Staphylococcus aureus periprosthetic joint infection is notoriously difficult to treat and is often associated with septic death. Results from the V710 Phase IIB/III clinical trial and subsequent research have demonstrated that non-neutralizing antibodies against the iron-regulated surface determinant protein B (IsdB) facilitate bacterial entry into leukocytes, generating "Trojan horse" leukocytes and the dissemination of surgical-site infections. In contrast, anti-glucosaminidase (Gmd) antibodies, which mediate opsonophagocytosis of bacterial clusters, are associated with protection from S. aureus osteomyelitis in both mice and humans. To further test whether anti-IsdB antibodies are pathogenic and anti-Gmd antibodies are protective in infected patients, we performed a clinical pilot study of three healthy patients undergoing primary total hip arthroplasty and a patient undergoing revision arthroplasty for S. aureus infection to assess the feasibility of quantifying anti-IsdB and anti-Gmd antibody-secreting cells in blood and periprosthetic bone marrow. We also assessed anti-IsdB and anti-Gmd antibody levels in serum, blood plasma, bone marrow plasma, and cell culture supernatant collected after six days with and without a previously established memory B-cell stimulation cocktail. The frequencies of memory B-cells and antibody-secreting plasmablasts were measured using IgG ELISpot. Consistent with our hypothesis, we found large numbers of anti-IsdB antibody-secreting cells in unstimulated periprosthetic bone marrow from the infected patient, which increased with stimulation. However, these cells were only detectable in peripheral blood mononuclear cells following stimulation. Furthermore, anti-Gmd antibody-secreting cells were undetectable in the bone marrow of this infected patient at the time of revision surgery. This patient also had very high titers of anti-IsdB antibodies in serum, blood plasma, and bone marrow plasma. Anti-IsdB and anti-Gmd antibody-secreting cells were only detected in bone marrow for one of the uninfected patients. Collectively, the results are consistent with the hypothesis that anti-IsdB antibodies are associated with disease and related to the presence of IsdB-specific memory B-cells and plasmablasts. This case series demonstrates the utility of ELISpot to assess local vs. systemic antigen-specific humoral responses in total hip arthroplasty patients and support a larger study to test the hypothesis that anti-IsdB antibody-secreting cells proximal to S. aureus infected implants are associated with recalcitrant infections and septic death.

Post-acute sequelae of SARS-CoV-2 (SARS2) infection (PASC) is a heterogeneous condition, but the main viral drivers are unknown. Here, we use MENSA, Media Enriched with Newly Synthesized Antibodies, secreted exclusively from circulating human plasmablasts, to provide an immune snapshot that defines the underlying viral triggers. We provide proof-of-concept testing that the MENSA technology can capture the new host immune response to accurately diagnose acute primary and breakthrough infections when known SARS2 virus or proteins are present. It is also positive after vaccination when spike proteins elicit an acute immune response. Applying the same principles for long-COVID patients, MENSA is positive for SARS2 in 40% of PASC vs none of the COVID recovered (CR) patients without any sequelae demonstrating ongoing SARS2 viral inflammation only in PASC. Additionally, in PASC patients, MENSAs are also positive for Epstein-Barr Virus (EBV) in 37%, Human Cytomegalovirus (CMV) in 23%, and herpes simplex virus 2 (HSV2) in 15% compared to 17%, 4%, and 4% in CR controls respectively. Combined, a total of 60% of PASC patients have a positive MENSA for SARS2, EBV, CMV, and/or HSV2. MENSA offers a unique antibody snapshot to reveal the underlying viral drivers in long-COVID thus demonstrating the persistence of SARS2 and reactivation of viral herpes in 60% of PASC patients.

Background: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. Objective: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients biologics (PoBs). Methods: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. Results: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. Conclusion: The patients in the PoB group had reduced SARSCoV-2-specific antibody titers, neutralizing activity, and virus- specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways. (J Allergy Clin Immunol 2024;154:435-46.)

