IgG Epitopes Research Articles

Assessing immune factors in maternal milk and paired infant plasma antibody binding to human rhinoviruses.

Before they can produce their own antibodies, newborns are protected from infections by transplacental transfer of maternal IgG antibodies and after birth through breast milk IgA antibodies. Rhinovirus (RV) infections are extremely common in early childhood, and while RV infections often result in only mild upper respiratory illnesses, they can also cause severe lower respiratory illnesses such as bronchiolitis and pneumonia. We used high-density peptide arrays to profile infant and maternal antibody reactivity to capsid and full proteome sequences of three human RVs - A16, B52, and C11. Numerous plasma IgG and breast milk IgA RV epitopes were identified that localized to regions of the RV capsid surface and interior, and also to several non-structural proteins. While most epitopes were bound by both IgG and IgA, there were several instances where isotype-specific and RV-specific binding were observed. We also profiled 62 unique RV-C protein loop sequences characteristic of this species' capsid VP1 protein. Many of the RV-C loop sequences were highly bound by IgG from one-year-old infants, indicating recent or ongoing active infections, or alternatively, a level of cross-reactivity among homologous RV-C sites.

Targeted mutagenesis of the β-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the β-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

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.

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.

A novel precision-serology assay for SARS-CoV-2 infection based on linear B-cell epitopes of Spike protein.

The COVID-19 pandemic illustrates the need for serology diagnostics with improved accuracy. While conventional serology based on recognition of entire proteins or subunits thereof has made significant contribution to the antibody assessment space, it often suffers from sub-optimal specificity. Epitope-based, high-precision, serology assays hold potential to capture the high specificity and diversity of the immune system, hence circumventing the cross-reactivity with closely related microbial antigens. We herein report mapping of linear IgG and IgA antibody epitopes of the SARS-CoV-2 Spike (S) protein in samples from SARS-CoV-2 exposed individuals along with certified SARS-CoV-2 verification plasma samples using peptide arrays. We identified 21 distinct linear epitopes. Importantly, we showed that pre-pandemic serum samples contain IgG antibodies reacting to the majority of protein S epitopes, most likely as a result of prior infection with seasonal coronaviruses. Only 4 of the identified SARS-CoV-2 protein S linear epitopes were specific for SARS-CoV-2 infection. These epitopes are located at positions 278-298 and 550-586, just proximal and distal to the RBD, as well as at position 1134-1156 in the HR2 subdomain and at 1248-1271 in the C-terminal subdomain of protein S. To substantiate the applicability of our findings, we tested three of the high-accuracy protein S epitopes in a Luminex assay, using a certified validation plasma sample set from SARS-CoV-2 infected individuals. The Luminex results were well aligned with the peptide array results, and correlated very well with in-house and commercial immune assays for RBD, S1 and S1/S2 domains of protein S. We present a comprehensive mapping of linear B-cell epitopes of SARS-CoV-2 protein S, that identifies peptides suitable for a precision serology assay devoid of cross-reactivity. These results have implications for development of highly specific serology test for exposure to SARS-CoV-2 and other members of the coronaviridae family, as well as for rapid development of serology tests for future emerging pandemic threats.

High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

Study on Lowering the Group 1 Protease Allergens from House Dust Mites by Exposing to Todomatsu Oil Atmosphere

Group 1 protease allergens that persist in fecal particles from house dust mites (HDM) are the prevalent indoor aeroallergens and the primary triggers of dust mite allergy. Consequently, it is vital to discover a secure, efficient, affordable, and eco-friendly inhibitor to restrain these allergens. Herein, an assessment on the suppression of allergens Der f 1 and Der p 1 (predominant Group 1 allergens) with todomatsu oil derived from the remnants of Abies sachalinensis was performed using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and molecular docking analysis in silico. The results demonstrated that todomatsu oil effectively suppresses allergens Der f 1 and Der p 1 by lowering their allergen contents, and the decline rate rises with increasing todomatsu oil concentration. The formation of alkyl hydrophobic interactions, pi-sigma bonds, and hydrogen bonds have been observed between oil ingredients and allergens Der f 1 and Der p 1. Intriguingly, the majority of oil ingredients prefer to dock on hydrophobic amino acids. Additionally, oil ingredients docked to the cysteine protease site on Der f 1 or IgG epitope on Der p 1 were discovered. Notably, the binding affinity (BA) score and inhibition constant (Ki) revealed that bornyl acetate, the component with the maximum relative proportion in todomatsu oil, is included in the top five ingredients with the best inhibition effectiveness. As a result, todomatsu oil has been identified as an efficient inhibitor against Group 1 HDM allergens and a viable measure to improve indoor air quality.

