Functions and Clinical Relevance of Liver-Derived Immunoglobulins.

Liver is the largest internal organ of the body with vital functions. In addition to its endocrine and exocrine activities, liver also plays a pivotal role in the immune system, including haematopoietic functions. Liver parenchymal cells, which are epithelial cells, have been found to possess innate immune functions by expressing pattern-recognition receptors (PRRs), producing complement components, and secreting cytokines. Intriguingly, in recent years, it has been discovered that liver epithelial cells also produce immunoglobulins (Igs), which have long been thought to be produced exclusively by B cells. Notably, even liver epithelial cells from B lymphocyte-deficient mice, including SCID mice and µMT mice, could also produce Igs. Compelling evidence has revealed both the physiological and pathological functions of liver-derived Igs. For instance, liver epithelial cells-derived IgM can serve as a source of natural and specific antibodies that contribute to innate immune responses, while liver-produced IgG can act as a growth factor to promote cell proliferation and survival in normal hepatocytes and hepatocarcinoma. Similar to that in B cells, the toll-like receptor 9 (TLR9)-MyD88 signaling pathway is also actively involved in promoting liver epithelial cells to secrete IgM. Liver-derived Igs could potentially serve as biomarkers, prognostic indicators, and therapeutic targets in the clinical setting, particularly for liver cancers and liver injury. Nevertheless, despite significant advances, much remains unknown about the mechanisms governing Ig transcription in liver cells, as well as the detailed functions of liver-derived Igs and their involvement in diseases and adaptive immunity. Further studies are still needed to reveal these underlying, undefined issues related to the role of liver-derived Igs in both immunity and diseases.

Enzyme-Linked Immunosorbent Assay for Antibodies Against the Tumor-Associated Antigen-Derived Cytotoxic T-Lymphocyte Epitope.

Antibodies serve as crucial indicators of the immune system in clinical tests. In therapeutic cancer vaccines, IgG antibodies against target antigens are vital for immune monitoring. Additionally, assessing baseline antigen-specific immune responses before cancer vaccine administration is possible by measuring IgM and IgG antibodies against the target antigen. To this end, we have developed an enzyme-linked immunosorbent assay (ELISA) system that detects and quantifies serum levels of IgG and IgM antibodies against the WT1 cytotoxic T-lymphocyte epitope peptide. The assay immobilizes the epitope peptide in a microplate to capture antigen-specific antibodies. Here, this article presents the details of our ELISA system to detect and measure antibodies against a tumor-associated antigen-derived cytotoxic T-lymphocyte epitope with high reproducibility. Detecting these antibodies has novel significance in the context of emerging critical roles of B lineage-cells in tumor immunity.

Distinct groups of autoantigens as drivers of ocular adnexal MALT lymphoma pathogenesis.

Chronic B-cell receptor signals incited by cognate antigens are believed to play a crucial role in the pathogenesis of mucosa-associated lymphoid tissue lymphomas. We have explored the immunoglobulin variable regions (IGHV) expressed by 124 ocular adnexal MALT lymphomas (OAML) and tested the in vitro reactivity of recombinant IgM derived from 23 OAMLs. Six of 124 OAMLs (5%) were found to express a high-affinity stereotyped rheumatoid factor. OAMLs have a biased IGHV4-34 usage, which confers intrinsic super auto-antigen reactivity with poly-N-acetyllactosamine (NAL) epitopes, present on cell surface glycoproteins of erythrocytes and B cells. Twenty-one OAMLs (17%) expressed IGHV4-34-encoded B-cell receptors. Five of the 23 recombinant OAML IgMs expressed IGHV4-34, four of which bound to the linear NAL i epitope expressed on B cells but not to the branched NAL I epitope on erythrocytes. One non-IGHV4-34-encoded OAML IgM was also reactive with B cells. Interestingly, three of the 23 OAML IgMs (13%) specifically reacted with proteins of U1-/U-snRNP complexes, which have been implicated as cognate-antigens in various autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue disease. The findings indicate that local autoimmune reactions are instrumental in the pathogenesis of a substantial fraction of OAMLs.

