Neutralizing Antibody Assay Research Articles

Arbovirus in Solid Organ Transplants: A Narrative Review of the Literature.

The incidence of arbovirus infections has increased in recent decades. Other than dengue, chikungunya, and West Nile viruses, the data on arbovirus in solid organ transplant (SOT) are limited to case reports, and infections in renal transplant recipients account for most of the reported cases. Dengue and West Nile infections seem to be more severe with higher mortality in SOT patients than in the general population. Acute kidney injury is more frequent in patients with dengue and chikungunya although persistent arthralgia with the latter is less frequent. There is no clear relationship between arboviral infection and acute cellular rejection. Pre-transplant screening of donors should be implemented during increased arboviral activity but, despite donor screening and negative donor nucleic acid amplification test (NAT), donor derived infection can occur. NAT may be transiently positive. IgM tests lack specificity, and neutralizing antibody assays are more specific but not readily available. Other tests, such as immunohistochemistry, antigen tests, PCR, metagenomic assays, and viral culture, can also be performed. There are a few vaccines available against some arboviruses, but live vaccines should be avoided. Treatment is largely supportive. More data on arboviral infection in SOT are needed to understand its epidemiology and clinical course.

Complex immunogenicity assessment in caplacizumab-treated patients with immune-mediated thrombotic thrombocytopenic purpura who have received plasma exchange

BackgroundISTH guidelines for immune-mediated thrombotic thrombocytopenic purpura (iTTP) treatment recommend concurrent therapeutic plasma exchange (TPE), immunosuppressive therapy (IST), and caplacizumab. TPE can complicate anti-drug antibody (ADA) measurements by transferring pre-existing antibodies (pre-Abs) into patients via donor plasma and/or diluting treatment-emergent (TE) ADAs. ObjectivesTo assess the presence of ADAs in patients with iTTP who received caplacizumab. MethodsImmunogenicity data from patients with iTTP receiving caplacizumab once daily plus TPE/IST in four clinical trials (TITAN, HERCULES, Post-HERCULES, and a trial conducted in Japanese patients) in the clinical development program were analyzed. ADA and modified ADA (mADA) assays differentiated pre-Abs from TE ADAs. A functional neutralizing antibody (NAb) assay and neutralizing epitope characterization assay (NECA) assessed the presence of ADAs with neutralizing potential. The impact of ADAs on efficacy, pharmacokinetics/pharmacodynamics, and safety was evaluated. ResultsAmong 228 patients in four studies, prevalence of pre-Abs ranged from 17.1% (TITAN) to 56.7% (HERCULES), while TE ADA prevalence ranged from 3.1% (HERCULES) to 14.3% (Japanese study). The TE NAb-positive rate ranged from 0% (Japanese study) to 12% (Post-HERCULES) using the functional NAb assay and from 2.7% (Post-Hercules) to 14.3% (Japanese study) using NECA. The presence of these antibodies did not impact treatment efficacy or safety. ConclusionsA complex immunogenicity assay strategy was required to define the pre-Ab/TE ADA status of patients with iTTP treated with caplacizumab in a clinical trial setting. In addition to the wide range of pre-Abs observed, few patients had detectable TE ADAs or NAbs, neither of which impacted on efficacy/safety.

Improved efficacy of SARS-CoV-2 isolation from COVID-19 clinical specimens using VeroE6 cells overexpressing TMPRSS2 and human ACE2

The cell culture-based isolation of novel coronavirus SARS-CoV-2 from clinical specimens obtained from patients with suspected COVID-19 is important not only for laboratory diagnosis but also for obtaining live virus to characterize emerging variants. Previous studies report that monkey kidney-derived VeroE6/TMPRSS2 cells allow efficient isolation of SARS-CoV-2 from clinical specimens because these cells show stable expression of the receptor molecule monkey ACE2 and the serine-protease TMPRSS2. Here, we demonstrated that VeroE6 cells overexpressing human ACE2 and TMPRSS2 (Vero E6-TMPRSS2-T2A-ACE2 cells) are superior to VeroE6/TMPRSS2 for isolating SARS-CoV-2 from clinical specimens. These cells showed a 1.6-fold increase in efficiency in SARS-CoV-2 isolation, and were particularly effective for clinical specimens with a relatively low viral load (< 106 copies/mL). When using vesicular stomatitis virus (VSV) pseudoviruses (VSV/SARS-2pv) bearing the spike proteins of all of the tested SARS-CoV-2 strains, Vero E6-TMPRSS2-T2A-ACE2 cells showed a 2– to fourfold increase in infectivity. Furthermore, the results of virus titration and neutralization antibody assays using Vero E6-TMPRSS2-T2A-ACE2 cells were different from those using VeroE6/TMPRSS2, highlighting the importance of selecting appropriate cell culture systems to determine SARS-CoV-2 infectivity.

B-043 Q-nab™ Igg kit: verification and validation of a novel cell-free quantitative neutralizing antibody assay

Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.

