Antigenic Cross-reactivity Research Articles

Myocardial fibrosis in rheumatic heart disease: emerging concepts and clinical implications.

Rheumatic heart disease (RHD) remains a significant cardiovascular burden in the world even though it is no longer common in affluent countries. Centuries of history surrounding this disease provide us with a thorough understanding of its pathophysiology. Infections in the throat, skin, or mucosa are the gateway for Group A Streptococcus (GAS) to penetrate our immune system. A significant inflammatory response to the heart is caused by an immunologic cascade triggered by GAS antigen cross-reactivity. This exaggerated immune response is primarily responsible for cardiac dysfunction. Recurrent inflammatory processes damage all layers of the heart, including the endocardium, myocardium, and pericardium. A vicious immunological cycle involving inflammatory mediators, angiotensin II, and TGF-β promotes extracellular matrix remodeling, resulting in myocardial fibrosis. Myocardial fibrosis appears to be a prevalent occurrence in patients with RHD. The presence of myocardial fibrosis, which causes left ventricular dysfunction in RHD, might be utilized to determine options for treatment and might also be used to predict the outcome of interventions in patients with RHD. This emerging concept of myocardial fibrosis needs to be explored comprehensively in order to be optimally utilized in the treatment of RHD.

Deglycosylation of Excretory-Secretory Antigens of the Second-Stage Larvae of Toxocara cati Improves Its Efficacy in the Diagnosis of Human Toxocariasis.

Toxocariasis is an important zoonotic infection, especially in tropical areas. One of the significant challenges in the serodiagnosis of human toxocariasis is the cross-reaction of Toxocara antigens with other parasites due to their relatively similar glycan structures. Removing the glycan structure from Toxocara excretory-secretory (TES) antigens may increase the efficacy of these antigens in the diagnosis of toxocariasis. The current study aimed to assess the efficacy of deglycosylated Toxocara cati excretory-secretory (dTES) antigens for the serodiagnosis of human toxocariasis. Toxocara ES antigens were prepared from T. cati second-stage larvae and deglycosylated using sodium hydroxide (NaOH). The TES antigens, along with the dTES antigens, were used in an ELISA as well as a western blotting system for the detection of anti-Toxocara antibodies. Sera samples collected from 30 confirmed cases of toxocariasis, 30 patients with other diseases, and 30 healthy subjects were evaluated by both systems. The sensitivity of TES and dTES ELISA for the diagnosis of human toxocariasis was 96.67% (95% CI = 82.78-99.92) and 93.33% (95% CI = 77.93-99.18), respectively, while the specificity of dTES (88.33%; 95% CI = 77.43-95.18) increased significantly compared to the TES (80.00%; 95% CI = 67.67-89.22). The sensitivity of both antigens was 100% (95% CI = 88.43-100) by the western blotting system. Moreover, the specificity of TES and dTES antigens was 95% (95% CI = 86.08-98.96) and 98.33% (95% CI = 91.06-99.96), respectively, when using the western blotting system. Results of the current study indicate that the chemical removal of the glycan epitopes of T. cati ES antigens significantly reduces cross-reactivity rates with other parasitic infections. Considering the findings of the present study, the dTES antigens seem to be suitable antigens for the serodiagnosis of human toxocariasis.

The prevalence of IgG antibodies against milk and milk antigens in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). The pathophysiology of MS is complex and is said to be influenced by multiple environmental determinants, including diet. We and others have previously demonstrated how consumption of bovine milk can aggravate disease severity in MS patients, which can be explained by molecular mimicry between milk antigens and those expressed within the CNS. In this study we set out to identify alternatives to drinking cow milk which might be less detrimental to MS patients who have a genetic predisposition towards developing antibody titers against bovine milk antigens that cross-react with CNS antigens. To this end, we screened 35 patients with MS and 20 healthy controls for their IgG reactivity against an array of animal-sourced milk, plant-based alternatives as well as individual antigens from bovine milk. We demonstrate that MS patients have a significantly higher IgG response to animal-sourced milk, especially cow milk, in comparison to healthy donors. We also show that the reactivity to cow milk in MS patients can be attributed to reactivity against different bovine milk antigens. Finally, our correlation data indicate the co-existence of antibodies to individual bovine milk antigens and their corresponding cross-reactive CNS antigens. Taken together, we suggest screening of blood from MS patients for antibodies against different types of milk and milk antigens in order to establish a personalized diet regimen.

