Conformational Epitopes Research Articles

Allergenicity and Conformational Diversity of Allergens

Allergens are substances that cause abnormal immune responses and can originate from various sources. IgE-mediated allergies are one of the most common and severe types of allergies, affecting more than 20% of the population in Western countries. Allergens can be subdivided into a limited number of families based on their structure, but this does not necessarily indicate the origin or the route of administration of the allergen, nor is the molecular basis of allergenicity clearly understood. This review examines how understanding the allergenicity of proteins involves their structural characterization and elucidates the study of conformational diversity by nuclear magnetic resonance spectroscopy. This article also discusses allergen cross-reactivity and the mechanisms by which IgE antibodies recognize and bind to allergens based on their conformational and linear epitopes. In addition, we outline how the pH, the proteolytic susceptibility and the endosomal degradation affect the outcome of allergic reactions, and how this is correlated with conformational changes and secondary structure rearrangement events. We want to emphasize the importance of considering structural diversity and dynamics, proteolytic susceptibility and pH-dependent factors to fully comprehend allergenicity.

Antibody reactive immunomes of Ehrlichia chaffeensis and E. canis are diverse and defined by conformational antigenic determinants.

For decades, the defined antibody reactive proteins of Ehrlichia chaffeensis and E. canis were limited to a small group with linear antibody epitopes. Recently, our laboratory has utilized an immunomics-based approach to rapidly screen and identify undefined Ehrlichia chaffeensis and E. canis antigenic proteins and antibody epitopes. In this study, we analyzed the remaining portion (~50%) of the E. chaffeensis and E. canis proteomes (n = 444 and n = 405 proteins, respectively), that were not examined in previous studies, to define the complete immunomes of these important pathogens. Almost half of the E. chaffeensis proteins screened (196/444) reacted with antibodies in convalescent HME patient sera, while only 43 E. canis proteins reacted with CME dog sera. New major immunoreactive proteins were identified in E. chaffeensis (n = 7) and E. canis (n = 1), increasing the total number of E. chaffeensis (n = 14) and E. canis proteins (n = 18) that exhibited antibody reactivity comparable to well-defined major antigenic proteins (TRP120 and TRP19). All of the E. chaffeensis but only some E. canis major immunoreactive proteins contained major conformation-dependent antibody epitopes. The E. chaffeensis immunoreactive proteins were generally small (< 250 amino acids; ~27kDa) and the E. canis proteins were slightly larger (> 320 amino acids; ~35 kDa). The majority of these new Ehrlichia major immunoreactive proteins were predicted to be type I secreted effectors, some of which contained transmembrane domains. Characterization of the immunomes of E. chaffeensis and E. canis and understanding the host specific Ehrlichia immune responses will facilitate identification of protective antigens and define the biophysical epitope characteristics vital to effective vaccine development for the ehrlichioses.

Design of an Ara h 2 hypoallergen from conformational epitopes.

Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapysuggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Effects of unfolding treatment assisted glycation on the IgE/IgG binding capacity and antioxidant activity of ovomucoid.

Ovomucoid is the immune-dominant allergen in the egg white of hens. Due to its structure based on nine disulfide bonds as well as its resistance to heat and enzymatic hydrolysis, the allergenicity of this food protein is difficult to decrease by technological processes. We sought to reduce its allergenicity through the Maillard reaction. The unfolding of ovomucoid with L-cysteine-mediated reduction was used to increase accessibility to conformational and linear epitopes by modifying the secondary and tertiary structures of the allergen. Glycation with different saccharides revealed the beneficial effect of maltose glycation on the IgG-binding capacity reduction. By determining the better glycation conditions of unfolded ovomucoid, we produced ovomucoid with reduced IgE binding capacity due to the glycation sites (K17, K112, K129, and K164) on epitopes. Moreover, after simulated infant and adult gastrointestinal digestion, the unfolded plus glycated ovomucoid showed higher ABTS˙+ scavenging activity, O2˙- scavenging activity, ˙OH scavenging activity, Fe2+ chelating activity, and a FRAP value; in particular, for ˙OH scavenging activity, there was a sharp increase of more than 100%.