Correlation of Anti-Phosphatidylserine/Prothrombin and Anti-Phosphatidylserine Antibodies with Pregnancy Outcomes.

BACKGROUND: The objective of this study was to investigate the clinical relevance of anti-prothrombin antibodies (aPT) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in relation to pregnancy outcomes and coagulation parameters, as well as immune markers. METHODS: We retrospectively analyzed 477 pregnant women with experienced at least one spontaneous miscarriage who were tested for aPT and aPS/PT antibodies, and compared their clinical characteristics, coagulation indicators, immune biomarkers, and pregnancy outcomes to assess the diagnostic accuracy of these antibodies. RESULTS: We found that the aPT IgG and the aPS/PT IgM were independently associated with increased risk of pregnancy loss, with odds ratios (ORs) of 1.055 (95% confidence interval [CI]: 1.009-1.103, p = 0.017) and 1.041 (95% CI: 1.015-1.067, p = 0.002), respectively. Moreover, we found that the aPS/PT IgM had a higher diagnostic performance than the aPT IgG, as indicated by the AUC of 0.663 and 0.593, respectively. The pregnancy loss rate was positively correlated with the level of aPS/PT IgM, while the aPT IgG is not. We also found that in the pregnancy loss group, aPT IgG showed negative correlations with prothrombin time (PT); aPS/PT IgM showed positive correlations with aPS/PT IgG. However, none of aPT IgG, aPT IgM, aPS/PT IgM, or aPS/PT IgG was related to other adverse pregnancy outcomes, such as preterm delivery, fetal growth restriction (FGR), or preeclampsia (PE). CONCLUSION: Our findings suggest that aPT IgG and aPS/PT IgM are independent risk factors for pregnancy loss, especially aPS/PT IgM, which has a positive linear correlation with pregnancy loss.

Schnitzler Syndrome: Insights into Its Pathogenesis, Clinical Manifestations, and Current Management.

Schnitzler syndrome is a rare disorder characterized by a chronic urticarial rash associated with immunoglobulin M (IgM) monoclonal gammopathy. Schnitzler syndrome shares strong clinicopathologic similarities with monogenic IL-1-mediated autoinflammatory disorders and is now considered an acquired adult-onset autoinflammatory disease. The spectacular effect of interleukin-1 inhibitors demonstrates the key role of this cytokine in the pathogenesis of the disease. However, the physiopathology of Schnitzler syndrome remains elusive, and the main question regarding the relationship between autoinflammatory features and monoclonal gammopathy is still unanswered. The purpose of this narrative review is to describe what is currently known about the pathogenesis of this peculiar disease, as well as to address its diagnosis and management.

Anti-pig antibodies in swine veterinarian serum: Implications for clinical xenotransplantation.

Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non-human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin-29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti-Gal IgM were present in all samples and lower levels of anti-SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non-human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non-Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.

KSHV infection of B cells primes protective T cell responses in humanized mice.

Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.

A Paper-Based Multiplexed Serological Test to Monitor Immunity against SARS-COV-2 Using Machine Learning.

The rapid spread of SARS-CoV-2 caused the COVID-19 pandemic and accelerated vaccine development to prevent the spread of the virus and control the disease. Given the sustained high infectivity and evolution of SARS-CoV-2, there is an ongoing interest in developing COVID-19 serology tests to monitor population-level immunity. To address this critical need, we designed a paper-based multiplexed vertical flow assay (xVFA) using five structural proteins of SARS-CoV-2, detecting IgG and IgM antibodies to monitor changes in COVID-19 immunity levels. Our platform not only tracked longitudinal immunity levels but also categorized COVID-19 immunity into three groups: protected, unprotected, and infected, based on the levels of IgG and IgM antibodies. We operated two xVFAs in parallel to detect IgG and IgM antibodies using a total of 40 µL of human serum sample in <20 min per test. After the assay, images of the paper-based sensor panel were captured using a mobile phone-based custom-designed optical reader and then processed by a neural network-based serodiagnostic algorithm. The serodiagnostic algorithm was trained with 120 measurements/tests and 30 serum samples from 7 randomly selected individuals and was blindly tested with 31 serum samples from 8 different individuals, collected before vaccination as well as after vaccination or infection, achieving an accuracy of 89.5%. The competitive performance of the xVFA, along with its portability, cost-effectiveness, and rapid operation, makes it a promising computational point-of-care (POC) serology test for monitoring COVID-19 immunity, aiding in timely decisions on the administration of booster vaccines and general public health policies to protect vulnerable populations.

