XAV-19, a Swine Glyco-Humanized Polyclonal Antibody Against SARS-CoV-2 Spike Receptor-Binding Domain, Targets Multiple Epitopes and Broadly Neutralizes Variants.

Amino acid substitutions and deletions in the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBMs), in direct contact sites with the angiotensin converting enzyme-2 (ACE-2). Therefore, in Spike/ACE-2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study on Vero E6 cells conducted over 1 month, no mutation was associated with the addition of increasing doses of XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE-2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (COVID-19) including the different variants of concern identified so far.

Sequential immunization of macaques elicits heterologous neutralizing antibodies targeting the V3-glycan patch of HIV-1 Env.

Broadly neutralizing antibodies (bNAbs) against HIV-1 develop after prolonged virus and antibody coevolution. Previous studies showed that sequential immunization with a V3-glycan patch germline-targeting HIV-1 envelope trimer (Env) followed by variant Envs can reproduce this process in mice carrying V3-glycan bNAb precursor B cells. However, eliciting bNAbs in animals with polyclonal antibody repertoires is more difficult. We used a V3-glycan immunogen multimerized on virus-like particles (VLPs), followed by boosting with increasingly native-like Env-VLPs, to elicit heterologous neutralizing antibodies in nonhuman primates (NHPs). Structures of antibody/Env complexes after prime and boost vaccinations demonstrated target epitope recognition with apparent maturation to accommodate glycans. However, we also observed increasing off-target antibodies with boosting. Eight vaccinated NHPs were subsequently challenged with simian-human immunodeficiency virus (SHIV), and seven of eight animals became infected. The single NHP that remained uninfected after viral challenge exhibited one of the lowest neutralization titers against the challenge virus. These results demonstrate that more potent heterologous neutralization resulting from sequential immunization is necessary for protection in this animal model. Thus, improved prime-boost regimens to increase bNAb potency and stimulate other immune protection mechanisms are essential for developing anti­HIV-1 vaccines.

Comparison of seven heterophile antibody assays for laboratory diagnosis of infectious mononucleosis in pediatric patients.

Heterophile antibody assays have been used to aid the diagnosis of infectious mononucleosis caused by the Epstein-Barr virus. Seven commercially available assays currently widely utilized in clinical laboratories were compared in this study. Variable performance characteristics and assay times are observed, and these pieces of data may assist clinical laboratories in assay selection and result interpretation.

Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Dsg2 Upregulation as a Rescue Mechanism in Pemphigus.

In pemphigus vulgaris (PV), autoantibodies directed against the desmosomal cadherin desmoglein (Dsg) 3 cause loss of intercellular adhesion. It is known that Dsg3 interactions are directly inhibited by autoantibody binding and that Dsg2 is upregulated in epidermis of PV patients. Here, we investigated whether heterophilic Dsg2-Dsg3 interactions occur and would modulate PV pathogenesis. Dsg2 was upregulated in PV patients' biopsies and in a human ex vivo pemphigus skin model. Immunoprecipitation and cell-free atomic force microscopy (AFM) experiments demonstrated heterophilic Dsg2-Dsg3 interactions. Similarly, in Dsg3-deficient keratinocytes with severely disturbed intercellular adhesion Dsg2 was upregulated in the desmosome containing fraction. AFM revealed that Dsg2-Dsg3 heterophilic interactions showed binding frequency, strength, Ca2+-dependency and catch-bond behavior comparable to homophilic Dsg3-Dsg3 or homophilic Dsg2-Dsg2 interactions. However, heterophilic Dsg2-Dsg3 interactions had a longer lifetime compared to homophilic Dsg2-Dsg2 interactions and PV autoantibody-induced direct inhibition was significantly less pronounced for heterophilic Dsg2-Dsg3 interactions compared to homophilic Dsg3 interactions. In contrast, a monoclonal anti-Dsg2 inhibitory antibody reduced heterophilic Dsg2-Dsg3 and homophilic Dsg2-Dsg2 binding to the same degree and further impaired intercellular adhesion in Dsg3-deficient keratinocytes. Taken together, the data demonstrate that Dsg2 undergoes heterophilic interactions with Dsg3, which may attenuate autoantibody-induced loss of keratinocyte adhesion in pemphigus.

Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout).

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

Relapse of Nephrotic Syndrome after Adrenocorticotropic Hormone-Induced Remission: Implications of Adrenocorticotropic Hormone Antibodies.

BACKGROUND: Prolonged use of corticosteroids continues to be the mainstay in the management of most proteinuric glomerulopathies, but is limited by extensive side effects. Alternative medications such as adrenocorticotropic hormone (ACTH) have been recently used to treat refractory glomerulopathies and have shown superior outcomes when compared with steroids. However, the clinical responsiveness to ACTH therapy varies considerably with a number of patients exhibiting de novo or acquired resistance. The underlying mechanism remains unknown. METHODS: A patient with steroid-dependent focal segmental glomerulosclerosis (FSGS) developed severe steroid side effects impacting quality of life and was converted to repository porcine ACTH therapy. Immediate response in the form of remission of nephrotic syndrome was noted followed by relapse in 10 weeks. Suspecting the role of some ACTH-antagonizing factors, the patient's serum was examined. RESULTS: Immunoblot-based antibody assay revealed high titers of de novo IgG antibodies in the patient's serum that were reactive to the porcine corticotropin with negligible cross-reactivity to human corticotropin. In vitro, in cultured B16 melanoma cells that express abundant melanocortin receptors, addition of the patient's serum substantially abrogated the porcine corticotropin triggered signaling activity of the melanocortinergic pathway, marked by phosphorylation of glycogen synthase kinase 3ß, thus suggesting a mitigating effect on the biological functionality of porcine corticotropin. CONCLUSION: ACTH is a useful alternative therapeutic modality for refractory proteinuric glomerulopathies like FSGS. However, as quintessential therapeutic biologics, natural ACTH, regardless of purity and origin, is inevitably antigenic and may cause the formation of neutralizing antibodies in some sensitive patients, followed by resistance to ACTH therapy. It is imperative to develop ACTH analogues with less immunogenicity for improving its responsiveness in patients with glomerular diseases.

Xenoantibodies and Complement Activity Determinations by Flow Cytometry in Pig-to-Primate Xenotransplantation.

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

Studying Xenograft Rejection of Bioprosthetic Heart Valves.

Millions of patients with valvular heart disease have benefitted from heart valve replacement since the procedure was first introduced in the 1960s; however, there are still many patients who get early structural valve deterioration (SVD) of their bioprosthetic heart valves (BHV). BHV are porcine, bovine, or equine tissues that have been glutaraldehyde fixed to preserve the tissue and presumably make the tissue immunologically inert. These glutaraldehyde-fixed BHV with anti-calcification treatments last long periods of time in older adults but develop early SVD in younger patients. The consensus at present is that the early SVD in younger patients is due to more "wear and tear" of the valves and higher calcium turnover in younger patients. However, as younger patients likely have a more robust immune system than older adults, there is a new hypothesis that BHV xenografts may undergo xenograft rejection, and this may contribute to the early SVD seen in younger patients.At present, the technology to noninvasively study in vivo whether an implanted BHV in a human patient is undergoing rejection is not available. Thus, a small animal discordant xenotransplant model in young rodents (to match the young patient getting a pig/bovine/equine BHV) was developed to study whether the hypothesis that glutaraldehyde-fixed BHV undergo xenograft rejection had any merit. In this chapter, we describe our model and its merits and the results of our investigations. Our work provides clear evidence of xenograft rejection in glutaraldehyde-fixed tissue, and our small animal model offers an opportunity to study this process in detail.

Interpretation of EBV serology for human body material donors: Is there a need for early antigen IgG and heterophile antibodies testing?

