An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, which results in its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule bound M87 in the crystal, but in solution recruited additional LL-37 molecules, consistent with a protein trap neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain, and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

DENV-Mimetic Polymersome Nanoparticles Bearing Multi-Epitope Lipopeptides Antigen as the Next-Generation Dengue Vaccine

Dengue remains a severe threat to public health. The safety and efficacy of the licensed dengue vaccine is not clinically satisfactory, which necessitate the need of new approach in designing an effective dengue vaccine without eliciting adverse reaction. Herein, we have designed a lipidated multi-epitope peptide vaccine (LipoDV) that can elicit highly targeted humoral and cell-mediated immune responses. To improve its immunogenicity, LipoDV was presented on the surface of MPLA-functionalized polymersome nanoparticles (PNs-LipoDV-MPLA). The as-constructed vaccine delivery platform resembles the structural morphology of DENV owing to its spherical nanoscale particle size and surface immunostimulatory properties given by LipoDV and MPLA that emulating the functional role of DENV E and prM/M proteins respectively. A proof-of-concept study demonstrated that BALB/c mice immunized with PNs-LipoDV-MPLA induced a stronger antigen-specific antibody response with an enhanced cell-mediated immunity as characterized by the elevated IFN-γ secretion in comparison to other tested vaccine candidates which possess a lesser structural trait of DENV. The DENV-mimicking nanoparticles vaccine exhibited negligible toxicity as analyzed by hemolytic test, MTT assay, histopathological examination and abnormal toxicity test on immunized mice. Collectively, our study provides a strong foundation in designing an effective peptide-based vaccine delivery platform against DENV infection.

CHARM: COVID-19 Health Action Response for Marines – association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and then bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were asked to enroll in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p=0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic subjects; however, the difference was not statistically significant (p=0.06) for symptomatic subjects (N pool: 44.4%; M pool: 25.9%). In asymptomatic participants IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but symptomatic participants, IFN-γ responses were more frequent to the S pool (75.0%) than N pool (33.3%) and M pool (33.3%). The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-γ and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

Distinct serotypes of streptococcal M proteins mediate fibrinogen-dependent platelet activation and pro-inflammatory effects

Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.

Will Mass Spectrometry Replace Current Techniques for Both Routine Monitoring and MRD Detection in Multiple Myeloma?

In recent years, mass spectrometry has been increasingly used for the detection of monoclonal proteins in serum. Mass spectrometry is more analytically sensitive than serum protein electrophoresis and immunofixation, can help distinguish therapeutic monoclonal antibodies from M-proteins, and can detect the presence of post-translational modifications. Mass spectrometry also shows promise as a less-invasive, peripheral-blood-based test for detecting minimal residual disease in multiple myeloma. Studies comparing the clinical utility of mass spectrometry to current blood- and bone-marrow-based techniques have been conducted. Although still primarily limited to research settings, clinical laboratories are starting to adopt this technique for patient care. This review will discuss the current status of mass spectrometry testing for multiple myeloma, the benefits and challenges of this technique, and how it may be incorporated into clinical practice in the future.

Design of M protein immunogens to elicit broadly reactive antibodies against Streptococcus pyogenes

M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (Strep A) are immunodominant targets of opsonizing antibodies. However, the antigenic sequence variability of the M protein into >220 M types has limited its utility as a vaccine immunogen, as antibody recognition is usually type-specific. At present no vaccine against Strep A exists. Unlike type-specific antibodies, C4BP binds type-promiscuously to M proteins. We recently showed that this was due to a three-dimensional (3D) pattern of amino acids that is conserved in numerous M types. We hypothesized that M protein immunogens biased towards the 3D pattern and away from variable sequences would evoke a broadly protective response. We show here that an immunogen containing only 34 amino acids of M2 protein retained C4BP-binding and was sufficient to evoke antibodies that were cross-reactive and opsonophagocytic against multiple M types. These proof-of-principle experiments provide significant evidence that an essential Strep A virulence trait (i.e., C4BP binding) can be targeted in the design of an immunogen that evokes a broadly protective response.

