Immune Response Assays Research Articles

Early, robust mucosal secretory IgA but not IgG response to SARS-CoV-2 spike in oral fluid is associated with faster viral clearance and COVID-19 symptom resolution.

High priority efforts are underway to support the development of novel mucosal COVID-19 vaccines, such as the US Government's Project NextGen and the Center for Epidemic Preparedness Innovations' goal to respond to the next pandemic with a new vaccine in 100 days. However, there is limited consensus about the complementary role of mucosal immunity in disease progression and how to evaluate immunogenicity of mucosal vaccines. This study investigated the role of oral mucosal antibody responses in viral clearance and COVID-19 symptom duration. Participants with PCR-confirmed SARS-CoV-2 infection provided oral fluid for testing with SARS-CoV-2 antibody multiplex assays, nasal swabs for RT-PCR and symptom information at up to eight follow-ups from April 2020 to February 2022. High and moderate oral fluid anti-spike (S) secretory IgA (SIgA) post infection was associated with significantly faster viral clearance and symptom resolution across age groups with effect sizes equivalent to having COVID-19 vaccine immunity at the time of infection. Those with high and moderate anti-S SIgA cleared the virus 14 days (95% CI: 10-18) and recovered 9-10 days (95% CI: 6-14) earlier. Delayed and higher anti-S IgG was associated with significantly longer time to clearance and recovery. Experiencing symptoms longer than four weeks was associated with lower anti-RBD SIgA 15-30 days after infection onset (p<0.001). Robust mucosal SIgA early post infection appears to support faster clearance of SARS-CoV-2 and recovery from COVID-19 symptoms. This research underscores the importance of harmonizing mucosal immune response assays to evaluate new mucosal vaccines.

Investigation of The Effect of Metal-based Medicine Arsenicum album on Humoral Immune Response in SRBC-immunized Mice.

In complementary and alternative medicinal systems, the Arsenicum album in ultra-high dilution was used in various therapeutic conditions, considering its effects on the body's immune system, including the COVID-19 pandemic. However, scientific evidence regarding its immunomodulatory effects is insufficient. The current study aimed to investigate the immunomodulatory effects of Arsenicum album in an experimental mouse model. Immunomodulatory activity of potentized dilutions of Arsenicum album i.e., 6C, 30C, 200C in BALB/c mice was evaluated by humoral antibody titer and delayedtype hypersensitivity assays wherein a fixed concentration (0.5 ml of 1× 109 cells/ml) of freshly prepared sheep RBC was administered as a foreign antigen to generate primary and secondary antibodies. Arsenicum album showed significant immunomodulatory activity by increasing primary antibody titer evaluated on day 21 of the treatment in all the dilutions as compared to SRBC and vehicle control group in humoral immune response assay without showing any effect on delayed-type hypersensitivity. The results of this preliminary study indicate that oral administration of Arsenicum album has the potential to augment primary humoral response at all dilutions. Hence, the possibility of using the Arsenicum album could be explored to treat immunological conditions, infections, etc., as an alternative therapy alongwith modern medicines.

Strain heterogeneity in a non-pathogenic Aspergillus fungus highlights factors associated with virulence

Fungal pathogens exhibit extensive strain heterogeneity, including variation in virulence. Whether closely related non-pathogenic species also exhibit strain heterogeneity remains unknown. Here, we comprehensively characterized the pathogenic potentials (i.e., the ability to cause morbidity and mortality) of 16 diverse strains of Aspergillus fischeri, a non-pathogenic close relative of the major pathogen Aspergillus fumigatus. In vitro immune response assays and in vivo virulence assays using a mouse model of pulmonary aspergillosis showed that A. fischeri strains varied widely in their pathogenic potential. Furthermore, pangenome analyses suggest that A. fischeri genomic and phenotypic diversity is even greater. Genomic, transcriptomic, and metabolic profiling identified several pathways and secondary metabolites associated with variation in virulence. Notably, strain virulence was associated with the simultaneous presence of the secondary metabolites hexadehydroastechrome and gliotoxin. We submit that examining the pathogenic potentials of non-pathogenic close relatives is key for understanding the origins of fungal pathogenicity.

GC/HRMS Analysis of E-Liquids Complements In Vivo Modeling Methods and can Help to Predict Toxicity.