Population immunity to pneumococcal serotypes in Kilifi, Kenya, before and 6 years after the introduction of PCV10 with a catch-up campaign: an observational study of cross-sectional serosurveys

In Kilifi (Kenya), a pneumococcal conjugate vaccine (PCV10) was introduced in 2011 in infants (aged <1 year, 3 + 0 schedule) with a catch-up campaign in children aged 1-4 years. We aimed to measure the effect of PCV10 on population immunity. In this observational study, repeated cross-sectional serosurveys were conducted in independent random samples of 500 children younger than 15 years every 2 years between 2009 and 2017. During these surveys, blood samples were collected by venesection. Concentrations of anti-capsular IgGs against vaccine serotypes (VTs) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, and against serotypes 6A and 19A, were assayed by ELISA. We plotted the geometric mean concentrations (GMCs) by birth year to visualise age-specific antibody profiles. In infants, IgG concentrations of 0·35 μg/mL or higher were considered protective. Of 3673 volunteers approached, 2152 submitted samples for analysis across the five surveys. Vaccine introduction resulted in an increase in the proportion of young children with protective IgG concentrations, compared with before vaccine introduction (from 0-33% of infants with VT-specific levels over the correlate of protection in 2009, to 60-94% of infants in 2011). However, among those vaccinated in infancy, GMCs of all ten VTs had waned rapidly by the age of 1, but rose again later in childhood. GMCs among children aged 10-14 years were consistently high over time (eg, the range of GMCs across survey rounds were between 0·45 μg/mL and 1·00 μg/mL for VT 23F and between 2·00 μg/mL and 3·11 μg/mL for VT 19F). PCV10 in a 3 + 0 schedule elicited protective IgG levels during infancy, when disease risk is high. The high antibody levels in children aged 10-14 years might indicate continued exposure to vaccine serotypes due to residual carriage or to memory responses to cross-reactive antigens. Despite rapid waning of IgG after vaccination, disease incidence among young children in this setting remains low, suggesting that lower thresholds of antibody, or other markers of immunity (eg, memory B cells), may be needed to assess population protection among children who have aged past infancy. Gavi, the Vaccine Alliance; Wellcome Trust.

Unravelling Antigenic Cross-Reactions toward the World of Coronaviruses: Extent of the Stability of Shared Epitopes and SARS-CoV-2 Anti-Spike Cross-Neutralizing Antibodies.

The human immune repertoire retains the molecular memory of a very great diversity of target antigens (epitopes) and can recall this upon a second encounter with epitopes against which it has previously been primed. Although genetically diverse, proteins of coronaviruses exhibit sufficient conservation to lead to antigenic cross-reactions. In this review, our goal is to question whether pre-existing immunity against seasonal human coronaviruses (HCoVs) or exposure to animal CoVs has influenced the susceptibility of human populations to SARS-CoV-2 and/or had an impact upon the physiopathological outcome of COVID-19. With the hindsight that we now have regarding COVID-19, we conclude that although antigenic cross-reactions between different coronaviruses exist, cross-reactive antibody levels (titers) do not necessarily reflect on memory B cell frequencies and are not always directed against epitopes which confer cross-protection against SARS-CoV-2. Moreover, the immunological memory of these infections is short-term and occurs in only a small percentage of the population. Thus, in contrast to what might be observed in terms of cross-protection at the level of a single individual recently exposed to circulating coronaviruses, a pre-existing immunity against HCoVs or other CoVs can only have a very minor impact on SARS-CoV-2 circulation at the level of human populations.