The incidence of prosthetic joint infection (PJI) following elective primary total knee arthroplasty (TKA) is very low but serious risk remains. To identify unknown risk factors, we completed a natural history study of IgG specific for Staphylococcus aureus antigens previously phenotyped as protective (anti‐Atl) and pathogenic (anti‐Isd). Twenty‐five male and 25 female optimized patients 50–85 years of age and BMI 24–39 undergoing primary TKA were prospectively enrolled. Blood sampling was performed preoperatively, postoperative Day 1, and at 2, 6, and 12 weeks, to assess serum cytokine, anti‐staphylococcal IgG levels and anti‐tetanus toxoid IgG measured via custom Luminex assay. Clinical, demographic, and PROMIS‐10 data were collected with outcomes to 2 years postop. All participants completed the study and 2‐year follow‐up. No patients were readmitted or noted to develop a surgical site infection or serious adverse event, and patient‐reported outcomes were improved. Serology revealed a highly significant decrease in six out of eight antibody titers against specific S. aureus antigens on Day 1 (p < 0.0001), five of which normalized to preoperative levels within 2 weeks. These changes were commensurate with a decrease and recovery of anti‐tetanus toxoid titers, and a 20% drop in hemoglobin 13.8 ± 1.7 at preop to 11.1 ± 1.8 mg/dL on Day 1 (p < 0.0001). After TKA, a significant decrease in humoral immunity commensurate with blood loss and hemodilution was recorded. This decrease in circulating anti‐staphylococcal antibodies in the early postop period may represent a periprosthetic joint infection risk factor for patients.

The Orthopaedic Research Society's Research Interest Group completed its international consensus meeting (ICM) on musculoskeletal infections (MSKI) following the 2023 Annual Meeting. The work products from this ICM include the 65 questions with recommendation and rationale, and the voting results from the 72 delegates. There are also five Consensus Articles in this issue of the Journal of Orthopaedic Research from the ICM Sections: Host Immunity, Established Infection-Treatment, Clinical Questions not addressed by the prior MSKI ICMs, In Vitro, and Animal Models. This Introduction summarizes the 3-year Delphi process used by the ICM with timelines and critical milestones. It also highlights several challenges that had to be addressed, and a large body of work that remains.

Background: mRNA SARS-CoV-2 vaccines are effective in reducing severe COVID-19 in the general population. However, a comprehensive study of the postvaccination immune response to SARS-CoV-2 has not been done in patients on dupilumab, benralizumab, or mepolizumab. Aim: Assess the post-vaccination SARS-CoV-2-specific immune responses in patients on dupilumab, benralizumab, or mepolizumab. Methods: We enrolled healthy controls and patients with severe asthma or atopic dermatitis on dupilumab, benralizumab, or mepolizumab in a prospective, observational trial. All subjects had an initial 2-dose SARS-CoV-2 mRNA vaccination. We used a multiplexed immunoassay to measure antibody levels, a pseudotyped virus luciferase-based assay and a life virus infection inhibition assay to quantify their neutralization activity, and spectral flow cytometry to quantify antigen-specific lymphocytes (using a SARS-CoV-2 protein tetramer-based detection for B cells and activation-induced markers for T cells after 24h incubation with viral peptides). Results: We collected samples from 46 healthy controls and 57 patients on biologics during a 6-month follow-up after the second vaccine dose. Compared to healthy controls, patients on biologics had lower levels of SARS-CoV-2 antibodies (p=0.001), pseudovirus neutralization (p=0.0002), live virus neutralization (p=0.0069), and frequencies of Spike-specific B cells (p=0.02). There were no significant differences in antigen-specific CD4 (p=0.26) or CD8 (p=0.70) T cells between the groups. Conclusion: Post-vaccination SARS-CoV-2 antibody and B cell but not T cell responses are lower in patients on biologics than in healthy controls.