Tobacco smoking is a major risk factor for disease development, with the user inhaling various chemicals known to be toxic. However, many of these chemicals are absent before tobacco is "burned". Similar, detailed data have only more recently being reported for the e-cigarette with regards to chemicals present before and after the e-liquid is "vaped." Here, zebrafish were dosed with vaped e-liquids, while C57-BL/6J mice were vaped using nose-cone only administration. Preliminary assessments were made using e-liquids and GC/HRMS to identify chemical signatures that differ between unvaped/vaped and flavored/unflavored samples. Oxidative stress and inflammatory immune cell response assays were then performed using our in vivo models. Chemical signatures differed, e.g., between unvaped/vaped samples and also between unflavored/flavored e-liquids, with known chemical irritants upregulated in vaped and unvaped flavored e-liquids compared with unflavored e-liquids. However, when possible respiratory irritants were evaluated, these agents were predominantly present in only the vaped e-liquid. Both oxidative stress and inflammatory responses were induced by a menthol-flavored but not a tobacco-flavored e-liquid. Thus, chemical signatures differ between unvaped versus vaped e-liquid samples and also between unflavored versus flavored e-liquids. These flavors also likely play a significant role in the variability of e-liquid characteristics, e.g., pro-inflammatory and/or cytotoxic responses.

DNA damage response-related immune activation signature predicts the response to immune checkpoint inhibitors: from gastrointestinal cancer analysis to pan-cancer validation.

DNA damage response (DDR) deficiency has emerged as a prominent determinant of tumor immunogenicity. This study aimed to construct a DDR-related immune activation (DRIA) signature and evaluate the predictive accuracy of the DRIA signature for response to immune checkpoint inhibitor (ICI) therapy in gastrointestinal (GI) cancer. A DRIA signature was established based on two previously reported DNA damage immune response assays. Clinical and gene expression data from two published GI cancer cohorts were used to assess and validate the association between the DRIA score and response to ICI therapy. The predictive accuracy of the DRIA score was validated based on one ICI-treated melanoma and three pan-cancer published cohorts. The DRIA signature includes three genes (CXCL10, IDO1, and IFI44L). In the discovery cancer cohort, DRIA-high patients with gastric cancer achieved a higher response rate to ICI therapy than DRIA-low patients (81.8% vs. 8.8%; P < 0.001), and the predictive accuracy of the DRIA score [area under the receiver operating characteristic curve (AUC) = 0.845] was superior to the predictive accuracy of PD-L1 expression, tumor mutational burden, microsatellite instability, and Epstein-Barr virus status. The validation cohort demonstrated that the DRIA score identified responders with microsatellite-stable colorectal and pancreatic adenocarcinoma who received dual PD-1 and CTLA-4 blockade with radiation therapy. Furthermore, the predictive performance of the DRIA score was shown to be robust through an extended validation in melanoma, urothelial cancer, and pan-cancer. The DRIA signature has superior and robust predictive accuracy for the efficacy of ICI therapy in GI cancer and pan-cancer, indicating that the DRIA signature may serve as a powerful biomarker for guiding ICI therapy decisions.

Exploring Antigenic Properties and Immune Responses in Bufo himalayanus: Implications for Conservation and Biodiversity Protection

Background: The objective of the study is to investigate antigenic property of soluble antigens of Nematodes Cosmocercoides dukae isolated from infected Bufo himalayanus (Class Amphibia, order: Anura) and to investigate the nature of protective immune response in laboratory rats. Bufo himalayanus is a species found in the districts of Darjeeling, Sikkim and adjoining hills of the sub-Himalayan belt, at an altitude of 6000 ft. above the sea level and known to be heavily infested by natural gut dwelling nematode infections. The present study is a small step towards protection and conservation of this amphibian species.&#x0D; Results: study of morphology of the nematode parasite confirmed it to be Cosmocercoides dukae. A crude whole body homogenate (antigenic protein) of the parasite was injected in healthy non-infected rats, spleen histo-architecture of treated rats differed from control rats. Humoral immune response assay by Gel Diffusion Precipitin (GDP) Test revealed that the parasite antigen invoked very little to no response. Study of the crude nematode antigenic protein extract concentration was 6.09 mg/ml and protein profile assay by SDS-PAGE showed seven major and several minor bands.&#x0D; Discussion: Though humoral immune response assay of parasite in experimental rats evoked very little or no immune response, this would not mean absence of antibodies, rather the antibody titers were presumably very low. It is to be noted that spleen of treated rats showed a distinctive histological feature. GDP test with infected toad serum also showed negative result. But protein profile assay of antigenic protein by SDS-PAGE showed many bands of proteins, which could be isolated and tested for further characterization.&#x0D; Conclusion: In order to maintain the Himalayan biodiversity, this amphibian species needs protection from such natural infective nematode parasites. If we are able to focus any light to this area, this knowledge might help the future research workers to plan for the protection of this species.