Abstract PR08: TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development

Abstract Background: KRAS mutations drive tumorigenesis in adenocarcinomas of the lung (30-40%), colon (40-50%) and pancreas (&amp;gt;90%). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with KRAS-mutated advanced solid tumors. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. Here, we describe the pre-clinical development of TCR1020, a human TCRab specific for KRAS G12V restricted to HLA-A*11:01. Methods: In vitro stimulation assays were used to generate CD8+ T cell responses specific for a decamer epitope of KRAS G12V (VVVGAVGVGK) using blood specimens collected from A*11:01+ healthy donors. TCR1020 was identified by TCRab RNA sequencing of flow cytometrically sorted peptide/HLA multimer+ CD8 T cells. Transgenic expression of TCR1020 was attained via lentiviral transduction of reporter cell lines and gene-edited (TCRabnull) primary T cells. TCR1020 antigen specificity, affinity and cross-reactivity was assessed using in vitro coculture, limiting peptide dilution, and combinatorial peptide scanning assays. TCR1020 reactivity to G12V/A*11:01+ cell lines was assessed using in vitro cytokine release and cytotoxicity assays, as well as mouse xenograft tumor models. Results: TCR1020 exhibits specificity to the cognate KRAS G12V epitope with cross-reactivity to G12C but not wild-type KRAS. TCR1020 has a measured functional avidity (EC50) of 262 pM. TCR1020-redirected primary CD8+ T cells promote cytolysis of A*11:01+ tumor cell lines of lung, colonic or pancreatic origin harboring endogenous KRAS G12V mutations. The adoptive transfer of TCR1020-engineered primary CD8+ T cells exhibits potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. TCR1020 demonstrates CD8 co-receptor independence, and CD4+ T cells expressing TCR1020 express cytokines and lyse G12V/A*11:01+ tumor cell lines. In-depth characterization of the TCR1020 peptide recognition motif using alanine/glycine scanning libraries reveals a unique antigen binding motif with 5 critical amino acid residues excluding peptide/HLA anchor residues. Combinatorial peptide library scanning identifies five A*11:01-restricted peptide candidates within the human proteome with potential cross-reactivity to TCR1020, none of which activate TCR1020-engineered T cells over a wide range of concentrations. TCR1020 exhibits no evidence of reactivity against cell lines derived from healthy human tissues nor alloreactivity against a panel of lymphoblastoid cell lines encompassing a wide array of HLA class I alleles. Conclusion: TCR1020 exhibits high potency and exquisite antigen cognate specificity, and it is under clinical development for the treatment of A*11:01+ patients with KRAS G12V+ advanced solid tumors. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR08.

Potential contribution of Helicobacter pylori proteins in the pathogenesis of type 1 gastric neuroendocrine tumor and urticaria. In silico approach.

Helicobacter pylori has been linked to several diseases such as chronic urticaria, gastritis, and type 1 gastric neuroendocrine tumors (type 1 gNET). Although these diseases seem to have different mechanisms, their relationship with H. pylori suggests a common inflammatory pathway. To identify potential cross-reactive antigens between H. pylori and humans involved in chronic urticaria and type 1 gNET. Alignment was carried out among human proteins associated with urticaria (9 proteins), type 1 gNET (32 proteins), and H. pylori proteome. We performed pairwise alignment among the human and H. pylori antigens with PSI-BLAST. Modeling based on homology was done with the Swiss model server and epitope prediction with the Ellipro server. Epitopes were located on a 3D model using PYMOL software. The highest conserved sequence was found between the human HSP 60 antigen and the H. pylori chaperonin GroEL with an identity of 54% and a cover of 92%, followed by the alpha and gamma enolases and two H. pylori phosphopyruvate hydratase, both with an identity and cover of 48% and 96%, respectively. The H/K ATPase (Chain A) showed high identity with two H. pylori proteins (35.21% with both P-type ATPase), but with low cover (only 6%). We observed eight linear and three discontinuous epitopes for human HSP 60 and three lineal and one discontinuous epitope for both alpha-enolase and gamma enolase, high conserved with H. pylori sequences. Some type 1 gNET antigens shared potential cross-reactive epitopes with H. pylori proteins, suggesting that molecular mimicry could be a mechanism that explains the relationship between the infection and this disease. Studies evaluating the functional impact of this relationship are needed.

Human immune response against filarial HSP70 and its role in the diagnosis of lymphatic filariasis

A sensitive and specific diagnostic kit is crucial for the detection of human lymphatic filariasis at the early stage of infection as the existing diagnostic tools are inefficient and expensive. In the present study, we have cloned and expressed Brugia malayi HSP70 (BmHSP70) protein and characterized it as a potential antigen for diagnosis of the asymptomatic microfilariae stage of Wuchereria. bancrofti infection using ELISA, western blot, and bioinformatics tools. The antigenic efficacy of BmHSP70 was also compared with ScHSP70. The BmHSP70 and ScHSP70 peptide showed highly antigenic in nature and they showed immunogenic cross-reactivity endemic normal (EN) < chronic (CH) < microfilaraemic (MF) in IgG, IgG1, and IgG4 ELISA. IgG4-specific immunoblotting of BmHSP70 with MF sera further explicated its stage-specific antigenic cross-reactivity. These antigens (ScHSP70 and BmHSP70) showed a positive immunogenic correlation with the number of MF in blood samples. Thus, proposing BmHSP70 as a potential immunodiagnostic antigen against lymphatic filariasis. A triplet of GGMP tetrapeptide specific to the filarial HSP70 was also identified which was absent in human HSP70. In terms of sensitivity and specificity of antigens, these results suggest that recombinant BmHSP70 is a good antigen and could be used to diagnose early-stage of microfilariae infection.

