent plasma or serum samples availableand those that did not complete any additional follow-up visits besides the convalescent visit were excluded.

[0184] At enrollment, detailed questionnaires were administered to parents or guardiansto document participant and family health history. Medical charts were abstracted to obtain details regarding hospital admission, symptoms present, culture results, laboratory and imaging results, and hospitalization outcomes. Colonization cultures (CultureSwab Liquid Stuart, Becton Dickinson, Franklin Lakes, NJ) were obtained from the anterior nares, axillae, and inguinal folds of each participant. S. aureus was identified from culture swabs by standard methods as previously described. An acute blood sample was drawn at enrollment and a convalescent blood sample was drawn 4-8 weeks after in participants with SSTI or invasive infection. A follow-up survey was administered at the convalescent visit to document the clinical course of the infection and to identify epidemiologic factors associated with S. aureus infection. All participants were followed longitudinally with surveys administered by mail, telephone, or e-mail every 3 months for 1 year to document interval infections and antimicrobial use. A recurrent infection was defined as a clinically diagnosed abscess, boil, cellulitis, or S. aureus culture-confirmed invasive infection (e.g., bacteremia, bone or joint infection). Participant information was collected and managed in REDCap electronic data capture tools hosted at Washington University in St. Louis.

[0185] Anti-Hla Neutralization Antibody assay: Rabbit erythrocyte are highly sensitiveto lysis by purified Hla in vitro. Hla lysis of rabbit erythrocytes with an effective concentration of 50% (EC50) of 30-200ng / mL is confirmed for each lot of erythrocytes. Sera is diluted three- fold for a total of 8 dilutions starting at 1:2 dilution and incubated for 15 minutes with 2nM purified Hla at room temperature.2.5×105of washed erythrocytes are added for 1 hour in a slow shaker at room temperature in sterile, 96-well U-bottom plates. All specimens were tested in duplicate and in the presence of erythrocytes and without erythrocytes as background control. Cells and debris are centrifuged at 2000rpm for 10 minutes and supernatant measured for hemolysis (450nm, Tecan Infinite M200 Pro). A reference standard curve was generated with two-fold serial dilutions of a monoclonal antibody against Hla (E11.10) for each run (4-6 plates / run). The log of the E11.10 standard reciprocal dilution is graphed against specific lysis readouts and the EC50 titer defined as the reciprocal dilution which neutralizes the toxicity of Hla by 50%. Individual patient samples are then log-transformed and values extrapolated based on the E11.10 EC50 titer to determine the Nab titer dilution that correlates with 50% lysis by thestandard. Background values are subtracted from each sample value prior to analysis. Negative control wells contained PBS without virus and positive control wells contained Triton X-100 without virus were run with each set of plates. The lower limit of detection of the assay was set as the mean of the negative control antigen wells plus two standard deviations for all tested specimens.

[0186] Anti-Hla Binding Antibody assay: High-binding 364-well plates (FisherScientific) are coated with purified Hla at 1μg / mL in PBS overnight at 4°C. Plates are blocked with 0.1% BSA in PBS for 1 hour at room temperature. Serum sample dilutions added starting at 1:2 dilution and then two-fold for 24 dilutions in duplicate for 2 hours at room temperature. All specimens were tested in duplicate. Plates are washed three times with 0.05% Tween 20 / PBS prior to the addition of HRP-conjugated goat anti-human antibody (Southern Biotech) at 1:20,000 dilution for 45 min at room temperature. Plates are then washed five times, developed with TMB substrate (ThermoFisher) for 15 minutes and reactions are stopped with H2SO4. Absorbance (OD450) is measured on a Tecan plate reader and data analysis performed using GraphPad Prism to determine half-maximal titers of each sample.

[0187] Statistical Analysis: Descriptive statistics were used to characterize participantcharacteristics. Comparison of anti-Hla NAb titers was performed using one-way analysis of variance (ANOVA) to compare continuous values between cohorts and Tukey’s multiple comparison test if significant. For longitudinal analyses, paired t-test was used to compare mean acute and convalescent anti-HlaNAb titers. Fold induction titer was calculated as the ratio of the convalescent titer to the acute titer. For titers that had zero-values, the fold induction titer was inputted as the convalescent titer for acute values of zero, the acute titer for convalescent values of zero, or one for cases where both the acute and convalescent titers were zero. Then, values were transformed using base-10 and utilized logistic regression to determine odds ratios of recurrence. All tests of significance were 2-tailed, p-values <0.05 were considered significant. Analysis was performed using GraphPad Prism version 10.2.1 (GraphPad Software, Boston, MA) or R version 4.3.3.

[0188] Analysis of human serum response to S. aureus Hla: Serum titer to Hla. ELISAbased methodology is utilized to define the serum titer (log transformed) of IgG antibody to S. aureus Hla. Recombinant Hla is utilized to coat the ELISA plate, after which a dilution range of human serum is incubated in serial wells to enable anti-Hla binding to recombinant Hla. SpecificIgG to Hla is detected using standard methods with an HRP-conjugated antibody to human IgG. Curves are generated representing the absorbance per well across the dilution series for each serum sample. The half maximal titer is defined based on absorbance and log transformed for analysis in the immunoscore.