IgE and IgG4 Epitopes of Dermatophagoides and Blomia Allergens before and after Sublingual Immunotherapy

Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

Supplementary figures High-throughput IgG Epitope mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design

Supplementary figures High-throughput IgG Epitope mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design

Changes in IgE and IgG4 Epitopes of Peanut Allergens Following Peanut Oral Immunotherapy

Oral immunotherapy (OIT) with Peanut (Arachis hypogaea) allergen powder-dnfp (PTAH) is a promising treatment to desensitize peanut allergic patients. Our aim was to assess changes in IgE and IgG4 binding to peanut allergens and their epitopes recognized by peanut allergic patients undergoing OIT with PTAH in the US.

Linear epitope mapping of the humoral response against SARS-CoV-2 in two independent African cohorts

Profiling of the antibody responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) proteins in African populations is scarce. Here, we performed a detailed IgM and IgG epitope mapping study against 487 peptides covering SARS-CoV-2 wild-type structural proteins. A panel of 41 pre-pandemic and 82 COVID-19 RT-PCR confirmed sera from Madagascar and Senegal were used. We found that the main 36 immunodominant linear epitopes identified were (i) similar in both countries, (ii) distributed mainly in the Spike and the Nucleocapsid proteins, (iii) located outside the RBD and NTD regions where most of the reported SARS-CoV-2 variant mutations occur, and (iv) identical to those reported in European, North American, and Asian studies. Within the severe group, antibody levels were inversely correlated with the viral load. This first antibody epitope mapping study performed in patients from two African countries may be helpful to guide rational peptide-based diagnostic assays or vaccine development.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

Allergenic food protein consumption is associated with systemic IgG antibody responses in non-allergic individuals

Although food-directed immunoglobulin E (IgE) has been studied in the context of allergies, the prevalence and magnitude of IgG responses against dietary antigens are incompletely characterized in the general population. Here, we measured IgG binding against food and environmental antigens obtained from allergen databases and the immune epitope database (IEDB), represented in a phage displayed library of 58,233 peptides. By profiling blood samples of a large cohort representing the average adult Israeli population (n= 1,003), we showed that many food antigens elicited systemic IgG in up to 50% of individuals. Dietary intake of specific food protein correlated with antibody binding, suggesting that diet can shape the IgG epitope repertoire. Our work documents abundant systemic IgG responses against food antigens and provides a reference map of the exact immunogenic epitopes on a population scale, laying the foundation to unravel the role of food- and environmental antigen-directed antibody binding in disease contexts.

IgE and IgG4 Epitope Mapping of Food Allergens with a Peptide Microarray Immunoassay.

Peptide microarrays are a powerful tool to identify linear epitopes of food allergens in a high-throughput manner. The main advantages of the microarray-based immunoassay are as follows: the possibility to assay thousands of targets simultaneously, the requirement of a low volume of serum, the more robust statistical analysis, and the possibility to test simultaneously several immunoglobulin subclasses. Among them, the last one has a special interest in the field of food allergy, because the development of tolerance to food allergens has been associated with a decrease in IgE and an increase in IgG4 levels against linear epitopes. However, the main limitation to the clinical use of microarray is the automated analysis of the data. Recent studies mapping the linear epitopes of food allergens with peptide microarray immunoassays have identified peptide biomarkers that can be used for early diagnosis of food allergies and to predict their severity or the self-development of tolerance. Using this approach, we have worked on epitope mapping of the two most important food allergens in the Spanish population, cow's milk, and chicken eggs. The final aim of these studies is to define subsets of peptides that could be used as biomarkers to improve the diagnosis and prognosis of food allergies. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of food allergens and data acquisition and analysis of IgE and IgG4 binding epitopes.

P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesNon-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.MethodsThe Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.ResultsMice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P <.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.ConclusionMice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.

Novel Approaches for Inhibiting the Indoor Allergen Der f 2 Excreted from House Dust Mites by Todomatsu Oil Produced from Woodland Residues.