BACKGROUND:Diagnostic immunoassays for Lyme disease have several limitations including: 1) not all patients seroconvert; 2) seroconversion occurs later than symptom onset; and 3) serum antibody levels remain elevated long after resolution of the infection.INTRODUCTION:MENSA (Medium Enriched for Newly Synthesized Antibodies) is a novel diagnostic fluid that contains antibodies produced in vitro by circulating antibody-secreting cells (ASC). It enables measurement of the active humoral immune response.METHODS:In this observational, case-control study, we developed the MicroB-plex Anti-C6/Anti-pepC10 Immunoassay to measure antibodies specific for the Borrelia burgdorferi peptide antigens C6 and pepC10 and validated it using a CDC serum sample collection. Then we examined serum and MENSA samples from 36 uninfected Control subjects and 12 Newly Diagnosed Lyme Disease Patients.RESULTS:Among the CDC samples, antibodies against C6 and/or pepC10 were detected in all seropositive Lyme patients (8/8), but not in sera from seronegative patients or healthy controls (0/24). Serum antibodies against C6 and pepC10 were detected in one of 36 uninfected control subjects (1/36); none were detected in the corresponding MENSA samples (0/36). In samples from newly diagnosed patients, serum antibodies identified 8/12 patients; MENSA antibodies also detected 8/12 patients. The two measures agreed on six positive individuals and differed on four others. In combination, the serum and MENSA tests identified 10/12 early Lyme patients. Typically, serum antibodies persisted 80 days or longer while MENSA antibodies declined to baseline within 40 days of successful treatment.DISCUSSION:MENSA-based immunoassays present a promising complement to serum immunoassays for diagnosis and tracking therapeutic success in Lyme infections.

Interleukin-27 is a pleiotropic cytokine whose functions during bacterial infections remain controversial, and its role in patients with S. aureus osteomyelitis is unknown. To address this knowledge gap, we completed a clinical study and observed elevated serum IL-27 levels (20-fold higher, P < 0.05) in patients compared with healthy controls. Remarkably, IL-27 serum levels were 60-fold higher in patients immediately following septic death than in uninfected patients (P < 0.05), suggesting a pathogenic role of IL-27. To test this hypothesis, we evaluated S. aureus osteomyelitis in WT and IL-27Rα−/− mice with and without exogenous IL-27 induction by intramuscular injection of rAAV-IL-27p28 or rAAV-GFP, respectively. We found that IL-27 was induced at the surgical site within 1 day of S. aureus infection of bone and was expressed by M0, M1 and M2 macrophages and osteoblasts but not by osteoclasts. Unexpectedly, exogenous IL-27p28 (~2 ng·mL−1 in serum) delivery ameliorated soft tissue abscesses and peri-implant bone loss during infection, accompanied by enhanced local IL-27 expression, significant accumulation of RORγt+ neutrophils at the infection site, a decrease in RANK+ cells, and compromised osteoclast formation. These effects were not observed in IL-27Rα−/− mice compared with WT mice, suggesting that IL-27 is dispensable for immunity but mediates redundant immune and bone cell functions during infection. In vitro studies and bulk RNA-seq of infected tibiae showed that IL-27 increased nos1, nos2, il17a, il17f, and rorc expression but did not directly stimulate chemotaxis. Collectively, these results identify a novel phenomenon of IL-27 expression by osteoblasts immediately following S. aureus infection of bone and suggest a protective role of systemic IL-27 in osteomyelitis.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

Within 8 weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop the recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection.