Titers of IgG and IgA against SARS-CoV-2 proteins and their association with symptoms in mild COVID-19 infection.

Humoral immunity in COVID-19 includes antibodies (Abs) targeting spike (S) and nucleocapsid (N) SARS-CoV-2 proteins. Antibody levels are known to correlate with disease severity, but titers are poorly reported in mild or asymptomatic cases. Here, we analyzed the titers of IgA and IgG against SARS-CoV-2 proteins in samples from 200 unvaccinated Hospital Workers (HWs) with mild COVID-19 at two time points after infection. We analyzed the relationship between Ab titers and patient characteristics, clinical features, and evolution over time. Significant differences in IgG and IgA titers against N, S1 and S2 proteins were found when samples were segregated according to time T1 after infection, seroprevalence at T1, sex and age of HWs and symptoms at infection. We found that IgM + samples had higher titers of IgG against N antigen and IgA against S1 and S2 antigens than IgM - samples. There were significant correlations between anti-S1 and S2 Abs. Interestingly, IgM + patients with dyspnea had lower titers of IgG and IgA against N, S1 and S2 than those without dyspnea. Comparing T1 and T2, we found that IgA against N, S1 and S2 but only IgG against certain Ag decreased significantly. In conclusion, an association was established between Ab titers and the development of infection symptoms.

Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity.

Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in murine myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity FcγRI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity.

Potential Usefulness of IgA for the Early Detection of SARS-CoV-2 Infection: Comparison With IgM.

Serological testing can be a powerful complementary approach to achieve timely diagnosis of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection, along with nucleic acid detection. Immunoglobulin (Ig) A antibodies are less frequently utilized to detect SARS-CoV-2 infection than IgM and IgG antibodies, even though IgA antibodies play an important role in protective immunity against SARS-CoV-2. This review discusses the differences in kinetics and assay performance between IgA and IgM antibodies and the factors influencing antibody responses. It highlights the potential usefulness of analyzing IgA antibodies for the early detection of SARS-CoV-2 infection. The early appearance of IgA and the high sensitivity of IgA-based immunoassays can aid in diagnosing coronavirus disease 2019. However, because of cross-reactivity, it is important to recognize the only moderate specificity of the early detection of SARS-CoV-2 IgA antibodies against spike antigens. Either the analysis of antibodies targeting the nucleocapsid antigen or a combination of antibodies against the nucleocapsid and spike antigens may strengthen the accuracy of serological evaluation.

Prevalence of anti-lymphocyte IgM autoantibodies driving complement activation in COVID-19 patients.

Introduction: COVID-19 patients can develop autoantibodies against a variety of secreted and membrane proteins, including some expressed on lymphocytes. However, it is unclear what proportion of patients might develop anti-lymphocyte antibodies (ALAb) and what functional relevance they might have. Methods: We evaluated the presence and lytic function of ALAb in the sera of a cohort of 85 COVID-19 patients (68 unvaccinated and 17 vaccinated) assigned to mild (N=63), or moderate/severe disease (N=22) groups. Thirty-seven patients were followed-up after recovery. We also analyzed in vivo complement deposition on COVID-19 patients' lymphocytes and examined its correlation with lymphocyte numbers during acute disease. Results: Compared with healthy donors (HD), patients had an increased prevalence of IgM ALAb, which was significantly higher in moderate/severe disease patients and persisted after recovery. Sera from IgM ALAb+ patients exhibited complement-dependent cytotoxicity (CDC) against HD lymphocytes. Complement protein C3b deposition on patients' CD4 T cells was inversely correlated with CD4 T cell numbers. This correlation was stronger in moderate/severe disease patients. Discussion: IgM ALAb and complement activation against lymphocytes may contribute to the acute lymphopenia observed in COVID-19 patients.