In this report we evaluated a diagnostic algorithm, proposed by the Belgian Superior Health Council, to detect acute and past Epstein-Barr virus (EBV) infections by means of serology in donors of human body material for transplantation. The available EBV serology parameters were tested on eighty serum samples on three random access analysers: Architect i2000 SR, Liasion XL and BioPlex 2200. The EBV sero-status was determined according to the proposed algorithm and results were compared between the different analysers. Seventy one % of the samples gave concordant interpretations on the three analysers. Most of the discordant results were attributable to early antigen (EA) IgG. The knowledge of the EA IgG and heterophile antibodies (HA) IgM status provided only limited added value and was only useful to distinguish between a very early acute infection and false positivity of viral capsid antigen IgM. The diagnostic algorithm proposed by the Belgian Superior Health Council is merely directive and each individual lab remains responsible for the interpretation and implementation of test combinations for the detection of EBV infections. Our study shows the limited added value of testing for EA IgG and HA IgM, based both on clinical and technical performance.

PTH Immunoassay Interference Due to Human Anti-Mouse Antibodies in a Subject With Obesity With Normal Parathyroid Function.

CONTEXT: Immunoassay interference has been most often found with prolactin measurement. However, only few data exist on immunoassay interference for other hormones. CASE DESCRIPTION: A 36-year-old woman with obesity (body mass index, 31 kg/m2) had regularly attended our endocrine unit for type 2 diabetes therapy. When she was included as a control subject in a study for obesity management, detailed laboratory testing was performed, including PTH. In the absence of clinical symptoms, she presented with normal calcium, phosphate, and vitamin D levels. However, the PTH levels were >5000 ng/L. These results were obtained using the Roche Elecsys electrochemiluminescence assay. Repeated measurements with this assay (mouse antibody) led to the same findings. However, using an Euroimmun assay (goat antibody), the exact PTH values were measured at 18.0 ng/L. After pretreatment with a heterophilic antibody blocking reagent, the results of the Roche assay had decreased to a normal level. This phenomenon was explained by the detection of human anti-mouse antibodies in the proband's serum. CONCLUSIONS: In cases of prolactin immunoassay interference, endogenous antibodies will bind to the hormone in vivo, resulting in complexes of a high molecular weight that are less efficiently cleared by the kidneys and, thus, accumulate in the blood. In contrast, the PTH values >5000 ng/L detected in our subject most likely had resulted from the specific interference of the human anti-mouse antibodies present in the proband's serum with the assay antibody, resulting in artificial stimulation of the Roche assay detection system ex vivo.

Heterophile antibody interference affecting multiple Roche immunoassays: A case study.

BACKGROUND: Analysis of many clinically important analytes is dependent on antibody-based assays. However, depending on the design, these assays are vulnerable to interference from endogenous molecules including circulating antibodies and free biotin. In this case report, we describe a patient whose laboratory findings from immunoassay based methodologies, are inconsistent with the clinical presentation. CASE PRESENTATION: A 14-year-old male was referred to Pediatric Endocrinology for suspected hyperthyroidism based on critically elevated free thyroxine (fT4) levels although clinical assessment was inconsistent with hyperthyroidism. Because repeat testing was discrepant, Endocrinology questioned the validity of the results prompting consultation with the laboratory to investigate the source of the inconsistent findings. Review of discordant results revealed that fT4 levels measured in laboratories utilizing Roche instrumentation were critically high, while results from laboratories using alternative platforms (i.e. Siemens Centaur) were within normal limits. CONCLUSION: After a comprehensive evaluation which included testing of paired specimens on multiple platforms, measurement of serially diluted specimens and a formal evaluation for the presence of heterophile antibodies, it was determined that a heterophile antibody interference was the most likely cause of the aberrant results in this patient.

Heterologous Antibodies Adsorption in Xenotransplantation of a Landrace Piglet Kidney Into a Rhesus Monkey.