Induction of Cellular and Humoral Immune Responses Is Associated with Durable Remissions in MRD+ AML-Patients after Maintenance Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Maintaining remission in AML after intensive chemotherapy continues to be a challenge, specifically in patients with measurable residual disease (MRD+). Immunotherapy could be an effective way to induce durable disease control for patients in remission. This phase 2 study used an allogenic leukemia-derived dendritic cell vaccine to induce an effective anti-leukemic response to control or eradicated MRD, leading to durable disease control. (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients, in complete remission (CR1/CRi), MRD + and ineligible for allo-HSCT at inclusion, were scheduled to receive 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Patients aged 34-79 (median 60 years) had an ELN2017 risk score assessed of 13 favorable, 6 intermediate and 1 adverse, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). Patients were vaccinated after a median of 8.9 months (range 3 to 60.8 Mo) after reaching CR1/CRi. MRD was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses on bone marrow samples. Immune responses were evaluated on peripheral blood samples taken before and during treatment by a) IFNγ ELISPOT using three antigens (WT1, PRAME and RHAMM), known to be expressed by vididencel, by b) flow cytometry, using a single 40-marker panel, and c) skin biopsies taken 2 days after the first, fourth and sixth dose to assess immune responses in the skin. Evaluation of relapse-free and overall survival was done at data cut-off (3 rd July 2023). Results. All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received only one booster vaccination. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, after a median follow-up of 24.8 Mo (range 6.6- 54.8) (N=20) 14 patients are still alive with 12 being relapse-free for 24.4 to 54.7 months. The median RFS and OS (not shown) have not yet been reached (Figure 1A). Estimated RFS and OS at 12 months is 73% (range 51-86%) and 87% (range 67-95%), respectively, whereas the 24 months RFS is 59% (range 35-77%) and OS is 73% (range 50-87%). Induction of at least one vaccine induced response (VIR), as measured by IFNγ ELISPOT, was observed in 17 out of 20 patients, with a sustained vaccine induced response (sVIR) in 9 patients. There is a clear correlation (LogRank p=0.03) between number of VIRs and overall survival (Figure 1B), with best survival in patients with at least 3 VIRs. Of these patients, 4 out of 7 had shown MRD conversion (MRD -). Analysis of the immune cell composition at baseline and after vaccination revealed that higher baseline levels of B-cells (p &amp;lt;0.05) , cDC1, cDC2 were observed in patients remaining in CR. Vaccination also improved immune cell frequencies of dendritic cells with highest levels correlating with longer RFS (p&amp;lt;0.05) and OS (p&amp;lt;0.05). Immune histochemistry on skin biopsies confirmed the large influx of CD4+ and CD8+ T-cells as well as antigen presenting cells, in all patients explaining the clinical observation of swelling, warmth and redness in almost all patients. Conclusion/discussion. Vaccination with vididencel resulted in durable disease control, reflected by 2-year RFS and OS of 59% and 73%, respectively and the emerging plateau in the corresponding Kaplan Meier curves. Immune response assays indicate the induction of a strong immune response, with induction of specific T-cell responses towards tumor associated antigens, increased frequencies of B-cells and dendritic cells, all correlating with longer RFS and OS. The locally skin induced immune response leading to influx of a plethora of immune cells, confirmed the mode of action of immune priming through intradermal administration of the cancer vaccine. These results warrant further studies combining vididencel with SOC such as hypomethylating agents in AML maintenance.