Prophylactic antineoplastic activity of Toxoplasma gondii RH derived antigen against ehrlich solid carcinoma with evidence of shared antigens by comparative immunoblotting

BackgroundWith cancer cases escalation, an urgent request to develop novel combating strategies arise. Pathogen-based cancer-immunotherapy is getting more consideration. Autoclaved parasitic antigens seem promising candidates, taking steadily their first steps. Our aim was to examine the prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine (ATV) and to test for the shared antigen theory between Toxoplasma gondii and cancer cells.MethodsMice were immunized with ATV followed by Ehrlich solid carcinoma (ESC) inoculation. Tumor weight, volume, histopathology, and immunohistochemistry for CD8+ T cells, Treg cells and VEGF were assessed. In addition, the proposed shared antigen theory between parasites and cancer was also verified using SDS-PAGE and immunoblotting.ResultsResults revealed powerful prophylactic activity of ATV with 13.3% inhibition of ESC incidence, significant reduction in tumor weight and volume in ATV vaccinated mice. Immunologically, significantly higher CD8+T cells and lower FOXP3+ Treg cells surrounded and infiltrated ESC in ATV immunized mice with higher CD8+T/Treg cells ratio and significant antiangiogenic effect. Moreover, SDS-PAGE and immunoblotting showed four shared bands between Ehrlich carcinoma and ATV of approximate molecular weights 60, 26, 22 and 12.5 KDa.ConclusionExclusively, we demonstrated a prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine against ESC. Moreover, to the best of our knowledge this is the first report highlighting the existence of cross-reactive antigens between Toxoplasma gondi parasite and cancer cells of Ehrlich carcinoma.

Antigen specificity and cross-reactivity drive functionally diverse anti-Aspergillus fumigatus T cell responses in cystic fibrosis.

BACKGROUNDThe fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODSWe used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTSWe show that clonally expanded, high-avidity A. fumigatus-specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSIONOur data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDINGGerman Research Foundation (DFG), under Germany's Excellence Strategy (EXC 2167-390884018 "Precision Medicine in Chronic Inflammation" and EXC 2051-390713860 "Balance of the Microverse"); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Pre-sensitization with tuberculosis and non-tuberculosis mycobacteria and its impact on TB incidence in different age groups: Chengalpattu BCG trial revisited

Individuals pre-sensitized with Mycobacterium tuberculosis (MTB), non-tuberculosis mycobacteria, and its impact on TB incidence are relatively unexplored in a high TB burden setting. We conducted secondary data analysis of a double-blind, randomized Chengalpattu BCG trial, India. Induration to Purified Protein Derivative (PPD)-S and PPD-B were proxies for exposures to MTB and M. intracellulare respectively. Optimum cut-off for PPD-S and B were determined using Receiver-Operating Characteristic curves and induration ≥12 mm for PPD-S and ≥10 mm for PPD-B were considered strong reaction. Incidence rates of culture positive pulmonary TB per 100,000 person-years (PY) were calculated. Of 270,043 individuals with skin test results, children <14 years (n = 109,383, 64% showed strong-reaction to PPD-B and 17% to PPD-S) and adults between 25 and 34 years (n = 40,292, 98% were strong reactors to PPD-B and 73% to PPD-S). Overall incidence rate was lower in individuals with PPD-S < PPD-B (136, 95% CI: 130–141/100,000 PY) compared to individuals with PPD-S > PPD-B (447, 95% CI: 427–468/100,000 PY). Incidence rates of culture positive pulmonary TB was affected by early age of exposure to cross-reactive mycobacterial antigens represented by PPD-B and exposure to MTB represented by PPD-S during adolescence and early adulthood.

Tubulointerstitial nephritis and uveitis syndrome post-COVID-19.

Tubulointerstitial nephritis and uveitis syndrome post-COVID-19.