Artificial intelligence-driven reverse vaccinology for Neisseria gonorrhoeae vaccine: Prioritizing epitope-based candidates.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. The increasing prevalence of this disease worldwide, the rise of antibiotic-resistant strains, and the difficulties in treatment necessitate the development of a vaccine, highlighting the significance of preventative measures to control and eradicate the infection. Currently, there is no widely available vaccine, partly due to the bacterium's ability to evade natural immunity and the limited research investment in gonorrhea compared to other diseases. To identify distinct vaccine candidates, we chose to focus on the uncharacterized, hypothetical proteins (HPs) as our initial approach. Using the in silico method, we first carried out a comprehensive assessment of hypothetical proteins of Neisseria gonorrhoeae, encompassing assessments of physicochemical properties, cellular localization, secretary pathways, transmembrane regions, antigenicity, toxicity, and prediction of B-cell and T-cell epitopes, among other analyses. Detailed analysis of all HPs resulted in the functional annotation of twenty proteins with a great degree of confidence. Further, using the immuno-informatics approach, the prediction pipeline identified one CD8+ restricted T-cell epitope, seven linear B-cell epitopes, and seven conformational B-cell epitopes as putative epitope-based peptide vaccine candidates which certainly require further validation in laboratory settings. The study accentuates the promise of functional annotation and immuno-informatics in the systematic design of epitope-based peptide vaccines targeting Neisseria gonorrhoeae.

In silico analysis of molecular mimicry between human aquaporin 3, Aspergillus fumigatus aquaporin and aquaporins from allergic sources.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that has a significant impact on quality of life. The immune response and allergy symptoms in AD are triggered by the recognition of specific allergens by IgE antibodies. Cross-reactivity can lead to auto-IgE responses, potentially worsening AD symptoms. Our research aimed to enhance our understanding of allergenic sources, including A. fumigatus, and their role in AD. We focused on molecular mimicry between human AQP3 and A. fumigatus aquaporin. In our in-silico analysis, we compared the amino acid sequences of human aquaporin 3 (AQP3) and A. fumigatus aquaporin with 25 aquaporins from various allergenic sources, sourced from the UniProt and NCBI databases. Phylogenetic relationship analysis and homology-based modeling were conducted. We identified conserved antigenic regions located within the 3D structures. The global identity levels among the studied aquaporins averaged 32.6%. One antigenic site exhibited a remarkable local region, with a conserved identity of 71.4%. We categorized the aquaporins into five monophyletic clades (A-E), with group B showing the highest identity (95%), including six mammalian aquaporins, including AQP3. When comparing A. fumigatus aquaporins, the highest identity was observed with Malassezia sympodialis at 35%. Both human and A. fumigatus aquaporins have three linear and three discontinuous epitopes. We identified potential linear and conformational epitopes of AQP3, indicating a possible molecular mimicry between humans and A. fumigatus aquaporins. This suggests autoreactivity and potential cross-reactivity, although further validation using in vitro and in vivo experiments is required.

Development of amyloid beta-directed antibodies against Alzheimer's disease: Twists and turns.

Alzheimer's disease (AD) is a severe and progressive neurodegenerative disease, and the treatment options that are currently available are limited. The amyloid cascade hypothesis has had a significant influence in explaining the pathology underlying AD. Inhibiting the production and aggregation of amyloid-beta (Aβ) and promoting its clearance have been important strategies in the development of anti-AD drugs over the past two decades. Specifically, Aβ directed antibodies have been highly anticipated, but drug development has been fraught with obstacles and challenges. Antibodies targeting the C-terminal or central region of Aβ, such as ponezumab, solanezumab, and crenezumab, primarily bind to Aβ monomers, yet no significant clearance of brain plaques or slowing of disease progression has been observed in clinical trials. Antibodies targeting the N-terminal region of Aβ, including aducanumab, lecanemab, and donanemab, primarily bind to aggregated forms of Aβ, and have shown efficacy in clearing brain plaques and slowing early-stage AD progression in clinical trials. However, clinical trials of gantenerumab, which targets conformational epitopes in the N-terminal and central sequences of Aβ and which selectively binds to aggregated forms, have failed, raising some new questions about the Aβ hypothesis. Advances in research on the pathological mechanisms of AD and advances in early diagnostic techniques may shift the time window for drug intervention and offer a potential pathway for developing effective drugs to delay the onset and progression of AD in the future.