A clinical trial of therapeutic vaccination in lymphoma with serial tumor sampling and single-cell analysis

AbstractIn situ vaccination (ISV) triggers an immune response to tumor-associated antigens at 1 tumor site, which can then tackle the disease throughout the body. Here, we report clinical and biological results of a phase 1/2 ISV trial in patients with low-grade lymphoma, combining an intratumoral toll-like receptor 9 (TLR9) agonist with local low-dose radiation and ibrutinib (an inhibitor of B- and T-cell kinases). Adverse events were predominately low grade. The overall response rate was 50%, including 1 complete response. All patients experienced tumor reduction at distant sites. Single-cell analyses of serial fine needle aspirates from injected and uninjected tumors revealed correlates of clinical response, such as lower CD47 and higher major histocompatibility complex class II expression on tumor cells, enhanced T-cell and natural killer cell effector function, and reduced immune suppression from transforming growth factor β and inhibitory T regulatory 1 cells. Although changes at the local injected site were more pronounced, changes at distant uninjected sites were more often associated with clinical responses. Functional immune response assays and tracking of T-cell receptor sequences provided evidence of treatment-induced tumor-specific T-cell responses. Induction of immune effectors and reversal of negative regulators were both important in producing clinically meaningful tumor responses. The trial was registered at www.clinicaltrials.gov as #NCT02927964.

Phage-derived anti-idiotype and anti-YTE antibodies in development of MK-1654 pharmacokinetic and immune response assays.

Background: MK-1654 is a fully human monoclonalantibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodiescan deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.

Deep neural networks predict class I major histocompatibility complex epitope presentation and transfer learn neoepitope immunogenicity.

Identifying neoepitopes that elicit an adaptive immune response is a major bottleneck to developing personalized cancer vaccines. Experimental validation of candidate neoepitopes is extremely resource intensive and the vast majority of candidates are non-immunogenic, creating a needle-in-a-haystack problem. Here we address this challenge, presenting computational methods for predicting class I major histocompatibility complex (MHC-I) epitopes and identifying immunogenic neoepitopes with improved precision. The BigMHC method comprises an ensemble of seven pan-allelic deep neural networks trained on peptide-MHC eluted ligand data from mass spectrometry assays and transfer learned on data from assays of antigen-specific immune response. Compared with four state-of-the-art classifiers, BigMHC significantly improves the prediction of epitope presentation on a test set of 45,409 MHC ligands among 900,592 random negatives (area under the receiver operating characteristic = 0.9733; area under the precision-recall curve = 0.8779). After transfer learning on immunogenicity data, BigMHC yields significantly higher precision than seven state-of-the-art models in identifying immunogenic neoepitopes, making BigMHC effective in clinical settings.

The role of Bcl-2 family genes in the physiology and immune response of the Pinctada fucata martensii

In the process of pearl production, Pinctada fucata martensii has a severe immune response, even leading to death. To effectively improve pearl production, researchers are committed to reducing immunological rejection through molecular biology methods. In previous studies, the apoptosis pathways were found to play an important function in the immune response of P. f. martensii. Bcl-2 family genes, the key genes in the apoptosis pathway, play a key role in regulating apoptotic pathways in cells. In the study, we cloned two genes of the P. f. martensii Bcl-2 family and named them respectively as PmBak/PmBcl-2. Fluorescence quantification of the pearl oysters' tissues showed that PmBak expression was highest in the hepatopancreas while PmBcl-2 expression was highest in the gill; nevertheless, different developmental stages showed that Bcl-2 family genes were highest in the blastula. Immune response assay using LPS, PGN, and poly I: C revealed that two genes were involved in the immune response; however, they showed relatively consistent expression patterns. To gain insight into the mechanism of action of the two genes, it was demonstrated by yeast two-hybrid experiments that PmBak and PmBcl-2 protein can interact with each other, while a subcellular localization assay revealed that Pmbak acts on mitochondria after apoptosis induction. In this study, we demonstrated the involvement of the Bcl-2 family of genes in the P. f. martensii immune response and explored the mechanism of the two genes. The study therefore provides insight into the mechanism of the apoptotic pathway in the immune response process of P. f. martensii.

Condyloma acuminata: An evaluation of the immune response at cellular and molecular levels.