Subunit Vaccine Targeting Phosphate ABC Transporter ATP-Binding Protein, PstB, Provides Cross-Protection against Streptococcus suis Serotype 2, 7, and 9 in Mice

Streptococcus suis is a significant pathogen in pigs and a newly emerging zoonotic agent in humans. The presence of multiple serotypes and strains with diversified sequence types in pig herds highlights the need for the identification of broadly cross-reactive universal vaccine antigen targets, capable of providing cross-protection against S. suis infection. Subunit vaccines based on the conserved proteins shared between different S. suis serotypes are potential candidates for such a universally protective vaccine. In the present study, phosphate ABC transporter ATP-binding protein PstB (PstB), an immunogenic protein of the S. suis bacterium, was expressed and purified, and then subjected to cross-protection evaluation in mice. The PstB protein showed nearly 100% amino acid similarity across a panel of 31 S. suis isolates representing different serotypes, which were collected from different countries. A recombinant PstB (rPstB) protein (S. suis serotype 2) was recognized by rabbit sera specific to this serotype, and induced high levels of IFN-γ and IL-4 in mice immunized with the recombinant protein. These cytokines are considered important for protection against S. suis infection. Immunization of mice with rPstB resulted in an 87.5% protection against challenge with S. suis serotype 2 and 9 strains, suggesting a high level of cross-protection for S. suis serotypes 2 and 9. A lower protection rate (62.5%) was observed in mice challenged with the S. suis serotype 7 strain. These data demonstrate that PstB is a promising target antigen for development as a component of a universal subunit vaccine against multiple S. suis serotypes.

Abstract B003: Cross-reacting antigens between the intestinal microbiome and tumor neoantigen are associated with immune checkpoint inhibitor response in a gastrointestinal tumor cohort

Abstract Background: Immune checkpoint inhibitor (ICI) is a revolutionary therapy that promotes the activation of effector T cells against tumors. Despite the potential for long-term benefit, a large proportion of patients does not respond to ICI therapy. One reason for this heterogenic response is due to the high variability in the intestinal microbiota among different patients. The abundance of certain bacteria has been associated with different responses and fecal meta transplant from responding donors was shown to rescue a beneficial phenotype in refractory patients. The presence of cross-reactive antigens (CRAgs) between the gut microbiome and tumor neoantigens has been proposed as a potential mechanism that could link the intestinal microbiota with ICI response. However, empirical evidence of CRAg’s role in ICI therapy is limited to a few studies. Methods: We investigated ICI response in a cohort of 22 stage IV gastrointestinal (GI) tumor patients in which whole-exome and gut metagenomic sequencing data were available. This cohort included patients with gastric cancer (n=11), esophageal carcinoma (n=6), and colon cancer (n=5). For each patient, we characterized the tumor neoantigen load based on epitopes that show high affinity and presentability in HLA molecules. Neoantigens were matched to the intestinal microbiome to identify corresponding CRAgs. CRAg quality, which ranks CRAgs according to their immunogenic potential, was defined based on matches in immunogenic protein or epitope databases. Results: We found the total number of CRAgs varied per tumor type, with a higher abundance in colon cancer (median µ=619) followed by esophageal (µ =287.5) and gastric (µ = 243). Interestingly, colon cancer patients showed a better progression-free survival (PFS) curve followed by esophageal and gastric cancer patients, respectively. We also observed that CRAg load was better associated with longer PFS (p=.03) than neoantigen load (p=0.07). Moreover, CRAg’s quality showed to be an important predictor of PFS. Patients with a high proportion of CRAgs matching at least one of the quality databases showed better PFS (p=0.02). Further, a taxa-specific analysis indicated that commensal CRAgs load was a better predictor of PFS (p&amp;lt;0.008) than relative abundance. Altogether, our work supports a mechanistic link between the microbiome, neoantigens, and immuno-therapy. Citation Format: Ravin Poudel, Die Dai, Fang Li, Xiaochen Yin, Yan Kou, Antonio L. C. Gomes. Cross-reacting antigens between the intestinal microbiome and tumor neoantigen are associated with immune checkpoint inhibitor response in a gastrointestinal tumor cohort [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B003.

Dengue Fever with Corona Virus Disease 2019: Is it a “Double Trouble” with Concurrent Both Diseases or Single Disease with “Polyhedron” Nature and Antigenic Cross Reactivity?