Single Molecule Real-Time Sequencing of the M Protein (SMaRT M-Seq): Toward Personalized Medicine Approaches in Monoclonal Gammopathies

Introduction In patients affected by monoclonal gammopathies, tumoral B cells or plasma cells secrete a monoclonal antibody (termed M protein), which can be used to track the presence of the tumor itself. Moreover, the M protein can directly cause potentially life-threatening organ damage, which is dictated by the specific, patient's unique clonal light and/or heavy chain, as in patients affected by immunoglobulin light chain (AL) amyloidosis. Yet, the current paradigm in the diagnosis and management of these conditions treats the M protein as a simple tumor biomarker to be identified/quantified. Patients' specific M protein sequences remain mostly undefined and molecular mechanisms underlying M-protein related clinical manifestations are largely obscure. Methods By combining the unbiased amplification of expressed immunoglobulin genes with long-read, single molecule real-time DNA sequencing and bioinformatics analyses, we have established a method to identify the full-length sequence of the variable region of expressed immunoglobulin genes and to rank the obtained sequences based on their relative abundance, thus enabling the identification of the full-length variable sequence of M protein genes from a high number of patients analysed in parallel. Results The assay, which we termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), has undergone an extensive technical validation. Sequencing of contrived bone marrow samples generated through serial dilutions of plasma cell lines into control bone marrow, as well as sequencing of bona fide bone marrow samples from AL patients and comparison with gold-standard techniques of immunoglobulin gene sequencing showed: 100% sequence-accuracy at the individual base-pair level; High repeatability (CV<0.8% for sequencing of pentaplicates) in defining the molecular clonal size (i.e. the fraction of total immunoglobulin sequences coinciding with the clonal sequence); A high sensitivity in identifying clonal immunoglobulin sequences (10 -3 when employing low-coverage sequencing on multiple, pooled samples). Noteworthy, SMaRT M Seq was applied to a cohort of 86 consecutive patients with AL amyloidosis (17 κ and 69 λ; median BMPC infiltration 9%, IQR 6-13%; median dFLC 176 mg/L, IQR 75-370 mg/L), including cases with small clonal burden and M protein which was undetectable with conventional M protein studies. A full-length sequence of the variable region of the clonal light chain was obtained in all patients (median molecular clonal size of 88.3%, IQR: 70.7 - 93%). The most common κ germline genes were IGKV1-33 and IGKV4-01 (24% each of the 17 κ AL patients), and the most common λ germline genes were IGLV6-57 (26% of the 69 λ AL patients), IGLV2-14 (17%), IGLV3-01 (17%) and IGLV1-44 (10%). The most frequent λ and κ germline genes together (IGLV6-57, IGLV2-14, IGLV3-01, IGLV1-44, IGKV1-33 and IGKV4-01) accounted for 66% of all the clones. Germline gene usage correlated with selected clinical features. Sequence information was then exploited to improve mass spectrometry-based amyloid typing on fat pad aspirates and to enable the sensitive detection of clonotypic sequences using short-read DNA sequencing of the involved light chain isotype (up to 10 -7 dilution). Conclusions We have established SMaRT M-Seq as a novel valuable assay to reliably identify the full-length variable sequence of M proteins. SMaRT M-Seq has undergone extensive technical validation, showing high accuracy, repeatability and sensitivity. The latter is determined by the number of reads analyzed per sample. This is in turn dictated by the sequencing output of the employed sequencing platform, and by the number of pooled samples analyzed in a given sequencing round, thus proving to be scalable. Even when analyzing multiple samples on a sequencing platform with low sequencing output, the achieved sensitivity of SMaRT M-Seq significantly exceeds the requirements for the identification of clonal B cells/plasma cells in patients with AL amyloidosis. Sequencing disease-associated M proteins from large cohorts of patients has the potential to uncover molecular mechanisms of M protein-related clinical manifestations which have remained largely unexplored so far, and could enable approaches of personalized medicine for the sensitive detection of patients' specific M proteins at diagnosis and after anti-clonal therapy. Disclosures Milani: Celgene: Other: Travel support; Janssen-Cilag: Honoraria. Fazio: Janseen: Honoraria. Petrucci: GSK: Honoraria, Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Janssen-Cilag: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board; Karyopharm: Honoraria, Other: Advisory Board. Palladini: Pfizer: Honoraria; Siemens: Honoraria; Janssen Global Services: Honoraria, Other: advisory board fees. Nuvolone: Janssen-Cilag: Honoraria; Oncopeptides, Inc.: Research Funding.