Condyloma acuminata (CA) is a benign proliferative disease mainly affecting in non-keratinized epithelia. Most cases of CA are caused by low-risk human papillomavirus (HPV), mainly HPV 6 and 11. The aim of the current study was to highlight the candidate genes and pathways associated with immune alterations in individuals who did not spontaneously eliminate the virus and, thus, develop genital warts. Paraffin-embedded condyloma samples (n = 56) were analyzed by immunohistochemistry using antibodies against CD1a, FOXP3, CD3, CD4, CD8, and IFN-γ. The immunomarkers were chosen based on the evaluation of the innate and adaptive immune pathways using qPCR analysis of 92 immune-related genes, applying a TaqMan Array Immune Response assay in HPV 6 or HPV 11 positive samples (n = 27). Gene expression analysis revealed 31 differentially expressed genes in CA lesions. Gene expression validation revealed upregulation of GZMB, IFNG, IL12B, and IL8 and downregulation of NFATC4 and IL7 in CA samples. Immunohistochemical analysis showed increased FOXP3, IFN-γ, CD1a, and CD4 expression in CA than in the control tissue samples. In contrast, CD3 and CD8 expression was decreased in CA lesion samples. Increased levels of pro-inflammatory cytokines in HPV-positive patients compared with HPV-negative patients seem to reflect the elevated immunogenicity of HPV-positive CA lesions. Host defense against HPV begins during the early stages of the innate immune response and is followed by activation of T lymphocytes, which are mainly represented by CD4+ and regulatory T cells. The low CD8+ T cell count in CA may contribute to this recurrent behavior. Additional studies are needed to elucidate the mechanism of host defense against HPV infection in CA.

Persistence of immunity against hepatitis A in Brazilian children vaccinated with a single dose of inactivated virus vaccine.

In 2014, the Brazilian National Immunization Program implemented the universal vaccination against the hepatitis A virus (HAV) for children aged 12 months and older, applying a single dose of the inactivated virus vaccine. It is essential to carry out follow-up studies in this population, aiming to verify the longevity of HAV immunological memory. This study evaluated the humoral and cellular immune response of a cohort of children vaccinated between 2014 and 2015, and further investigated between 2015 and 2016, and who had their initial antibody response assessed after the single dose. A second evaluation took place in January 2022. We examined 109 children out of the 252 that took part in the initial cohort. Seventy (64.2%) of them had anti-HAV IgG antibodies. Cellular immune response assays were performed in 37 anti-HAV-negative and 30 anti-HAV-positive children. Production of interferon-gamma (IFN-y) stimulated with the VP1 antigen was demonstrated in 34.3% of these 67 samples. Of the 37 negative anti-HAV samples, 12 (32.4%) produced IFN-y. Among the 30 anti-HAV-positive, 11 (36.7%) produced IFN-y. In total, 82 (76.6%) children presented some type of immune response against HAV. These findings demonstrate the persistence of immunological memory against HAV in the majority of children vaccinated between 6 and 7 years with a single dose of the inactivated virus vaccine.

Evaluation of the host immune response assay SeptiCyte RAPID for potential triage of COVID-19 patients

Tools for the evaluation of COVID-19 severity would help clinicians with triage decisions, especially the decision whether to admit to ICU. The aim of this study was to evaluate SeptiCyte RAPID, a host immune response assay (Immunexpress, Seattle USA) as a triaging tool for COVID-19 patients requiring hospitalization and potentially ICU care. SeptiCyte RAPID employs a host gene expression signature consisting of the ratio of expression levels of two immune related mRNAs, PLA2G7 and PLAC8, measured from whole blood samples. Blood samples from 146 adult SARS-CoV-2 (+) patients were collected within 48 h of hospital admission in PAXgene blood RNA tubes at Hospital del Mar, Barcelona, Spain, between July 28th and December 1st, 2020. Data on demographics, vital signs, clinical chemistry parameters, radiology, interventions, and SeptiCyte RAPID were collected and analyzed with bioinformatics methods. The performance of SeptiCyte RAPID for COVID-19 severity assessment and ICU admission was evaluated, relative to the comparator of retrospective clinical assessment by the Hospital del Mar clinical care team. In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity: critical vs. mild (AUC = 0.93, p < 0.0001), critical vs. moderate (AUC = 0.77, p = 0.002), severe vs. mild (AUC = 0.85, p = 0.0003), severe vs. moderate (AUC = 0.63, p = 0.05). This discrimination was significantly better (by AUC or p-value) than could be achieved by CRP, lactate, creatine, IL-6, or D-dimer. Some of the critical or severe cases had “early” blood draws (before ICU admission; n = 33). For these cases, when compared to moderate and mild cases not in ICU (n = 37), SeptiCyte RAPID had AUC = 0.78 (p = 0.00012). In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity as defined by the WHO COVID-19 Clinical Management Living Guidance of January 25th, 2021. Measurements taken early (before a patient is considered for ICU admission) suggest that high SeptiScores could aid in predicting the need for later ICU admission.