Introduction: Corona virus disease 2019 (COVID-2019) disease caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2, is having propensity of pulmonary and extrapulmonary involvement. Although dengue virus has a predominant extrapulmonary effect, pulmonary involvement is less common. COVID-19–dengue overlap is a mixture of both diseases sharing few similarities. Methods: A multicentric observational study conducted from May 2021 to October 2021, in MIMSR Medical College and Venkatesh Hospital, Latur, India, included 300 COVID-19 cases with dengue NS1 or dengue immunoglobulin M (IgM) positive, with lung involvement documented and categorized on high-resolution computerized tomography (CT) thorax at the entry point. We have recorded demographic parameters as age, gender, comorbidity, and laboratory parameters such as total white blood cell count, platelet count, lactate dehydrogenase (LDH), C-reactive protein (CRP), interleukin-6 (IL-6), dengue serology, and use of BIPAP/NIV in COVID-19 cases in critical care settings in all study cases. CT severity scoring was done as per universally accepted standard mild if score < 7, moderated if score 8–15, and severe if score > 15. The final radiological outcome as presence or absence of fibrosis with clinical recovery was documented. All cases were subjected to dengue immunoglobulin G (IgG) antibody titers and dengue IgM/IgG antibody titer analysis after 12 weeks of discharge from the hospital after clinical recovery. Statistical analysis is done by using Chi-square test. Results: In a study of 300 COVID-19 pneumonia cases, COVID-19–dengue overlap was documented in 16.3% (49/300) of cases. Predominant age groups between 18–95 years and age ≥ 50 years were 60% (180/300), age < 50 were 40% (120/300). In the gender distribution in the study group, male population was 70.3% (211/300) and females were 29.7% (89/300). The main symptoms in the study group were shortness of breath in 79% of cases, fever in 71%, cough especially dry in 48%, and fatigability in 79%, tachycardia in 72%, tachypnea in 24%, and oxygen desaturation on 6 minutes walk in 29%. Positive dengue serology was documented as per CT severity scoring in 26/42 mild CT severity cases, 16/92 in moderate CT severity cases, and 7/166 in severe CT severity cases (P < 0.00001). Hematological parameters were having a significant association in COVID-19 cases with and without dengue overlap such as abnormal white blood cell count (P < 0.0076) and abnormal platelet count (P < 0.00001). Clinical parameters like hypoxia have a significant association in COVID-19 cases with and without dengue overlap (P < 0.00001). “Inflammatory markers” analysis such as interleukin 6 (IL-6) (P < 0.00001), CRP (P < 0.00001), and LDH (P < 0.00001) has documented significant association in COVID-19 cases with and without dengue overlap. In a study of 49 cases of COVID-19–dengue overlap, post-COVID lung fibrosis was documented in 1 case while 251 COVID-19 patients with negative dengue serology documented post-COVID lung fibrosis in 45 cases (P < 0.004). In a study of 49 cases of COVID-19–dengue overlap, actual serological assessment in dengue IgM/IgG and COVID-19 antibody titers was documented in significant association (P < 0.00001). Conclusion: COVID-19–dengue overlap is very frequently documented in tropical settings and disease of concern in critical care settings as the natural trend of this entity is different and has an impact on clinical outcome if diagnosis is delayed. COVID-19 pneumonia with dengue fever behaves like “two sides of the same coin” or the “polyhedron” nature of COVID-19 due to antigenic cross reactivity. Rationality for coexistent pathology is still undetermined.

Abstract PR019: Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence

Abstract Background: KRAS mutations drive tumorigenesis in &amp;gt;90% of pancreatic ductal adenocarcinomas (PDAC). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with metastatic KRAS-mutated solid tumors including PDAC. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. TCR therapies utilizing HLA class I-restricted TCRs often fail to harness CD4+ T cell activity vital for sustained anti-tumor immune responses. Here, we characterize HLA class I-restricted TCRs isolated from healthy donors and vaccinated patients targeting KRAS G12D or G12V, the most prevalent mKRAS variants observed in PDAC tumors. Methods: mKRAS-specific TCR sequences were identified from healthy donors or vaccinated PDAC patients enrolled on IRB-approved clinical studies at the University of Pennsylvania. Transgenic expression of synthetic TCRs was attained via lentiviral transduction of reporter cell lines or gene-edited (TCRabnull) primary CD8+ or CD4+ T cells. Limiting peptide dilution and combinatorial peptide library assays were used to assess antigen specificity, affinity and cross-reactivity. TCR reactivity to mKRAS cell lines was assessed by FACS analysis, in vitro cytotoxicity assays (4h 51Cr-release, xCELLigence Real Time Cell Analysis), and mouse xenograft tumor models. Results: TCRs exhibit specificity to the cognate KRAS G12D or G12V epitope without cross-reactivity to wild-type KRAS. TCRs are of high affinity and promote cytolysis of HLA-matched tumor cell lines harboring endogenous KRAS mutations irrespective of histologic origin. The adoptive transfer of gene-edited (TCRabnull) primary CD8+ T cells expressing synthetic TCRs exhibit potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. Select TCRs retain functional avidity independent of the CD8 co-receptor as determined by multimer binding and limiting peptide dilution assays. Disruption of the CD8:HLA binding interface via the introduction of D227A and T228A mutations within the HLA a3 domain has limited to no effect on TCR-mediated CD8+ T cell cytolytic activity. Transgenic expression of co-receptor independent TCRs by CD4+ T cells confers cytolytic activity against KRAS-mutated cell lines in both short- and long-term cytotoxicity assays, and flow cytometric analysis confirms antigen-specific expression of CD107a, IFN-g, TNF-a and IL-2 in a subset of CD4+ T cells. Conclusion: This study identifies TCRs with high therapeutic potency for the clinical development of TCR therapy trials targeting mKRAS in PDAC patients. Select TCRs exhibit CD8 co-receptor independence. CD4+ T cells redirected with co-receptor independent TCRs exhibit cytotoxic activity and express Th1 cytokines which may serve to enhance the persistence and anti-tumor activity of adoptive TCR therapy targeting mKRAS. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR019.