In silico immunoinformatics based prediction and designing of multi-epitope construct against human rhinovirus C

Human rhinovirus C (HRV-C) is an RNA virus infecting human respiratory tract. It is associated with complexities like asthma, chronic obstructive pulmonary disease, and respiratory damage. HRV-C has many serotypes. Till date there is no vaccine. Despite some limitations, corticosteroids, bronchodilators, and common cold medicines are used to treat HRV-C infections. Here, we have used immunoinformatics approach to predict suitable cytotoxic T-cell, helper T-cell and linear B-cell epitopes from the most antigenic protein. VP2 protein of Rhinovirus C53 strain USA/CO/2014-20993 was found to be most antigenic. The multi-epitope construct was designed using the best CTL, HTL and linear B-cell epitopes and attaching them with adjuvant and linkers. Interferon-gamma inducing epitopes and conformational B-cell epitopes were also predicted from the construct. Physicochemical and structural properties of the construct were satisfactory. Binding pockets were identified that could be the targets for designing effective inhibitors. Molecular docking revealed strong binding affinity of the construct with human Toll-like receptors 2 and 4. Normal mode analysis divulged stability of the docked complex. Codon optimization, in silico cloning and immune simulation analysis demonstrated suitability of the construct. These findings are likely to aid in vitro studies for developing vaccine against HRV-C.

Potential and limitations of epitope mapping and molecular targeting in Hymenoptera venom allergy.

Hymenoptera venom (HV) allergy can lead to life threatening conditions by specific IgE (sIgE)-mediated anaphylactic reactions. The knowledge about major allergens from venom of different clinically relevant species increased in the last decades, allowing the development of component-resolved diagnostics in which sIgE to single allergens is analysed. Despite these advances, the precise regions of the allergens that bind to IgE are only known for few HV allergens. The detailed characterization of IgE epitopes may provide valuable information to improve immunodiagnostic tests and to develop new therapeutic strategies using allergen-derived peptides or other targeted approaches. Epitope-resolved analysis is challenging, since the identification of conformational epitopes present in many allergens demands complex technologies for molecular analyses. Furthermore, functional analysis of the epitopeś interaction with their respective ligands is needed to distinguish epitopes that can activate the allergic immune response, from those that are recognized by irrelevant antibodies or T cell receptors from non-effector cells. In this review, we focus on the use of mapping and molecular targeting approaches for characterization of the epitopes of the major venom allergens of clinically relevant Hymenoptera species. The screening of the most relevant allergen peptides by epitope mapping could be helpful for the development of molecules that target major and immunodominant epitopes blocking the allergen induced cellular reactions as novel approach for the treatment of HV allergy.

In silico epitope prediction of Borrelia burgdorferi sensu lato antigens for the detection of specific antibodies

Despite many years of research, serodiagnosis of Lyme disease still faces many obstacles. Difficulties arise mainly due to the low degree of amino acid sequence conservation of the most immunogenic antigens among B. burgdorferi s.l. genospecies, as well as differences in protein production depending on the environment in which the spirochete is located. Mapping B-cell epitopes located on antigens allows for a better understanding of antibody-pathogen interactions which is essential for the development of new and more effective diagnostic tools. In this study, in silico B-cell epitope mapping was performed to determine the theoretical diagnostic potential of selected B. burgdorferi s.l. proteins (BB0108, BB0126, BB0298, BB0689, BB0323, FliL, PstS, SecD, EF-Tu). Bioinformatics software predicted 35 conserved linear and 31 conformational epitopes with the degree of identity among B. burgdorferi s.l. of at least 85%, which may prove to be useful in the development of a new tool for the diagnosis of Lyme disease.