Screening for Monoclonal Gammopathy of Undetermined Significance: A Population-Based Randomized Clinical Trial. First Results from the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) Study

Background: Cancer screening is performed worldwide for several malignancies. Monoclonal gammopathy of undetermined significance (MGUS) precedes multiple myeloma (MM) and related lymphoproliferative disorders (LP). However, less than 5% of all MM patients are diagnosed during their precursor state and individuals who develop MM while being monitored for MGUS have better overall survival and fewer complications, compared to MM patients diagnosed without knowledge of MGUS. Thus, population-based screening for MGUS could identify candidates for early treatment of MM/LPs. To evaluate whether systematic screening is beneficial, we performed the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study, the first population-based screening study for MGUS that includes a randomized clinical trial (RCT) of follow-up and treatment strategies. Methods: All living residents of Iceland on September 9th, 2016 who were born before 1976 (N=148,708) were invited to participate. Of those, 80,759 (54.3%) provided informed consent for screening. Serum samples were collected from participants alongside clinical blood sampling in the Icelandic health service between September 2016 and the end of 2020. All samples were shipped to the Binding Site in Birmingham, UK, for screening. Samples were tested for M-proteins by capillary zone electrophoresis and immunofixation electrophoresis performed to confirm and characterize suspected M-proteins. Free light chains (FLCs) were measured using the FreeLite® assay. Individuals with a previous diagnosis of MM/LPs/MGUS (N=237) were excluded. Per protocol and informed consent, participants with MGUS were randomized to one of the three study arms: Arm 1 where participants are not contacted; Arm 2 where individuals are followed based on current guidelines; and Arm 3 where individuals are followed with a more intensive diagnostic and monitoring strategy. Participants who progress are offered early treatment. All participants repeatedly answered questionnaires on quality of life and mental health. Results: A total of 75,422 participants (93.4%) provided a serum sample for screening. Of those, 3,725 (4.9%) had MGUS. The prevalence of MGUS was dependent on age with 2.3%, 6.2%, and 12.9% diagnosed in age groups 40-59, 60-79, and 80-103 years, respectively. The prevalence of MGUS was higher in males, 5.9% vs 4.1% (p<0.0001). Most individuals with MGUS had either low-risk (38%) or low-intermediate (36%) risk MGUS, followed by high-intermediate (26%) risk MGUS. High-risk MGUS was only present in 0.2% of MGUS cases (n=9). The RCT includes 3,487 newly diagnosed MGUS individuals with 1164, 1159, and 1164 individuals in arms 1, 2 and 3, respectively (Table). The median age at diagnosis was 69 years in arms 1 and 2, and 70 years in arm 3. Females constituted 45.9% and the isotypes were IgG (50%), IgA (10%), IgM (18%) and biclonal (8%). The median M-protein concentration was 0.34 g/dL. A total of 428 light-chain MGUS cases were randomized. The demographic distribution was well balanced between the three arms. After a median follow-up of 3 years, 194 patients in the RCT have been diagnosed with any LP: 9 in arm 1, 92 in arm 2, and 133 in arm 3 (p<0.001). The participants in arm 1 were diagnosed with smoldering Waldenström's macroglobulinemia (SWM)(N=2), WM (N=2), chronic lymphocytic leukemia (CLL) (N=1), and MM (N=4). Participants in arm 2 were diagnosed with amyloidosis (N=1), SWM (N=18), WM (N=2), CLL (N=2), non-Hodgkin lymphoma (NHL) (N=1), smoldering MM (SMM) (N=56), and MM (N=12). Participants in arm 3 were diagnosed with amyloidosis (N=2), SWM (N=22), CLL (N=5), NHL (N=6), SMM (N=82), and MM (N=16). The difference between study arms was statistically significant for all LPs combined, and for SWM, SMM, and MM (Table). Conclusion: In this large prospective population-based screening study including >75,000 screened persons, we have identified 3,725 individuals with monoclonal gammopathy. In the RCT, after 3 years of follow-up, we show that active screening identifies significantly higher number of individuals with full-blown malignancy and smoldering disease, illustrating the fact that early detection and intervention is achievable. Although our findings are encouraging, until final results of the iStopMM study become available, including data on survival and quality of life, we advise against systematic MGUS screening in healthy individuals. Figure 1 Figure 1. Disclosures Kristinsson: Amgen: Research Funding; Celgene: Research Funding. Kampanis: The Binding Site: Current Employment. Hultcrantz: Curio Science LLC: Consultancy; Daiichi Sankyo: Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Intellisphere LLC: Consultancy. Durie: Amgen: Other: fees from non-CME/CE services ; Amgen, Celgene/Bristol-Myers Squibb, Janssen, and Takeda: Consultancy. Harding: The Binding Site: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Landgren: Janssen: Research Funding; Celgene: Research Funding; Janssen: Honoraria; Janssen: Other: IDMC; Amgen: Honoraria; Takeda: Other: IDMC; Amgen: Research Funding; GSK: Honoraria.