BACKGROUND: To explore the adsorption of heterologous antibodies in 6 xenotransplants of Landrace piglet kidneys into rhesus monkeys. METHODS: The Landrace piglets and rhesus monkeys were used as donors and recipients, respectively. The donor kidney was the left kidney excised from each Landrace piglet and lavaged with University of Wisconsin solution through the renal artery and vein ex vivo. The renal arteriovenous end of the recipient was preserved. After anastomosis of the renal artery and vein with the arteriovenous end of the recipient for reperfusion, a cross-lymphocyte cytotoxicity test of the heterogeneous kidney was performed. RESULTS: All 6 Landrace piglet kidneys absorbed heterologous antibodies that were pre-existing in the rhesus macaques' kidneys. The cross-lymphocyte toxicity test was performed after the kidney were completely blackened. The cross-lymphocyte toxicity in all each heterogeneous kidney changed from strong positive to weak positive. CONCLUSIONS: Heterologous antibodies were adsorbed in xenotransplants of Landrace piglet kidneys into rhesus monkeys. Xenotransplanted kidney can adsorb heterologous antibodies and consume relevant complements, which is a good model for research of hyperacute rejection in xenotransplantation.

The complex functioning of the complement system in xenotransplantation.

The role of complement in xenotransplantation is well-known and is a topic that has been reviewed previously. However, our understanding of the immense complexity of its interaction with other constituents of the innate immune response and of the coagulation, adaptive immune, and inflammatory responses to a xenograft is steadily increasing. In addition, the complement system plays a function in metabolism and homeostasis. New reviews at intervals are therefore clearly warranted. The pathways of complement activation, the function of the complement system, and the interaction between complement and coagulation, inflammation, and the adaptive immune system in relation to xenotransplantation are reviewed. Through several different mechanisms, complement activation is a major factor in contributing to xenograft failure. In the organ-source pig, the detrimental influence of the complement system is seen during organ harvest and preservation, for example, in ischemia-reperfusion injury. In the recipient, the effect of complement can be seen through its interaction with the immune, coagulation, and inflammatory responses. Genetic-engineering and other therapeutic methods by which the xenograft can be protected from the effects of complement activation are discussed. The review provides an updated source of reference to this increasingly complex subject.

The desirable donor pig to eliminate all xenoreactive antigens.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

A false positive case of high-sensitivity cardiac troponin in a patient with acute chest pain: Analytical study of the interference.

We report a case of a heterophile antibodies interference in a new high-sensitivity troponin commercial immunoassay (cTNIH Siemens), observed in a patient with possible acute coronary syndrome (ACS). The analytical interference was investigated with standard laboratories procedures. The false positive result was found with different troponin methods and kits. We also investigated the protein sequence of cTnl and no sequence variants were detected. The discordance between clinical pictures and high concentration of cTnl, together with the collaboration between clinicians and laboratory staff avoided possible erroneous diagnosis and further invasive investigations to the patient.

Human chorionic gonadotropin suspected heterophile interference investigations in immunoassays: a recommended approach.

Background Heterophile antibody (HAb) interferences in immunoassays can cause falsely elevated hCG concentrations leading to incorrect diagnosis and treatments options. When results are not consistent with the clinical findings, hCG HAb interference investigation may be requested by the physician. A retrospective evaluation of the frequency of HAb interference was performed among cases of physician-requested investigations and the effectiveness of commercially available blocking reagents to detect HAb interference in two immunoassay systems was evaluated. Methods One hundred and thirteen physician requests for hCG HAb investigation from 2008 to 2017 were reviewed. The primary method used to measure hCG was the Beckman Coulter Access Total ßhCG (2008-2010) and the Roche Elecsys HCG+ß (2014-2017). HAb investigation included measurement by two immunoassays before and after treatment of samples with heterophile blocking reagents and serial dilution studies. Results Five cases of HAb and HAb-like interference were identified. The interference frequency was 6.7% for the Beckman assay and 2.9% for the Roche assay. The presence of HAb was detected using heterophile blocking reagents and an alternative method in three cases. The other two cases were detected due to discrepant results with an alternative method and non-linear serial dilutions (HAb-like). Conclusions HAb interference was observed in the Beckman and the Roche assays. The heterophile blocking reagents failed to detect 40% of interference cases. Blocking reagents should not solely be used for these investigations. Multiple strategies including the use of serial dilutions and using an alternative platform are critical when troubleshooting interferences in hCG immunoassays.