Nanobodies: a promising tool to perturb ApoE4 activity in Alzheimer’s disease pathology

AbstractBackgroundThe APOE‐ε4 allele encoding for apolipoprotein (apo) E4, an isoform of the APOE gene, is the strongest genetic risk factor for Alzheimer’s disease (AD). The exact mechanism by which apoE4 increases this risk is not fully understood. However, apoE4 has a very different structure than the other isoforms, apoE2 and apoE3, and it is suggested that this different structure contributes to the development of AD1. More efforts are still needed to further understand how apoE4 contributes to AD pathogenesis. Single domain antibodies or nanobodies are versatile tools to reveal new information on apoE4 and its pathological effects in AD.MethodsNanobodies are routinely obtained from immunized camelids, or from naïve libraries. Nanobodies need to be characterized by i.e. immunoprecipitation with apoE2, apoE3 and apoE4 and their affinity can be determined via an Octet device. Via laser‐induced photoporation, nanobodies can be delivered into living induced pluripotent stem cell derived APOE‐ε4 homozygous neurons. Nanobodies can be tagged with a fluorophore via click chemistry and derivates can be made by equipping them with a degradation or delocalization tag (cloning).ResultsLlama immunization will lead to a set of ApoE‐targeting nanobodies which will be further reduced to a couple of nanobodies after their further characterization (specificity and affinity). Given the high similarity between apoE isoforms, it is to be expected that some nanobodies will cross‐react with different isoforms. Because nanobodies recognize conformational epitopes it is likely that ApoE4 nanobodies can be generated.Delivery of the nanobodies into apoE4 expressing neurons allows to manipulate endogenous apoE4 and as such our approach does not require expression modulation of the target: the protein can be degraded or it can be trapped/relocated to different regions or organelles in cells. We aim to perturb apoE4 in a way that typical AD pathologies seen in AD neurons are ameliorated and as such gain more insight about the pathophysiology of apoE4 effects in AD.ConclusionNanobodies are excellent tools to uncover new biological information concerning apoE4.

Standardization of food allergen ladders for optimal effectiveness and patient safety.

The use of extensively heated (EH) milk and egg products, and dietary advancement therapies such as milk and egg ladders is increasingly common for the management of milk and egg allergies. Although the majority of patients with milk and egg allergies will outgrow their allergies, the ability to tolerate extensively hydrolyzed forms of these allergens is an early indicator of developing long-term tolerance. The denaturation of conformational epitopes during the heating process reduces the allergenicity of these proteins, which makes patients who are EH tolerant more likely to tolerate progressively more of these proteins.

Selective targeting and protection against toxic amyloid‐beta oligomers by PMN310, a monoclonal antibody rationally designed for greater therapeutic potency in Alzheimer’s disease

AbstractBackgroundA large body of evidence indicates that the most pathogenic species of amyloid‐beta (Aß) in Alzheimer’s disease (AD) consists of soluble toxic oligomers (AßO) as opposed to insoluble fibrils and monomers. Clinical results to date indicate that non‐selective antibodies that bind Aß monomers, oligomers and plaque fail to provide a therapeutic benefit. Partially selective antibodies that bind AßO and plaque have shown improved success but are associated with dose‐limiting adverse events (amyloid‐related imaging abnormalities; ARIA). These findings suggest that antibodies capable of selectively neutralizing toxic AßO may achieve improved efficacy and safety. Monoclonal antibody PMN310 was raised against a conformational epitope predicted by computational modeling to be exposed on toxic AßO but not monomers or fibrils. A summary of the selectivity and protective activity of PMN310 against toxic AßO in vitro and in vivo is presented.MethodThe binding selectivity of PMN310 was characterized and compared to that of other Aß‐directed antibodies by surface plasmon resonance (SPR) and immunohistochemistry (IHC). Its ability to neutralize the propagation and toxicity of AßO was assessed in vitro in a thioflavin‐T propagation assay and in cultures of primary rodent neurons, respectively. In vivo protective activity was tested in wild‐type mice injected intracerebroventricularly (ICV) with AßO and in the APP/L transgenic mouse model of AD.ResultIn SPR analysis, PMN310 showed selective binding to AßO over monomers and, compared to otherAß‐directed antibodies, was minimally impacted by monomer competition in binding to toxic oligomers from AD brain extract. In addition, PMN310 did not bind to Aß plaque or vascular deposits as determined by IHC suggesting a potentially reduced risk of ARIA compared to other Aß‐directed antibodies. In vitro, PMN310 inhibited AßO propagation and neuronal toxicity. In vivo, PMN310 prevented AßO‐induced loss of memory formation after ICV injection. Systemic administration to APP/L transgenic mice preserved memory and learning in the water maze task.ConclusionThe antibody PMN310 was shown to selectively bind toxic AßO and protect against their pathogenic activity in vitro. In two rodent models of AD, PMN310 protected memory function, suggesting that PMN310 may offer a new therapeutic option for the treatment or prevention of AD.