Infrared spectroscopy as a predictive tool for the severity of COVID-19 using patient's saliva: A strategy to avoid hyperinflammation.

Discriminate the severity level of COVID-19 disease is still a challenge. Here we investigate the capability of micro-infrared absorption spectroscopy (micro-FTIR) to probe COVID-19 severity level and predict hyperinflammation, correlating the assigned vibrational data to relevant biomolecules related to the immune system. Saliva of 184 patients was analysed by ELISA assay (Hepcidin) and micro-FTIR. Vibrational bands related to IgM and IgA can discriminate healthy from Severe individuals (sensitivity ≥ 0.749, specificity ≥ 0.945) and are less effective in discriminating Mild or Moderate individuals from the Severe group (sensitivity ≥ 0.628, specificity ≥ 0.867). Analysis of the second derivative of spectra probed increased levels of IL-6 in the saliva a key additional information for the degree of severity prediction. Because the model discriminates all the groups regarding the Severe group, it predicts an intense state of inflammation based on FTIR analysis. It is a powerful tool for predicting hyperinflammation conditions related to SARS-CoV-2 infection and may be an ally in implementing drugs or therapeutic approaches to manage COVID-19 in the Severe stage in healthcare facilities.

The single-dose Janssen Ad26.COV2.S COVID-19 vaccine elicited robust and persistent anti-spike IgG antibody responses in a 12-month Ugandan cohort.

Introduction: The study investigation examined the immune response to the Janssen Ad26.COV2.S COVID-19 vaccine within a Ugandan cohort, specifically targeting antibodies directed against spike (S) and nucleocapsid (N) proteins. We aimed to examine the durability and robustness of the induced antibody response while also assessing occurrences of breakthrough infections and previous anti-Spike seropositivity to SARS-CoV-2. Methods: The study included 319 specimens collected over 12 months from 60 vaccinees aged 18 to 64. Binding antibodies were quantified using a validated ELISA method to measure SARS-CoV-2-specific IgG, IgM, and IgA levels against the S and N proteins. Results: The results showed that baseline seropositivity for S-IgG was high at 67%, increasing to 98% by day 14 and consistently stayed above 95% for up to 12 months. However, S-IgM responses remained suboptimal. A raised S-IgA seropositivity rate was seen that doubled from 40% at baseline to 86% just two weeks following the initial vaccine dose, indicating sustained and robust peripheral immunity. An increase in N-IgG levels at nine months post-vaccination suggested breakthrough infections in eight cases. Baseline cross-reactivity influenced spike-directed antibody responses, with individuals harbouring S-IgG antibodies showing notably higher responses. Discussion: Robust and long lasting vaccine and infection-induced immune responses were observed, with significant implications for regions where administering subsequent doses poses logistical challenges.

Rational design and production of a chimeric antigen targeting Zika virus that induces neutralizing antibodies in mice.

The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome. Currently, there is no specific treatment or vaccine approved to combat this disease, and thus, developing safe and effective vaccines is a relevant goal. In this study, a multi-epitope protein called rpZDIII was designed based on a series of ZIKV antigenic sequences, a bacterial carrier, and linkers. The analysis of the predicted 3D structure of the rpZDIII chimeric antigen was performed on the AlphaFold 2 server, and it was produced in E. coli and purified from inclusion bodies, followed by solubilization and refolding processes. The yield achieved for rpZDIII was 11 mg/L in terms of pure soluble recombinant protein per liter of fermentation. rpZDIII was deemed immunogenic since it induced serum IgG and IgM responses in mice upon subcutaneous immunization in a three-dose scheme. Moreover, sera from mice immunized with rpZDIII showed neutralizing activity against ZIKV. Therefore, this study reveals rpZDIII as a promising immunogen for the development of a rationally designed multi-epitope vaccine against ZIKV, and completion of its preclinical evaluation is guaranteed.