Humoral and Cellular Immune Responses to Vector, Mix-and-Match, or mRNA Vaccines against SARS-CoV-2 and the Relationship between the Two Immune Responses.

ABSTRACTWe investigated how differences in age, sex, or vaccine type can affect humoral and cellular immune responses after vaccination with vector (ChAdOx1 nCoV-19), mix-and-match (first, ChAdOx1 nCoV-19, and second, BNT162b2), or mRNA (BNT162b2 or mRNA-1273) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Venous blood was collected from 573 subjects (vector, 396; mix-and-match, 96; and mRNA, 81) before the first vaccination (T0), 7 to 8 weeks (vector) or 3 to 4 weeks (mRNA) after the first vaccination (T1), and 3 to 4 weeks after the second vaccination (T2). The humoral and cellular immune responses were evaluated using Elecsys anti-SARS-CoV-2 (Roche), Alinity SARS-CoV-2 IgG II Quant (Abbott), cPass SARS-CoV-2 neutralization antibody detection (GenScript), and QuantiFERON SARS-CoV-2 (Qiagen) kits. At T1, the levels of the receptor-binding domain antibodies (RBD Ab) and neutralizing antibodies (NAb) decreased with aging, but interferon gamma release (IGR) levels increased. The RBD Ab, NAb, and IGR levels were higher in females than in males at T1 and T2. The NAb levels were higher in the mix-and-match and mRNA vaccine groups than in the vector vaccine group at T2. The RBD Ab and IGR levels were higher in the mRNA vaccine group than in the vector or mix-and-match vaccine groups at T2. The optimal cutoffs for RBD Ab and NAb, which were used to determine the presence of T cell responses, were 5.7 binding antibody units per milliliter (BAU mL−1) and 12.0 IU mL−1, respectively. Age, sex, and vaccine type affected the humoral and cellular immune responses, and T cell responses could be estimated from RBD Ab and NAb levels.IMPORTANCE There have been few studies that comprehensively evaluated factors affecting immune responses and the correlation between humoral and cellular immune responses after vector, mix-and-match, and mRNA vaccines against SARS-CoV-2. Therefore, we analyzed the effects of age, sex, and the different vaccine regimens on the immune responses to vaccination against SARS-CoV-2. The correlation between humoral and cellular immune responses and the cutoffs were derived for RBD antibodies and neutralizing antibodies to predict the presence of the cellular immune responses. In this comprehensive study, we demonstrated that there were differences in the immune responses induced after vaccination depending on the age and sex of an individual. Among the three vaccine regimens, the mix-and-match and mRNA vaccines induced the most robust immune responses. Finally, the proposed optimal cutoffs for RBD and neutralizing antibodies may be useful for predicting cellular immune responses when assays for cellular immune responses are not available.

Fabrication of Tissue-Engineered Cartilage Using Decellularized Scaffolds and Chondrocytes.

In this paper, we aim to explore the application value of tissue engineering for the construction of artificial cartilage in vitro. Chondrocytes from healthy porcine articular cartilage tissue were seeded on articular cartilage extracellular matrix (ACECM) scaffolds and cultivated. Type II collagen immunofluorescent staining was used to assess secretion from the extracellular matrix. Chondrocytes, which were mainly polygonal and cobblestone-shaped, were inoculated on ACECM-oriented scaffolding for 7 days, and the neo-tissue showed translucent shape and toughness. Using inverted and fluorescence microscopy, we found that chondrocytes on the scaffolds performed well in terms of adhesion and growth, and they secreted collagen type II. Moreover, the porcine ACECM scaffolds had good biocompatibility. The inflammatory cell detection, cellular immune response assay and humoral immune response assay showed porcine ACECM scaffolds were used for xenotransplantation without significant immune inflammatory response. All these findings reveal that ACECM-oriented scaffold is an ideal natural biomaterial for cartilage tissue engineering.