Estimating Selection Bias in Previous Monoclonal Gammopathy of Undetermined Significance Research - the Importance of Screening: Results from the Population-Based Screening Study Iceland Screens, Treats or Prevents Multiple Myeloma (iStopMM)

Introduction Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder preceding multiple myeloma and related disorders, present in 4.2% of the population over the age of 50. Although usually asymptomatic, MGUS has been associated with various health-related problems, including thrombosis, infections, fractures, neuropathy, and death. Because MGUS is asymptomatic, its diagnosis is typically incidental, during clinical workup for unrelated medical issues, and therefore most individuals remain undiagnosed. Consequently, MGUS cohorts in past studies may have suffered from more comorbidities than the actual population with MGUS. This might have introduced selection bias in previous studies on MGUS, which extent has not been studied in a systematic way. Therefore, previously reported associations between MGUS and various medical issues might not be as profound as formerly observed. The aim of this study was to compare characteristics of incidentally diagnosed MGUS versus MGUS diagnosed by systematic screening, with particular focus on demographics, comorbidities, and MGUS-related factors. Methods The study is based on the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study. iStopMM is a population-based screening study for MGUS and a randomized controlled trial of follow-up strategies that has included 54% (n = 80,759) of the Icelandic population above 40 years of age. In total, 75,422 participants were screened for MGUS by serum protein electrophoresis (SPEP) and free light chain (FLC) assay. Information on which individuals had incidentally diagnosed MGUS (clinical MGUS) prior to participation in iStopMM were gathered from the Icelandic cancer registry and laboratory results from Landspítali University Hospital and Læknasetrið, the only laboratories in Iceland that perform SPEP. M-protein concentration, MGUS isotype and FLC ratio were obtained from the original screening samples. Comorbidity data was acquired from two high-quality national registries: Hospital Discharge Register and Register of Primary Health Care Contacts, with >95% completeness and accuracy. MGUS diagnosed by screening was further classified into MGUS with or without M-proteins (light-chain MGUS). Those with clinical MGUS were used as the reference group in all analyses. Since all individuals with clinical MGUS had M-proteins, individuals with light-chain MGUS were excluded from this study. T-test and chi-square test were used for demographic comparison; linear regression adjusting for sex and age for continuous variables, and logistic regression adjusting for sex and age for comparison of categorical variables, regarding MGUS-related factors and comorbidities. Results The study cohort consisted of 3,300 individuals who had MGUS with M-proteins; 224 individuals with clinical MGUS and 3,076 with screened MGUS. The clinical MGUS group was significantly older (p <0.01), more likely to live in the capital area of Iceland (p 0.02), and had a 0.14 g/dL higher mean M-protein concentration (95% confidence interval [95% CI] 0.10-0.19 g/dL, p <0.001) than those with screened MGUS. Individuals with clinical MGUS were also 1.73 times more likely to have a comorbidity (odds ratio [OR] 1.73, 95% CI 1.14-2.72, p 0.01) than those with screened MGUS, reflected in a significantly higher mean number of comorbidities (2.79 vs. 2.09, p <0.001). Finally, clinical MGUS were significantly more likely to have arrhythmias (OR 1.45, p 0.05), chronic kidney diseases (OR 2.42, p <0.001), endocrine diseases (OR 1.82, p <0.001), heart failure (OR 2.60, p <0.001), neurological diseases (OR 3.07, p <0.001) or rheumatological diseases (OR 3.25, p <0.001). Discussion In this large population-based study including 75,000 screened individuals, we found clinical MGUS cases to be older, more likely to live in Iceland's capital area, and have a higher M-protein concentration than those found to have MGUS while screened on the iStopMM study. Individuals with clinical MGUS also had a higher number of underlying comorbidities and were 1.5-3.3 times more likely to suffer from arrhythmias, chronic kidney diseases, endocrine disorders, heart failure, neurological diseases, and rheumatological diseases. Our findings highlight the importance of screening studies to evaluate the true epidemiological and biological implications of MGUS and suggest selection bias in prior studies. Figure 1 Figure 1. Disclosures Kampanis: The Binding Site: Current Employment. Hultcrantz: Daiichi Sankyo: Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Curio Science LLC: Consultancy; Amgen: Research Funding; Intellisphere LLC: Consultancy. Durie: Amgen, Celgene/Bristol-Myers Squibb, Janssen, and Takeda: Consultancy; Amgen: Other: fees from non-CME/CE services . Harding: The Binding Site: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Landgren: Janssen: Honoraria; Janssen: Other: IDMC; Takeda: Other: IDMC; Janssen: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Amgen: Research Funding; GSK: Honoraria. Kristinsson: Amgen: Research Funding; Celgene: Research Funding.