Abstract C004: TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most immune-resistant tumor types. Emerging evidence suggests improved clinical outcomes for subsets of PDAC patients with neoantigen-specific T cell immunity. A low-moderate tumor mutational burden limits antigenic targets in PDAC tumors, but recurrent missense mutations within the KRAS proto-oncogene drive tumorigenesis in &amp;gt;90% of cases. We and others have validated mutant KRAS (mKRAS) epitopes as immunological targets of T cell receptor (TCR)-based therapies. TCRs directed against mKRAS offer broad therapeutic potential. However, TCR degeneracy poses a major challenge to the clinical development of therapeutic TCRs given the risk of unforeseen off-target immunotoxicity due to peptide cross-reactivity. Here, we investigate the specificity and cross-reactivity of four TCRs specific for mKRAS G12V restricted to the HLA-A3 superfamily of HLA class I alleles. Of the four TCRs, one is restricted to HLA-A*03:01 (TCRA3V) and three are restricted to HLA-A*11:01 (TCRA11Va, TCRA11Vb, TCRA11Vc). All TCRs exhibit reactivity to the decamer peptide VVVGAVGVGK, while TCRA11Vc also exhibits reactivity to the nonamer peptide VVGAVGVGK. In-depth characterization of each TCR peptide recognition motif using alanine/glycine scanning libraries revealed TCRA3V and TCRA11Va-c demonstrate unique antigen binding motifs with a range of 3-6 critical amino acid residues, suggesting that substantial changes within the peptide:TCR interface may be tolerated for epitope recognition in the context of HLA-A*03:01 or HLA-A*11:01. TCR fingerprinting via combinatorial peptide libraries identified peptide repertoires capable of inducing TCR activation. Candidate cross-reactive peptides encoded in the human proteome were identified using the UnitProtKB database and filtered by computational epitope binding (NetMHC 4.0), setting a predicted peptide binding threshold of ≤500 nM. Using this approach, we identified a range of 2-35 candidate cross-reactive peptides for TCRA3V and TCRA11Va-c. Candidate cross-reactive peptides were synthesized and screened for their ability to induce TCR activation, which identified a single potential off-target peptide recognized by TCRA3V. Evaluation of tumor cell lines expressing high endogenous levels of this candidate cross-reactive antigen failed to elicit TCR activation, suggesting limited natural processing and presentation of this epitope. No potential off-target peptides were identified for TCRA11Va-c. Overall, this study serves as a platform to identify TCRs with high therapeutic potency and limited off-target potential for the clinical development of TCR therapy trials. Citation Format: Rebecca B. Nadler, Adham Bear, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C004.

Bordetella pertussis whole cell immunization protects against Pseudomonas aeruginosa infections

Whole cell vaccines are complex mixtures of antigens, immunogens, and sometimes adjuvants that can trigger potent and protective immune responses. In some instances, such as whole cell Bordetella pertussis vaccination, the immune response to vaccination extends beyond the pathogen the vaccine was intended for and contributes to protection against other clinically significant pathogens. In this study, we describe how B. pertussis whole cell vaccination protects mice against acute pneumonia caused by Pseudomonas aeruginosa. Using ELISA and western blot, we identified that B. pertussis whole cell vaccination induces production of antibodies that bind to lab-adapted and clinical strains of P. aeruginosa, regardless of immunization route or adjuvant used. The cross-reactive antigens were identified using immunoprecipitation, mass spectrometry, and subsequent immunoblotting. We determined that B. pertussis GroEL and OmpA present in the B. pertussis whole cell vaccine led to production of antibodies against P. aeruginosa GroEL and OprF, respectively. Finally, we showed that recombinant B. pertussis OmpA was sufficient to induce protection against P. aeruginosa acute murine pneumonia. This study highlights the potential for use of B. pertussis OmpA as a vaccine antigen for prevention of P. aeruginosa infection, and the potential of broadly protective antigens for vaccine development.