[0189] Hla neutralizing antibody titer: A dilution range of human serum is incubated inserial wells with a fixed quantity of recombinant active Hla. These samples are then added to a fixed quantity of rabbit red blood cells. Lysis of the rabbit red blood cells is quantified by an absorbance-based assay (OD450); lysis is inversely proportional to Hla neutralization by the serum antibody. Two methods are utilized to quantify this data:

[0190] (a) Curves are generated representing the absorbance per well across the dilutionseries for each serum sample. The IC50 titer is defined based on absorbance and log transformed for analysis in the immunoscore.

[0191] (b) The absorbance curves are utilized to define a specific neutralizing antibodyconcentration IC50 in mcg / ml based on comparative analysis to the standard 7B8-hIgG1 antibody. FIG.14A shows a 7B8-HIgG1 standard curve and quantitation of three distinct human sera for Hla neutralizing antibody (FIG.14B).

Claims

CLAIMS What is claimed is:

1. A method of determining risk of a Staphylococcus infection in a subject,comprising: (a) providing data obtained from immunological tests from a biologicalsample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies; and (b) generating a score for the subject based on the data;wherein a score above a predetermined threshold value is indicative of the subject being at risk of the Staphylococcus infection.

2. A method of vaccinating a subject with a Staphylococcus vaccine comprising:(a) providing data obtained from immunological tests from a biologicalsample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies; (b) generating a score for the subject based on the data, wherein a score abovea predetermined threshold value is indicative of the subject being at risk of the Staphylococcus infection; and (c) administering a Staphylococcus vaccine to the subject determined to be atrisk of the Staphylococcus infection.

3. A method of predicting efficacy of a Staphylococcus vaccine in a subject,comprising:(a) providing data obtained from immunological tests from a biologicalsample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies; (b) generating a score for the subject based on the data; andwherein a score above a predetermined threshold value is indicative of the vaccine being efficacious for the subject.

4. A method of determining risk of recurrent Staphylococcus infection in a subject,comprising: (a) providing data obtained from immunological tests from a biologicalsample from the subject, wherein the immunological tests comprise a total amount of α-hemolysin (Hla) Immunoglobulin G (Ig) titer and an amount of Hla neutralizing antibodies; and (b) generating a score for the subject based on the data;wherein a score above a predetermined threshold value is indicative of the subject being at risk of recurrent Staphylococcus infection.

5. The method of any one of claims 1-4, wherein the Staphylococcus isStaphylococcus aureus.

6. The method of claim 5, wherein the Staphylococcus aureus is Methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-intermediate Staphylococcus aureus (VISA), or vancomycin-resistant Staphylococcus aureus (VRSA).

7. The method of any one of claims 1-4, wherein the Staphylococcus infection isselected from an invasive infection, a skin and soft tissue infections (SSTI) infection, skin boil, impetigo, cellulitis, staphylococcal scalded kin syndrome, food poisoning, nausea, vomiting, diarrhea, dehydration, low blood pressure, bacteremia, meningitis, endocarditis, pneumonia,toxic shock syndrome, fever, rash, confusion, muscle ache, stomach pain, septic arthritis, joint swelling, joint pain, and skin blister.

8. The method of claim 4, wherein a score below the predetermined thresholdindicate that the subject would be free of infection for about 3 months to 15 months.

9. The method of any one of claims 1-4, wherein the biological sample is a bloodsample.

10. The method of claim 9, wherein the blood sample is selected from whole blood,serum, and plasma.

11. The method of any one of claims 1-4, wherein the total Hla Ig titer and the Hlaneutralizing antibodies are measured, respectively, as a log of the total amount of Hla Ig titer and the amount of Hla neutralizing antibodies.

12. The method of any one of claims 1-4, wherein the score is a combined scoredetermined by weighting the data obtained from each of the immunological tests with a predefined coefficient to obtain a weighted value for each immunological test and adding the weighted values to obtain the combined score.

13. The method of claim 12, wherein:(a) the Staphylococcus infection is a SSTI; the immunological tests comprisea log of the total Hla Ig titer and the amount of Hla neutralizing antibodies; their respective weights are 0.036 and 0.266; and the predetermined threshold value is 0.473; or (b) the Staphylococcus infection is an invasive infection; the immunologicaltests comprise a log of the total Hla Ig titer and the amount of Hla neutralizing antibodies; their respective weights are 1.335 and -0.323; and the predetermined threshold value is 0.277.

14. The method of any one of claims 1-4, wherein (a) further comprises providing abiological sex of the subject, an age of the subject, and a history of S. aureus colonization of the subject.

15. The method of claim 14, wherein the score is a combined score determined byweighting the log of the total Hla Ig titer, the amount of Hla neutralizing antibodies, thebiological sex of the subject, the age of the subject, and the history of S. aureus colonization of the subject.