Abstract 2779: A live tumor fragment (LTF) platform with real-time imaging for immune response assays

Abstract Immuno-Oncology (IO) therapies provide remarkable clinical benefits. However, too few patients respond, and there are no diagnostic tools that predict IO response with high accuracy. Moreover, as more IO drugs and combinations are approved, selecting the best IO-based regimen for each patient will become more complex. To facilitate this selection, we are developing an ex vivo live tumor fragment (LTF) screening platform that retains representation of a patient’s tumor microenvironment (TME) including immune cells, enabling development of deep neural networks to predict IO response and thereby individualize therapy. Here we present preliminary data from our proof-of-concept platform that performs tissue fragmentation/sorting, liquid handling, drug treatment, high-resolution dynamic imaging, and multiplex immuno-assays. Human tumor excisions were obtained from the Univ. of Wisconsin (IRB approved), and CT26 tumors were grown in mice subcutaneously. Live tumors were cut into 300 x 300 µm fragments of 100 - 300 µm thickness, sorted into multi-well plates, and cultured for 48 or 72 h in the presence or absence of anti-PD1 (nivolumab or mouse equivalent), anti-PD1 plus anti-CTLA4 (ipilimumab or mouse equivalent), or concanavalin A (ConA) as a positive control. Cell viability was measured by ATP luminescence assay or flow cytometry. Motility of CD8+ cells was tracked in human LTF using camelid VHH anti-hCD8a-AF594 and 3D multiphoton microscopy for 30 min. Supernatant cytokines were measured using bead immunoassay and T cell markers by flow cytometry. T cells were retained within LTFs, and the proportion of lymphocytes in LTFs was independent of fragment thickness (1.6%/2.1% for 300 µm, 1.4%/2.3% for 200 µm and 1.4%/2.8% for 100 µm thickness, for CD4+ and CD8+ T cells, respectively). Total cell viability and T cell viability exceeded 80% at 48h, and 3D LTF structure remained intact for at least 48 h. We treated LTFs with anti-PD1, anti-PD1 plus anti-CTLA4, or ConA and confirmed the presence of IFN-γ and 10 (mouse) or 16 (human) other cytokines associated with immune activation in both the ConA and anti-PD1 treated samples, but not the control. In human LTFs, cytokine panel upregulation was observed for anti-PD1 vs. control (p=1.1e-5) and anti-PD1 plus anti-CTLA4 vs. anti-PD1 (p=3.7e-9). Using multiphoton microscopy and CD8-binding nanobodies, we observed vigorous CD8+ T cell motility in human LTFs, with a speed of 10 µm/min, which is comparable to that reported in vivo. Our LTF platform has an immuno-competent TME in which we can detect cellular and secreted immune response markers, compare alternative treatments, and track the surveillance activity of infiltrating T cells. Future work will further advance the platform, enabling clinical trials for training and validating deep neural networks to predict response to checkpoint inhibitors and other IO drugs. Citation Format: Tomasz Zal, Leung Kau Tang, Laura Hrycyniak, Anura Shrestha, Dinesh Joshi, Pichet Adstamongkonkul, Kelsey Tweed, Wesley A. Cox-Muranami, Sean Caenepeel, Hinco J. Gierman, Kevin Eliceiri, Kristina A. Matkowskyj, Sam J. Lubner, John Rafter, Mike Szulczewski, Maneesh Arora, Jonathan Oliner. A live tumor fragment (LTF) platform with real-time imaging for immune response assays [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 2779.

Optimization of immune receptor-related hypersensitive cell death response assay using agrobacterium-mediated transient expression in tobacco plants

BackgroundThe study of the regulatory mechanisms of evolutionarily conserved Nucleotide-binding leucine-rich repeat (NLR) resistance (R) proteins in animals and plants is of increasing importance due to understanding basic immunity and the value of various crop engineering applications of NLR immune receptors. The importance of temperature is also emerging when applying NLR to crops responding to global climate change. In particular, studies of pathogen effector recognition and autoimmune activity of NLRs in plants can quickly and easily determine their function in tobacco using agro-mediated transient assay. However, there are conditions that should not be overlooked in these cell death-related assays in tobacco.ResultsEnvironmental conditions play an important role in the immune response of plants. The system used in this study was to establish conditions for optimal hypertensive response (HR) cell death analysis by using the paired NLR RPS4/RRS1 autoimmune and AvrRps4 effector recognition system. The most suitable greenhouse temperature for growing plants was fixed at 22 °C. In this study, RPS4/RRS1-mediated autoimmune activity, RPS4 TIR domain-dependent cell death, and RPS4/RRS1-mediated HR cell death upon AvrRps4 perception significantly inhibited under conditions of 65% humidity. The HR is strongly activated when the humidity is below 10%. Besides, the leaf position of tobacco is important for HR cell death. Position #4 of the leaf from the top in 4–5 weeks old tobacco plants showed the most effective HR cell death.ConclusionsAs whole genome sequencing (WGS) or resistance gene enrichment sequencing (RenSeq) of various crops continues, different types of NLRs and their functions will be studied. At this time, if we optimize the conditions for evaluating NLR-mediated HR cell death, it will help to more accurately identify the function of NLRs. In addition, it will be possible to contribute to crop development in response to global climate change through NLR engineering.