SARS-CoV-2 vaccines confer great protection against COVID-19 in healthy adults, but it is unknown how patients with severe asthma and atopic dermatitis on biologic therapies respond despite animal data suggesting a lower response. We conducted a prospective observational trial from February to September 2021 and recruited adults with severe asthma or atopic dermatitis treated with benralizumab, mepolizumab or dupilumab, receiving SARS-CoV-2 mRNA vaccination. Patients with prior COVID-19 or significant immunosuppression were excluded. Blood samples were taken from 74 patients and 39 healthy controls and analyzed for SARS-CoV-2 antigen reactivities using a multiplex Luminex assay. Patients treated with biologic therapies had lower IgG levels to the SARS-CoV-2 receptor-binding domain compared to healthy adults 30 days after the second vaccination. Ninety days after the second vaccination, IgG levels for patients on biologic therapies remained significantly lower compared to healthy adults (average median fluorescence intensity 66,437 and 100,519, respectively; p-value 0.0276). Similar results were seen for Delta variant and Spike protein S1 titers. Our data suggest that patients with severe asthma or atopic dermatitis on biologic therapies have lower antibody levels after SARS-CoV-2 mRNA vaccination compared to healthy adults. It is unclear if these differences are due to the biologic therapies or confounding factors commonly seen in these patients. Nevertheless, as there is evidence that lower vaccine-specific titers afford less protection against COVID-19, clinicians should consider a booster vaccination in these patients as they may unknowingly remain at high risk for severe disease.