Tau maturation in clinicopathological spectrum of Lewy body and Alzheimer’s disease

AbstractBackgroundIn Alzheimer’s disease neuropathologic change (ADNC), maturation of neurofibrillary tangles and tau pathology are characterized by posttranslational modifications of tau. ANDC and Lewy body disease (LBD) commonly co‐exist in aging brains and there are overlapping clinical syndromes and heterogeneity in clinical features among cases with mixed ADNC and LBD pathology. Given this clinical and pathological overlap, we examined the postmortem expression of tau conformational epitopes that mark various stages of tangle maturation in clinicopathologic spectrum of Alzheimer’s disease (AD) and LBD.MethodAutopsy‐confirmed cases with diffuse neocortical LBD with/without ADNC (n = 59) were selected (Table1). We used digital histology methods to measure percent area occupied (%AO) with pathology in three regions (middle frontal, anterior cingulate, and superior temporal cortices) immunohistochemically stained with MJF‐R13 (α‐synuclein), NAB228 (beta‐amyloid), and tau monoclonal antibodies marking phosphorylated (AT8), early conformational (MC1, preferentially reactive in pre‐tangles), and C‐terminally truncated tau epitopes (TauC3, preferentially reactive in mature intracellular and ghost tangles). Linear models tested the group comparisons, and correlation between α‐synuclein or beta‐amyloid with outcome of average neocortical tau. In models, %AO were log‐transformed, and age and sex were included as covariates.Resultα‐synuclein was strongly correlated with early tau epitopes labelled with AT8 (β = 1.37, p&lt;0.001) and MC1 (β = 1.20, p&lt;0.001), but not with mature tau epitopes detected by TauC3 (p = 0.38). There were significant positive correlations between all tau epitopes and beta‐amyloid, including TauC3 immunoreactive tau (p≤0.013) (Figure1). In the mixed pathology group, those with clinical LBD had a positive association between α‐synuclein and MC1 (β = 1.61, p = 0.014) and AT8 (β = 1.67, p = 0.005) positive tau, while those with clinical AD had no correlation between α‐synuclein and tau epitopes. There was greater MC1 (β = 3.40, p&lt;0.001), AT8 (β = 3.59, p&lt;0.001), and TauC3 (β = 1.43, p = 0.005) pathology in patients with clinical AD compared to clinical LBD.ConclusionIn LBD, higher α‐synuclein is associated with greater phosphorylated and early tau conformations, but not later conformation of mature tau, which was selectively associated with beta‐amyloid pathology. Our findings suggest that tau pathology may modify the clinical expression of synucleinopathies and C‐terminal truncation of tau may be more closely related to AD pathophysiology than synucleinopathy.

IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy

BackgroundOral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants.ObjectiveHere we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials.MethodsPre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides.ResultsStatistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered.ConclusionAt a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut.