The Meloidogyne javanica effector Mj2G02 interferes with jasmonic acid signalling to suppress cell death and promote parasitism in Arabidopsis.

Plant‐parasitic nematodes can cause devastating damage to crops. These nematodes secrete effectors that suppress the host immune responses to enhance their survival. In this study, Mj2G02, an effector from Meloidogyne javanica, is described. In situ hybridization and transcriptional analysis showed that Mj2G02 was highly expressed in the early infection stages and exclusively expressed in the nematode subventral oesophageal gland cells. In planta RNA interference targeting Mj2G02 impaired M. javanica parasitism, and Mj2G02‐transgenic Arabidopsis lines displayed more susceptibility to M. javanica. Using an Agrobacterium‐mediated transient expression system and plant immune response assays, we demonstrated that Mj2G02 localized in the plant cell nuclei and could suppress Gpa2/RBP‐1‐induced cell death. Moreover, by RNA‐Seq and quantitative reverse transcription PCR analyses, we showed that Mj2G02 was capable of interfering with the host jasmonic acid (JA) signalling pathway. Multiple jasmonate ZIM‐domain (JAZ) genes were significantly upregulated, whereas the JAR1 gene and four JA‐responsive genes, MYC3, UPI, THI2.1, and WRKY75, were significantly downregulated. In addition, HPLC analysis showed that the endogenous jasmonoyl‐isoleucine (JA‐Ile) level in Mj2G02‐transgenic Arabidopsis lines was significantly decreased compared to that in wildtype plants. Our results indicate that the M. javanica effector Mj2G02 suppresses the plant immune response, therefore facilitating nematode parasitism. This process is probably mediated by a JA‐Ile reduction and JAZ enhancement to repress JA‐responsive genes.

A DNA-damage immune response assay combined with PET biomarkers predicts response to neo-adjuvant chemotherapy and survival in oesophageal adenocarcinoma

18F-fluorodeoxyglucose PET-CT may guide treatment decisions in patients with oesophageal adenocarcinoma (OAC). This study evaluated the added value of maximum standardised uptake value (SUVmax) to a novel DNA-damage immune response (DDIR) assay to improve pathological response prediction. The diagnostic accuracy of PET response and the prognostic significance of PET metrics for recurrence-free survival (RFS) and overall survival (OS) were assessed. This was a retrospective, single-centre study of OAC patients treated with neo-adjuvant chemotherapy from 2003 to 2014. SUVmax was recorded from baseline and repeat PET-CT after completion of pre-operative chemotherapy. Logistic regression models tested the additional predictive value of PET metrics combined with the DDIR assay for pathological response. Cox regression models tested the prognostic significance of PET metrics for RFS and OS. In total, 113 patients were included; 25 (22.1%) were DDIR positive and 88 (77.9%) were DDIR negative. 69 (61.1%) were PET responders (SUVmax reduction of 35%) and 44 (38.9%) were PET non-responders. After adding PET metrics to DDIR status, post-chemotherapy SUVmax (hazard ratio (HR) 0.75, p = 0.02), SUVmax change (HR 1.04, p = 0.003) and an optimum SUVmax reduction of 46.5% (HR 4.36, p = 0.021) showed additional value for predicting pathological response. The optimised SUVmax threshold was independently significant for RFS (HR 0.47, 95% CI 0.26–0.85, p = 0.012) and OS (HR 0.51, 95% CI 0.26–0.99, p = 0.047). This study demonstrated the additional value of PET metrics, when combined with a novel DDIR assay, to predict pathological response in OAC patients treated with neo-adjuvant chemotherapy. Furthermore, an optimised SUVmax reduction threshold for pathological response was calculated and was independently significant for RFS and OS.