SESSION TITLE: Late Breaking Insights In Management of Asthma and COPDSESSION TYPE: Original InvestigationsPRESENTED ON: 10/18/2022 09:15 am - 10:15 amPURPOSE: SARS-CoV-2 vaccines have greatly reduced the impact of the COVID-19 pandemic. However, immune responses and their ability to protect against SARS-CoV-2 infection and severe clinical outcomes vary amongst vaccinees. Understanding who remains at high risk for severe infection despite vaccination and who may need additional vaccine boosters is critical for the control of this and future pandemics. We recently reported a reduced humoral immune response after mRNA SARS-CoV-2 vaccination in patients with severe asthma or atopic dermatitis on biologic therapies three months after the second vaccination, compared to healthy controls. The purpose of this study is to characterize the immune response of these patients six months after vaccination.METHODS: We conducted a prospective observational trial from February 2021 to February 2022 and enrolled 77 adults with severe asthma or atopic dermatitis treated with benralizumab, mepolizumab or dupilumab, receiving a SARS-CoV-2 mRNA vaccination, in addition to 45 healthy controls. We analyzed pseudovirus neutralization against wild-type, Delta variant and Omicron variant SARS-CoV-2, using a pseudotyped lentivirus.RESULTS: After excluding patients with prior COVID-19 or significant immunosuppression, we analyzed 28 patients (5 patients on benralizumab, 20 patients on dupilumab, 3 patients on mepolizumab) in addition to 34 healthy controls at 6 months after vaccination. We found that patients with severe asthma or atopic dermatitis treated with biologics had lower pseudovirus neutralization titer at 6 months, compared to healthy controls. The mean 50% inhibitory dilution against wild-type SARS-CoV-2 among patients on biologics were lower at 2.313 log10 compared to 2.743 log10 in the healthy control group, p-value <0.0001. Additionally, the patients on biologics had lower neutralizing antibody titers against Delta variant and Omicron variant SARS-CoV-2.CONCLUSIONS: Our data shows that patients with severe asthma or atopic dermatitis on biologic therapies have lower neutralization titer after SARS-CoV-2 mRNA vaccination compared to healthy controls 6 months after the second vaccination. Large population studies have recently shown that severe or active asthma is associated with worse COVID-19 outcomes and several studies have shown that lower humoral immunity after vaccination is associated with less protection against disease. It is therefore critical to provide booster vaccinations to these vulnerable patients.CLINICAL IMPLICATIONS: Clinicians should encourage patients with severe asthma or atopic dermatitis on biologic therapies to receive SARS-CoV-2 booster vaccinations as they may unknowingly remain at high risk for severe disease.DISCLOSURES:No relevant relationships by Fabliha AnamNo relevant relationships by Suneethamma CheedarlaNo relevant relationships by Narayanaiah CheedarlaNo relevant relationships by John DaissNo relevant relationships by Natalie HaddadNo relevant relationships by Ian HentenaarNo relevant relationships by Fernando HolguinNo relevant relationships by Caroline KimNo relevant relationships by Pedro LamotheNo relevant relationships by Frances LeeNo relevant relationships by ANDREW NEISHNo relevant relationships by wendy neveuNo relevant relationships by Rahulkumar PatelNo relevant relationships by Carmen PolitoNo relevant relationships by Richard RamonellNo relevant relationships by Mayuran RavindranNo relevant relationships by John RobackNo relevant relationships by Martin RunnstromConsultant relationship with BLI, Inc. Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=RoyaltyConsultant relationship with Bristol Meyers Squibb/Celgene Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting feeSpeaker/Speaker's Bureau relationship with Bristol Meyers Squibb/Celgene Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=HonorariaConsultant relationship with GlaxoSmithKline Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting feeSpeaker/Speaker's Bureau relationship with GlaxoSmithKline Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=HonorariaConsultant relationship with Janssen Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting feeSpeaker/Speaker's Bureau relationship with Janssen Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=HonorariaConsultant relationship with Visterra Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting feeSpeaker/Speaker's Bureau relationship with Visterra Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=HonorariaConsultant relationship with Kyverna Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting feeNo relevant relationships by Sunita SharmaNo relevant relationships by Colin SwensonNo relevant relationships by Robert Swerlick SESSION TITLE: Late Breaking Insights In Management of Asthma and COPD SESSION TYPE: Original Investigations PRESENTED ON: 10/18/2022 09:15 am - 10:15 am PURPOSE: SARS-CoV-2 vaccines have greatly reduced the impact of the COVID-19 pandemic. However, immune responses and their ability to protect against SARS-CoV-2 infection and severe clinical outcomes vary amongst vaccinees. Understanding who remains at high risk for severe infection despite vaccination and who may need additional vaccine boosters is critical for the control of this and future pandemics. We recently reported a reduced humoral immune response after mRNA SARS-CoV-2 vaccination in patients with severe asthma or atopic dermatitis on biologic therapies three months after the second vaccination, compared to healthy controls. The purpose of this study is to characterize the immune response of these patients six months after vaccination. METHODS: We conducted a prospective observational trial from February 2021 to February 2022 and enrolled 77 adults with severe asthma or atopic dermatitis treated with benralizumab, mepolizumab or dupilumab, receiving a SARS-CoV-2 mRNA vaccination, in addition to 45 healthy controls. We analyzed pseudovirus neutralization against wild-type, Delta variant and Omicron variant SARS-CoV-2, using a pseudotyped lentivirus. RESULTS: After excluding patients with prior COVID-19 or significant immunosuppression, we analyzed 28 patients (5 patients on benralizumab, 20 patients on dupilumab, 3 patients on mepolizumab) in addition to 34 healthy controls at 6 months after vaccination. We found that patients with severe asthma or atopic dermatitis treated with biologics had lower pseudovirus neutralization titer at 6 months, compared to healthy controls. The mean 50% inhibitory dilution against wild-type SARS-CoV-2 among patients on biologics were lower at 2.313 log10 compared to 2.743 log10 in the healthy control group, p-value <0.0001. Additionally, the patients on biologics had lower neutralizing antibody titers against Delta variant and Omicron variant SARS-CoV-2. CONCLUSIONS: Our data shows that patients with severe asthma or atopic dermatitis on biologic therapies have lower neutralization titer after SARS-CoV-2 mRNA vaccination compared to healthy controls 6 months after the second vaccination. Large population studies have recently shown that severe or active asthma is associated with worse COVID-19 outcomes and several studies have shown that lower humoral immunity after vaccination is associated with less protection against disease. It is therefore critical to provide booster vaccinations to these vulnerable patients. CLINICAL IMPLICATIONS: Clinicians should encourage patients with severe asthma or atopic dermatitis on biologic therapies to receive SARS-CoV-2 booster vaccinations as they may unknowingly remain at high risk for severe disease. DISCLOSURES: No relevant relationships by Fabliha Anam No relevant relationships by Suneethamma Cheedarla No relevant relationships by Narayanaiah Cheedarla No relevant relationships by John Daiss No relevant relationships by Natalie Haddad No relevant relationships by Ian Hentenaar No relevant relationships by Fernando Holguin No relevant relationships by Caroline Kim No relevant relationships by Pedro Lamothe No relevant relationships by Frances Lee No relevant relationships by ANDREW NEISH No relevant relationships by wendy neveu No relevant relationships by Rahulkumar Patel No relevant relationships by Carmen Polito No relevant relationships by Richard Ramonell No relevant relationships by Mayuran Ravindran No relevant relationships by John Roback No relevant relationships by Martin Runnstrom Consultant relationship with BLI, Inc. Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Royalty Consultant relationship with Bristol Meyers Squibb/Celgene Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting fee Speaker/Speaker's Bureau relationship with Bristol Meyers Squibb/Celgene Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Honoraria Consultant relationship with GlaxoSmithKline Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting fee Speaker/Speaker's Bureau relationship with GlaxoSmithKline Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Honoraria Consultant relationship with Janssen Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting fee Speaker/Speaker's Bureau relationship with Janssen Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Honoraria Consultant relationship with Visterra Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting fee Speaker/Speaker's Bureau relationship with Visterra Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Honoraria Consultant relationship with Kyverna Please note: past 36 months Added 07/18/2022 by Ignacio Sanz, value=Consulting fee No relevant relationships by Sunita Sharma No relevant relationships by Colin Swenson No relevant relationships by Robert Swerlick