Genome characteristics of atypical porcine pestivirus from abortion cases in Shandong Province, China

BackgroundAtypical porcine pestivirus (APPV) is a novel, highly variable porcine pestivirus. Previous reports have suggested that the virus is associated with congenital tremor (CT) type A-II in piglets, and little information is available about the correlation between the virus and sow abortion, or on coinfection with other viruses. In China, reported APPV strains were mainly isolated from South China and Central China, and data about the APPV genome from northern China are relatively scarce.MethodsEleven umbilical cords, one placenta, and one aborted piglet, were collected from aborted sows of the same farm in Shandong Province of northern China. Nucleic acids were extracted from the above samples, and subsequently pooled for viral metagenomics sequencing and bioinformatics analysis. The viral coexistence status and complete genome characteristics of APPV in Shandong Province were determined.ResultsIn abortion cases, APPV was present with Getah virus, porcine picobirnavirus, porcine kobuvirus, porcine sapovirus, Po-Circo-like virus, porcine serum-associated circular virus, porcine bocavirus 1, porcine parvovirus 1, porcine parvovirus 3 and porcine circovirus 3, etc. The first complete genome sequence(11,556 nt) of APPV in Shandong Province of northern China, was obtained using viral metagenomics and designated APPV-SDHY-2022. Comparison with Chinese reference strains revealed that the polyprotein of APPV-SDHY-2022 shared 82.6-84.2%, 93.2-93.6%, and 80.7-85% nucleotide identity and 91.4-92.4%, 96.4-97.7%, and 90.6-92.2% amino acid identity with those of the Clade I, Clade II and Clade III strains, respectively. Phylogenetic analysis based on the complete polyprotein CDS and NS5A sequences concluded that APPV-SDHY-2022 belongs to Clade II. Analysis of the NS5A nucleotide sequences revealed homology of greater than 94.6% for the same isoform, 84.7-94.5% for different isoforms of the same clade and 76.8-81.1% for different clades. Therefore, Clade II was further divided into three subclades, and APPV-SDHY-2022 belonged to subclade 2.3. Members of Clade II have 20 unique amino acids in individual proteins, distinguishing them from Clade I and Clade III members. The E2 protein showed the greatest diversity of putative N-glycosylation sites with 9 patterns, and APPV-SDHY-2022 along with other Chinese APPV strains shared the conserved B-cell conformational epitope residues 39E, 70R, 173R, 190K and 191N of the E2 protein.ConclusionsWe reported viral coexistence and the first complete genome sequence of APPV from abortion cases and from Shandong Province. The new APPV isolate belongs to an independent branch of Clade II. Our results increase the molecular and epidemiological understanding of APPV in China.

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Phase I Study of the T-Cell Receptor-like Antibody, Hu8F4 in Patients with Relapsed and Refractory Myeloid Malignancies