16. The method of claim 15, wherein:(a) the Staphylococcus infection is a SSTI; the weight for male sex is -1.17;the weight for age is -0.178; weight for S. aureus colonization is -0.482; the weight for no S. aureus colonization is -2.16; the weight of the amount of Hla neutralizing antibodies is 0.179; the weight of the log of the total Hla Ig titer is 0.399; and the predetermined threshold value is 0.458; or (b) the Staphylococcus infection is an invasive infection; the weight for malesex is 0.660; the weight for age is 0.055; the weight for S. aureus colonization is -0.658; the weight for no S. aureus colonization is -1.65; the weight of the amount of Hla neutralizing antibodies is 1.21; the weight of the log of the total Hla Ig titer is -0.348; and the predetermined threshold value is 0.

517.

17. The method of any one of claims 1-4, wherein (a) further comprises providing abiological sex of the subject, an age of the subject, a history of S. aureus colonization of the subject, and status of SSTI in the subject’s household.

18. The method of claim 17, wherein the score is a combined score determined byweighting the amount of Hla neutralizing antibodies, the log of the total Hla Ig titer, the biological sex of the subject, the age of the subject, the history of S. aureus colonization of the subject, and status of SSTI in the subject’s household.

19. The method of claim 18, wherein:(a) the Staphylococcus infection is a SSTI; the weight for male sex is 1.07; theweight for age is -0.168; the weight for S. aureus colonization of the subject is -0.467; the weight for no S. aureus colonization is -2.20; the weight of the amount of Hla neutralizing antibodies is 0.080; the weight of the log of the total Hla Ig titer is 0.407; the weight for SSTI in the subject’s household is 14.5; the weight for no SSTI in the subject’s household is 13.6; and the predetermined threshold value is 0.520; or (b) the Staphylococcus infection is an invasive infection; and the weight formale sex is 0.625; the weight for age is 0.054; the weight for no S. aureus colonization is -1.75; the weight of the amount of Hla neutralizing antibodies is 1.26; the weight of the log of the total Hla Ig titer is -0.332; the weight for SSTI in the subject’s household is -1.42; the weight for no SSTI in the subject’s household is -0.857; and the predetermined threshold value is 0.

480.

20. The method of any one of claims 1-4, wherein the immunological tests furthercomprise a level of SpA neutralization titer, a response of T follicular helper cells to Staphylococcus(Tfh), a response of T regulatory cells to Staphylococcus (Treg), a ratio of Tfh over Treg (Tfh:Treg ratio), and a cytokine response profile of T cells.

21. The method of claim 20, wherein the score is a combined score determined byweighting the data obtained from each of the immunological tests with a predefined coefficient and adding the weighted data to obtain the combined score.

22. The method of claim 21, wherein the predefined coefficient is obtained from acontrol data set, wherein each of the immunological tests in the control dataset is ranked for the ability to predict risk of the Staphylococcus infection, predict efficacy of a Staphylococcus vaccine, determining risk of recurrent Staphylococcus infection, or predict whether the subject is at risk of SSTI or invasive Staphylococcus infection.

23. The method of any one of claims 1-22, wherein the score is generated by aprocessor using a machine learning model.

24. The method of claim 23, wherein the machine learning model comprises logisticregression.

25. The method of any one of claims 1-4, further comprising administering to thesubject a vaccine or a treatment for Staphylococcus.

26. The method of any one of claims 2-3, and 25, wherein the vaccine is StaphVax.

27. The method of any one of claims 2-3, and 25, wherein the vaccine comprises anantigen selected from CP5, CP8, ClfA(SA3Ag), CP5-CRM197, CP8-CRM197, MntC, ClfA(SA4Ag), Alpha-toxin,Panton-Valentine Leukocidin, Leukocidin AB, HlgAB, HlgBC, Surface protein A (SpA), enterotoxin B, and a live or attenuated S. aureus.

28. The method of claim 4, further comprising determining a prognosis of the subject,wherein a score above the predetermined threshold indicates a positive outcome selected from positive survival outcome, a positive response to a Staphylococcus treatment, reduced recurrent Staphylococcus infection.

29. The method of claim 4, further comprising administering a treatment to thesubject.

30. The method of any one of claims 25 and 29, wherein the treatment comprisesproviding an antibiotic to the subject.

31. The method of claim 30, wherein the antibiotic is selected from cefazolin,nafcillin, oxacillin, vancomycin, daptomycin, and linezolid.

32. The method of any one of claims 1-2, and 4, wherein a score below or equal to thepredetermined threshold value is indicative of the subject being at low risk of the Staphylococcus infection or recurrent Staphylococcus infection.

33. The method of claim 3, wherein a score below or equal to the predeterminedthreshold value is indicative of the vaccine being unlikely to be efficacious for the subject.

34. A system comprising: a processor in communication with a memory, the memoryincluding instructions, which, when executed, cause the processor to execute the method of any one of claims 1-4.

35. A kit comprising:(a) means for performing one or more immunological tests comprising at leastamount of Hla neutralizing antibodies and the total Hla Ig titer; (b) instructions for:(i) determining risk of a Staphylococcus infection of claim 1;(ii) vaccinating a subject with a Staphylococcus vaccine of claim 2;(iii) predicting efficacy of a Staphylococcus vaccine of claim 3; or(iv) determining risk of recurrent Staphylococcus infection of claim 4.

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