Background: Within eight weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no validated biomarkers predictive of recurrence during primary infection. This study demonstrates the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Methods: Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Serum and MENSA samples prepared during weeks 1, 2, and 4 following symptom-onset were measured for antibodies specific for ten C. difficile antigens.Results: Among the 46 C. difficile-infected patients, nine (19.5%) experienced recurrence within eight weeks of primary infection. Among the 37 non-recurrent patients, 23 had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred within the first week post-symptom onset, including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (19/37). In contrast, none of the patients who subsequently recurred after hospitalization produced antibodies specific for the three C. difficile toxin antigens. IgA antibodies for the toxin antigens demonstrated the greatest predictive power for protection from recurrence. Discussion: The development of IgG and/or IgA antibodies for three C. difficile toxins in serum and/or MENSA has prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence. Early identification of patients at-risk for recurrence can reduce costs and morbidity.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

BACKGROUND:Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes.METHODS:We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients.RESULTS:We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative.CONCLUSION:Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

ABSTRACT BACKGROUND Clostridioides difficile infections (CDI) have been a challenging and increasing serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitations OBJECTIVE Develop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI. MATERIALS AND METHODS Uninfected controls (n=95) and CDI patients (n=167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens. RESULTS Sixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum. DISCUSSION We describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The MENSA positive-only patients accounted for 30% and may be attributed to nascent antibody secretion in MENSA prior to seroconversion. Conversely, the serum positive-only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays. IMPORTANCE While there was considerable overlap between patients identified through MENSA and serum, both methods detected additional, unique patients. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Interleukin-27 is a pleiotropic cytokine whose reported functions during bacterial infections are debated as an area of active research. To address this, we investigated the role of IL-27 signaling during Staphylococcus aureus osteomyelitis. Clinically, we observed elevated serum IL-27 levels (20-fold higher, p<0.05) in patients with S. aureus osteomyelitis compared to uninfected patients undergoing elective total joint replacement. Remarkably, IL-27 serum levels immediately following septic death were 60-fold higher vs. uninfected patients (p<0.05), suggesting that IL-27 may be a biomarker of end-stage infection and/or cytokine storm. To test this, we hypothesized that IL-27 mediates bacterial clearance during the acute phase of S. aureus osteomyelitis, and subsequently suppresses inflammation to prevent cytokine storm and osteolysis during chronic infection. In mice, we observed that systemic IL-27 delivery by a recombinant adeno-associated viral vector (rAAV-IL-27) ameliorates surgical site soft tissue infection and peri-implant bone loss during the establishment of implant-associated S. aureus osteomyelitis. This effect was not observed in IL-27 receptor α knock-out mice, suggesting a direct role of IL-27/IL-27R signaling on immune and bone cell functions. Examination of IL-27-mediated immune responses via transcriptome analyses of infected tibiae demonstrated that IL-27 is a biphasic cytokine with IL-27/IL-27R activating immunostimulatory responses including Th17, IL-2, TLR, and iNOS signaling early, and subsequently suppressing these pathways during chronic infection. Ex vivo confirmation using murine macrophages revealed that IL-27 co-stimulates TLR signaling to increase the production of nitric oxide, and immunomodulatory cytokines such as IL-10, IL-21, IL-31, and TNF-β, but is not a chemokine. Author Summary Staphylococcus aureus is the most common pathogen in orthopaedic infections, and hard-to-treat (MRSA) strains cause >50% of these infections. Thus, there is an urgent need to develop immunotherapies to treat these life-threatening S. aureus infections. Currently, the role of multifunctional IL-27 on S. aureus osteomyelitis is unknown. In a clinical study, we observed that IL-27 is an important biomarker for identifying S. aureus osteomyelitis patients, and that elevated serum IL-27 levels correlated with adverse clinical outcomes, such as septic death. In our efforts to uncover the underlying mechanisms, we reveal that IL-27 is a biphasic cytokine, activating proinflammatory immune pathways, including Th17 responses, early during acute S. aureus osteomyelitis, and subsequently repressing them during the chronic phase to prevent cytokine storm and bone damage. These results indicate that immune modulation of IL-27/IL-27R signaling could be a viable therapeutic strategy in mitigating S. aureus osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)–scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