Background: Relapse after conventional chemotherapy for newly diagnosed AML is responsible for the majority of treatment failure. Relapsed (R/R) disease is frequently refractory to salvage attempts with further chemotherapy. The effectiveness of stem cell transplant and associated graft vs. leukemia effect implies an important role for immune-based therapy in producing long lasting remissions. Existing approaches using immunotherapy have failed to establish a suitable surface target or treatment paradigm that is effective in myeloid malignancies. Hu8F4 is a humanized T-cell receptor-like monoclonal antibody that binds to the conformational epitope of PR1 bound to HLA-A2, which is highly, differentially expressed on the surface of AML compared to normal progenitors. Methods: This is a first-in-human, Phase I dose escalation trial of Hu8F4 in patients (pts) with myeloid malignancies. Pts with R/R AML, MDS, CMML, and myeloid blast phase of CML with adequate organ function and PS ≤ 2 were eligible. Pts were treated on 7 escalating dose levels, ranging from 0.01 mg/kg to 10 mg/kg IV on D1 &amp; 15. Initial dose levels required 1 pt per dose (0.01, 0.03, 0.1, 0.3, 1), followed by 3 pts per dose (3, 10). Results: Fifteen pts with R/R AML have been enrolled, with a median age of 62 years (range, 23-77 years), including 7 females (47%). Pts had received a median of 4 prior therapies; 5 pts (33%) had a PS of 2. At enrollment, the median WBC was 2 (0.1 - 18.4), median BM blasts were 21% (0 - 76); 9 pts (60%) had complex karyotype and 4 (27%) had a TP53 mutation. All pts had &amp;gt; 98% surface expression of PR1 on the myeloid blasts. Hu8F4 C max ranged up to 160,000 ng/mL with t 1/2 of 48 hours and clearance of 2.61 hr*ng/mL at the highest dose. Weak anti-drug antibodies were observed after week 4 in 2 of 3 pts treated at 3 mg/kg. With a median follow up of 5 months (1.1 - 9.9), pts have received a median of 1 (1-4) cycle of therapy. No DLTs were observed at the 3 mg/kg cohort.; 4 additional pts who were on concomitant tacrolimus were enrolled at this level to assess safety with concurrent use, and no DLTs were observed. Three pts were enrolled at the 10 mg/kg level, and 1 experienced dose limiting thrombocytopenia, triggering expansion to 6 pts. Two pts had a decline in BM blasts and 4 had stable disease. There was rapid decline in peripheral blasts immediately after infusion of Hu8F4 on D1 and 15 with associated elevation in serum LDH in some pts and a rise in normal granulocytes, consistent with drug-mediated anti-leukemia effects. The pharmacokinetic parameters and transient blast reduction indicated a possible sink effect mediated by high levels of circulating blasts. SAEs documented on study were mostly disease-related and included infections, cytopenias, hemoptysis, pneumonia, and GI bleeding. Treatment related AEs were temporally related to the drug infusion, including hypotension (Grade 2: N=2), and rigors (Grade 2: N=10; Grade 1: N=1). Infusion reactions were observed at all dose levels, more common at 3 and 10 mg/kg, but were transient, and managed with steroids and antihistamines. All pts proceeded with their next dose without further issues. Neither cytokine release syndrome nor neurologic toxicities were observed. Correlative studies support antibody dependent cellular cytotoxicity and phagocytosis as important mechanisms of anti-AML activity, which was more effective at lower dose levels. Conclusion: Hu8F4, a first-in-class TCR mimic antibody, was well tolerated with expansion planned at more frequent dose intervals. On-target peripheral blast reduction temporally related to infusion suggests biological activity. Real-time pharmacokinetic data indicate a possible sink effect that may be overcome by a more frequent dosing strategy that is planned.

The utility of macadamia nut legumin Mac i 2 and its conformational epitopes in the diagnosis of macadamia nut allergy

The utility of macadamia nut legumin Mac i 2 and its conformational epitopes in the diagnosis of macadamia nut allergy

Novel and unique rheumatoid factors cross-react with viral epitopes in COVID-19

Rheumatoid factors (RFs), polyreactive antibodies canonically known to bind two conformational epitopes of IgG Fc, are a hallmark of rheumatoid arthritis but also can arise in other inflammatory conditions and infections. Also, infections may contribute to the development of rheumatoid arthritis and other autoimmune diseases. Recently, RFs only in rheumatoid arthritis were found to bind novel linear IgG epitopes as well as thousands of other rheumatoid arthritis autoantigens. Specific epitopes recognized by infection-induced polyreactive RFs remain undefined but could provide insights into loss of immune tolerance. Here, we identified novel linear IgG epitopes bound by RFs in COVID-19 but not rheumatoid arthritis or other conditions. The main COVID-19 RF was polyreactive, binding two IgG and multiple viral peptides with a tripeptide motif, as well as IgG Fc and SARS-CoV-2 spike proteins. In contrast, a rheumatoid arthritis-specific RF recognized IgG Fc, but not tripeptide motif-containing peptides or spike. Thus, RFs have disease-specific IgG reactivity and distinct polyreactivities that reflect the broader immune response. Moreover, the polyreactivity of a virus-induced RF appears to be attributable to a very short peptide motif. These findings refine our understanding of RFs and provide new insights into how viral infections may contribute to autoimmunity.