Antigenic Characterization and Pandemic Risk Assessment of North American H1 Influenza A Viruses Circulating in Swine.

The first pandemic of the 21st century was caused by an H1N1 influenza A virus (IAV) introduced from pigs into humans, highlighting the importance of swine as reservoirs for pandemic viruses. Two major lineages of swine H1 circulate in North America: the 1A classical swine lineage (including that of the 2009 H1N1 pandemic) and the 1B human seasonal-like lineage. Here, we investigated the evolution of these H1 IAV lineages in North American swine and their potential pandemic risk. We assessed the antigenic distance between the HA of representative swine H1 and human seasonal vaccine strains (1978 to 2015) in hemagglutination inhibition (HI) assays using a panel of monovalent antisera raised in pigs. Antigenic cross-reactivity varied by strain but was associated with genetic distance. Generally, the swine 1A lineage viruses that seeded the 2009 H1 pandemic were antigenically most similar to the H1 pandemic vaccine strains, with the exception of viruses in the genetic clade 1A.1.1.3, which had a two-amino acid deletion mutation near the receptor-binding site, which dramatically reduced antibody recognition. The swine 1B lineage strains, which arose from previously circulating (pre-2009 pandemic) human seasonal viruses, were more antigenically similar to pre-2009 human seasonal H1 vaccine viruses than post-2009 strains. Human population immunity was measured by cross-reactivity in HI assays to representative swine H1 strains. There was a broad range of titers against each swine strain that was not associated with age, sex, or location. However, there was almost no cross-reactivity in human sera to the 1A.1.1.3 and 1B.2.1 genetic clades of swine viruses, and the 1A.1.1.3 and 1B.2.1 clades were also the most antigenically distant to the human vaccine strains. Our data demonstrate that the antigenic distances of representative swine strains from human vaccine strains represent an important part of the rational assessment of swine IAV for zoonotic risk research and pandemic preparedness prioritization. IMPORTANCE Human H1 influenza A viruses (IAV) spread to pigs in North America, resulting in a sustained circulation of two major groups of H1 viruses in swine. We quantified the genetic diversity of H1 in swine and measured antigenic phenotypes. We demonstrated that the swine H1 lineages were significantly different from the human vaccine strains and that this antigenic dissimilarity increased over time as the viruses evolved in swine. Pandemic preparedness vaccine strains for human vaccines also demonstrated a loss in similarity with contemporary swine strains. Human sera revealed a range of responses to swine IAV, including two groups of viruses with little to no immunity. The surveillance and risk assessment of IAV diversity in pig populations are essential to detect strains with reduced immunity in humans and provide critical information for pandemic preparedness.

Differential patterns of cross-reactive antibody response against SARS-CoV-2 spike protein detected for chronically ill and healthy COVID-19 naïve individuals

Immunity to previously encountered viruses can alter response to unrelated pathogens. We reasoned that similar mechanism may also involve SARS-CoV-2 and thereby affect the specificity and the quality of the immune response against the virus. Here, we employed high-throughput next generation phage display method to explore the link between antibody immune response to previously encountered antigens and spike (S) glycoprotein. By profiling the antibody response in COVID-19 naïve individuals with a diverse clinical history (including cardiovascular, neurological, or oncological diseases), we identified 15 highly antigenic epitopes on spike protein that showed cross-reactivity with antigens of seasonal, persistent, latent or chronic infections from common human viruses. We observed varying degrees of cross-reactivity of different viral antigens with S in an epitope-specific manner. The data show that pre-existing SARS-CoV-2 S1 and S2 cross-reactive serum antibody is readily detectable in pre-pandemic cohort. In the severe COVID-19 cases, we found differential antibody response to the 15 defined antigenic and cross-reactive epitopes on spike. We also noted that despite the high mutation rates of Omicron (B.1.1.529) variants of SARS-CoV-2, some of the epitopes overlapped with the described mutations. Finally, we propose that the resolved epitopes on spike if targeted by re-called antibody response from SARS-CoV-2 infections or vaccinations can function in chronically ill COVID-19 naïve/unvaccinated individuals as immunogenic targets to boost antibodies augmenting the chronic conditions. Understanding the relationships between prior antigen exposure at the antibody epitope level and the immune response to subsequent infections with viruses from a different strain is paramount to guiding strategies to exit the COVID-19 pandemic.