Bone infection has received increasing attention in recent years as one of the main outstanding clinical problems in orthopaedic-trauma surgery that has not been successfully addressed. In fact, infection may develop across a spectrum of patient types regardless of the level of perioperative management, including antibiotic prophylaxis. Some of the main unknown factors that may be involved, and the main targets for future intervention, include more accurate and less invasive diagnostic options, more thorough and accurate debridement protocols, and more potent and targeted antimicrobials. The underlying biology dominates the clinical management of bone infections, with features such as biofilm formation, osteolysis and vascularisation being particularly influential. Based on the persistence of this problem, an improved understanding of the basic biology is deemed necessary to enable innovation in the field. Furthermore, from the clinical side, better evidence, documentation and outreach will be required to translate these innovations to the patient. This review presents the findings and progress of the AO Trauma Clinical Priority Program on the topic of bone infection.

Treatment of deep musculoskeletal infection (MSKI) begins with accurate identification of the offending pathogen, surgical excision/debridement, and a course of culture-directed antibiotics. Despite this, the incidence of recurrent infection continues to rise. A major contributor to this is inaccurate or negative initial cultures. Accurate identification of the main pathogen is paramount to treatment success. This is especially important in treating diabetic foot infections (DFIs) with limb salvage efforts. This study seeks to utilize standard culture, next-generation sequencing (NGS), and immunoassay for newly synthesized antibodies (NSA) to Staphylococcus aureus and Streptococcus agalactiae for diagnosis. This is a level II prospective observational study approved by our IRB. Thirty patients > 18 years of age who presented with a DFI and underwent surgical debridement or amputation by a single academic orthopedic surgeon from October 2018 to September 2019 were enrolled. Intraoperative samples were obtained from the base of the wound and sent for culture, NGS, and a peripheral blood sample was obtained at the time of diagnosis. NGS and culture were highly correlated for S. aureus (kappa = 0.86) and S. agalactiae (kappa = 1.0), NSA immunoassay and culture demonstrated a fair correlation for S. aureus (kappa = 0.18) and S. agalactiae (kappa = 0.67), and NGS and NSA immunoassay demonstrated fair correlation for S. aureus (kappa = 0.1667) and S. agalactiae (kappa = 0.67). Our study demonstrates a high concordance between culture and NGS in identifying the dominant pathogen in DFU. NGS may be a useful adjunct in DFI diagnosis.