House dust mite (HDM) is a globally ubiquitous domestic cause of allergic diseases. There is a pressing demand to discover efficient, harmless, and eco-friendly natural extracts to inhibit HDM allergens that are more likely to trigger allergies and challenging to be prevented entirely. This study, therefore, is aimed at assessing the inhibition of the allergenicity of major HDM allergen Der f 2 by todomatsu oil extracted from residues of Abies Sachalinensis. The inhibition was investigated experimentally (using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and in silico using molecular docking. The results showed that todomatsu oil inhibits the allergenicity of Der f 2 by reducing its amount instead of the IgG binding capacity of a single protein. Moreover, the compounds in todomatsu oil bind to Der f 2 via alkyl hydrophobic interactions. Notably, most compounds interact with the hydrophobic amino acids of Der f 2, and seven substances interact with CYS27. Contrarily, the principal compounds fail to attach to the amino acids forming the IgG epitope in Der f 2. Interestingly, chemical components with the lowest relative percentages in todomatsu oil show high-affinity values on Der f 2, especially β-maaliene (−8.0 kcal/mol). In conclusion, todomatsu oil has been proven in vitro as a potential effective public health strategy to inhibit the allergenicity of Der f 2.

Chikungunya Virus E2 Structural Protein B-Cell Epitopes Analysis.

The Togaviridae family comprises a large and diverse group of viruses responsible for recurrent outbreaks in humans. Within this family, the Chikungunya virus (CHIKV) is an important Alphavirus in terms of morbidity, mortality, and economic impact on humans in different regions of the world. The objective of this study was to perform an IgG epitope recognition of the CHIKV’s structural proteins E2 and E3 using linear synthetic peptides recognized by serum from patients in the convalescence phase of infection. The serum samples used were collected in the state of Sergipe, Brazil in 2016. Based on the results obtained using immunoinformatic predictions, synthetic B-cell peptides corresponding to the epitopes of structural proteins E2 and E3 of the CHIKV were analyzed by the indirect peptide ELISA technique. Protein E2 was the main target of the immune response, and three conserved peptides, corresponding to peptides P3 and P4 located at Domain A and P5 at the end of Domain B, were identified. The peptides P4 and P5 were the most reactive and specific among the 11 epitopes analyzed and showed potential for use in serological diagnostic trials and development and/or improvement of the Chikungunya virus diagnosis and vaccine design.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

Abstract 5199: Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes

Abstract While the role of T cells in cancer immunotherapy has been well studied and is relatively clear and well defined, there are few studies elucidating the more complicated involvement of B cells in anti-cancer processes. Preclinical data showed that IFx-Hu2.0 primes both innate and adaptive immune responses through the expression of Emm55, a bacterial protein. Recognition by antigen presenting cells (APC) is followed by interantigenic epitope spreading to tumor antigens expressed in transfected tumor cells. The current study was designed to determine the extent of epitope spreading through quantitating the IgM and IgG responses to known melanoma antigens. Plasma samples (pre-and post-intralesional injection of IFx-Hu2.0) from seven patients with unresectable stage III-IV cutaneous melanoma enrolled in a first-in-human, phase 1 open-label trial (NCT03655756) were analyzed and compared using PEPperCHIP® Melanoma Antigen Microarrays with 21 melanoma-associated antigens converted into linear 15 amino acid peptides with a peptide-peptide overlap of 13 amino acids. The resulting array contained 4,125 different peptides printed in duplicate along with multiple control peptides. The arrays were incubated with plasma samples [collected immediately prior to and four weeks (N=6) or two weeks (N=1) post treatment and three weeks post injection #2 (N=1)] at three dilutions, followed by staining with secondary and control antibodies. Readout with a LI-COR Odyssey Imaging System was followed by quantification of spot intensities and peptide annotation with PepSlide® Analyzer. For generation of IgM/IgG intensity ratios, a minimal baseline intensity of 200 fluorescence units was set. Changes in both IgM and IgG epitope recognition to melanoma-associated antigens was detected in all patients. Baseline and IFx-Hu2.0-induced antibody response profiles for all individuals were heterogeneous, with no dominant common antibody response, pointing to the unpredictability of specific epitope recognition and a relative independence from MHC haplotype requirements due to epitope spreading. Notwithstanding the signals from adjacent peptides, high numbers of newly recognized melanoma epitopes were observed for all patients and all plasma samples. These data suggest enhanced crosstalk between innate and adaptive immune cells, epitope presentation by APC to T and B cells and epitope spreading to multiple previously unrecognized tumor epitopes. IgM and IgG levels were maintained at four weeks post IFx-Hu2.0 administration suggesting continued primary recognition and maturation of effector immune response. B cells represent an important proportion of infiltrating lymphocytes in melanoma and are associated with good prognosis in most cancer types. Studies are underway to determine the relative contribution of B cell activity to anti-tumor effects seen with IFx-Hu2.0. Citation Format: Patricia Lawman, Michael Shamblott, Ashraf Dehlawi, James Bianco. Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5199.