Assessment of the 2023 ACR/EULAR antiphospholipid syndrome classification criteria in a Chinese cohort: Impact on clinical practice.

OBJECTIVES: To evaluate the effectiveness of the 2023 ACR/EULAR criteria for antiphospholipid syndrome (APS) in a Chinese cohort, and compare them with the Sapporo and revised Sapporo criteria. METHODS: A cohort comprising 436 patients diagnosed with APS and 514 control subjects was enrolled, including 83 with seronegative APS and 86 classified as antiphospholipid antibody (aPL) carriers. We assessed IgG and IgM anticardiolipin antibodies (aCL) and anti-ß2-glycoprotein I (aß2GPI) antibodies using ELISA, along with a systematic collection of lupus anticoagulant data. Subsequently, we compared the sensitivity and specificity across the three classification criteria. RESULTS: The 2023 ACR/EULAR criteria exhibited improved specificity at 98 %, surpassing the revised Sapporo (90 %) and original Sapporo (91 %) criteria. However, this came with decreased sensitivity at 82 %, in contrast to higher sensitivities in the revised Sapporo (98 %) and Sapporo (91 %) criteria. Examining individual components sheds light on the scoring system's rationale within the new criteria. The inclusion of microvascular thrombosis, cardiac valve disease, and thrombocytopenia improved the identification of nine patients previously classified as "probable APS". Insufficient scoring in 78 previously diagnosed APS individuals was linked to traditional risk factor evaluations for thrombotic events, the emphasis on determining whether obstetric events are linked to severe preeclampsia (PEC) or placental insufficiency (PI), and the lower scores assigned to IgM aCL and/or aß2GPI antibody. Seronegative APS remained a challenge, as non-criteria aPL and other methods were not included. CONCLUSIONS: The new criteria presented notable advancements in specificity. This study provides detailed insights into the strengths and possible challenges of the 2023 ACR/EULAR criteria, enhancing our understanding of their impact on clinical practice.

Optimized Enzyme-Linked Immunosorbent Assay for Anti-PEG Antibody Detection in Healthy Donors and Patients Treated with PEGylated Liposomal Doxorubicin.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG5000, as opposed to bovine serum albumin-PEG20000, and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population.

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

Autoantibodies against complement proteins in patients with antiphospholipid syndrome: Prevalence and clinical associations.

Seventy-seven patients with antiphospholipid syndrome were tested for autoantibodies against C1q, C3, FB, FH, and C4bp. Fifty-seven patients had at least one anti-complement antibody. IgM anti-FH positivity was associated with thrombosis when anti-C3 and anti-FB were, negatively or positively, associated with various noncriteria manifestations of antiphospholipid syndrome.

Engineered IgM and IgG cleaving enzymes for mitigating antibody neutralization and complement activation in AAV gene transfer.

Systemic dosing of adeno-associated viral (AAV) vectors poses potential risk of adverse side effects including complement activation triggered by anti-capsid immunity. Due to the multifactorial nature of toxicities observed in this setting, a wide spectrum of immune modulatory regimens are being investigated in the clinic. Here, we discover an IgM cleaving enzyme (IceM) that degrades human IgM, a key trigger in the anti-AAV immune cascade. We then engineer a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. IceMG cleaves B cell surface antigen receptors and inactivates phospholipase gamma signaling in vitro. Importantly, IceMG is more effective at inhibiting complement activation compared with an IgG cleaving enzyme alone. Upon IV dosing, IceMG rapidly and reversibly clears circulating IgM and IgG in macaques. Antisera from these animals treated with IceMG shows decreased ability to neutralize AAV and activate complement. Consistently, pre-conditioning with IceMG restores AAV transduction in mice passively immunized with human antisera. Thus, IgM cleaving enzymes show promise in simultaneously addressing multiple aspects of anti-AAV immunity mediated by B cells, circulating antibodies and complement. These studies have implications for improving safety of AAV gene therapies and possibly broader applications including organ transplantation and autoimmune diseases.