Performance evaluation of four kits for the detection of neutralizing antibody against SARS-CoV-2 in human serum

To evaluate four novel SARS-CoV-2 neutralizing antibody assay kits' application in neutralizing antibodies of population. Questionnaires from the voluntary participating researchers and selected the qualified questionnaires to analyse. For negative and positive coincidence rate, four novel SARS-Cov-2 neutralization antibody assay kits were tested. For within-run and between-run Precision verification study, four serum samples with two high and two low titer neutralizing antibodies were used to analyse. Based on the questionnaires, 175 qualified samples were divided into two groups. (1) negative neutralizing antibodies group: 31 samples had not been infected with the novel SARS-Cov-2 nor received the vaccine within the past one year; (2) positive neutralizing antibodies group: 144 samples were infected by COVID-19. There was 28 negative and 3 positive neutralizing antibodies of the individuals among the 31 negative samples which based on the questionnaires. The negative rates of 28 negative individules tested by GenScript, Vazyme and Hygeianey were 82.14 %, 60.71 % and 17.85 %, while the positive rates of the 147 positive samples were 93.87 %, 95.23 % and 100 %. The within-run coefficient of variations (C·V) of PBNAs, GenScript, Vazyme and Hygeianey were 11.49 %, 9.12 %, 7.97 % and 7.48 %, while the between-run coefficient of variations (C·V) were 21.37 %, 14.21 %, 12.29 % and 11.78 %. Due to the large within-run and between-run coefficient of variations, PBNAs was not suitable for large-scale promotion, while ELISAs could be leveraged for routine monitoring the titer of neutralizing antibodies against SARS-CoV-2.

Neutralizing Antibody Sample Testing and Report Harmonization

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.Graphical

Establishment and preliminary application of neutralizing antibody detection method for human respiratory syncytial virus

Objective: To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application. Methods: The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins). Results: Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient rs was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion: The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC).Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target.Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance wereachieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Lysophosphatidic acid promotes ESCC progression by increasing the level of CCL2 secreted by esophageal epithelial cells.

Lysophosphatidic acid (LPA) is a small bioactive lipid which acts as a potent regulator in various tumor progressions through six G-protein-coupled receptors (LPA1-LPA6). Our previous study demonstrated that the LPA-producing enzyme, autotaxin (ATX), was upregulated in esophageal squamous cell carcinoma (ESCC) and ATX high expression levels indicated a poor prognosis. Esophageal squamous cell carcinoma is a type of malignant tumor which originates from epithelial cells. Its progression can be affected by the interaction between cancer cells and normal cells. However, the impact of LPA on the interaction between esophageal epithelial cells and cancer cells in the development of ESCC remains uncertain. MTS and Edu assays were performed to determine ESCC cell proliferation in culture medium (CM) derived from LPA-stimulated esophageal epithelial cells (Het-1a). A wound healing assay, transwell migration and an invasion assay were performed to assess the metastatic ability of ESCC cells. Cytokine array analysis was conducted to detect the differentially secreted cytokines in CM. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized to uncover the pathways and cytokines that are influenced by LPA in ESCC. Immunohistochemical staining was employed to measure the expression of ATX and CCL2 in early-stage ESCC. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay and an antibody neutralization assay were employed to measure the mechanism of LPA-mediated communication between epithelial cells and cancer cells. Functional experiments showed that exposing ESCC cancer cells to CM from LPA-treated Het-1a results in promoting proliferation, migration, invasion and epithelial-mesenchymal transition processes. Using cytokine array analysis, we discovered that LPA triggers the release of multiple cytokines from epithelial cells. After screening of the TCGA and GEO databases, CCL2 was identified and found to be correlated with ATX expression in ESCC. Furthermore, CCL2 levels in both mRNA expression and secretion were observed to be upregulated in epithelial cells upon stimulation with LPA. Blocking CCL2 effectively reduced the pro-migration influence of CM derived from LPA-treated Het-1a. Mechanism studies have demonstrated that LPA activated the NF-κB signaling pathway through LPA1/3, ultimately causing an increase in CCL2 expression and secretion in Het-1a. Our findings, taken together, demonstrate that CM from LPA-treated esophageal epithelial cells plays a significant role in promoting the progression of ESCC, with CCL2 acting as the primary regulator.

Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies

Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease in-vitro BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs in vitro by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies in vitro using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays.“Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays in vitro”.

Profiling of Early Immune Responses to Vaccination Using THP-1-Derived Dendritic Cells.

The COVID-19 pandemic has made assessing vaccine efficacy more challenging. Besides neutralizing antibody assays, systems vaccinology studies use omics technology to reveal immune response mechanisms and identify gene signatures in human peripheral blood mononuclear cells (PBMCs). However, due to their low proportion in PBMCs, profiling the immune response signatures of dendritic cells (DCs) is difficult. Here, we develop a predictive model for evaluating early immune responses in dendritic cells. We establish a THP-1-derived dendritic cell (TDDC) model and stimulate their maturation in vitro with an optimal dose of attenuated yellow fever 17D (YF-17D). Transcriptomic analysis reveals that type I interferon (IFN-I)-induced immunity plays a key role in dendritic cells. IFN-I regulatory biomarkers (IRF7, SIGLEC1) and IFN-I-inducible biomarkers (IFI27, IFI44, IFIT1, IFIT3, ISG15, MX1, OAS2, OAS3) are identified and validated in vitro and in vivo. Furthermore, we apply this TDDC approach to various types of vaccines, providing novel insights into their early immune response signatures and their heterogeneity in vaccine recipients. Our findings suggest that a standardizable TDDC model is a promising predictive approach to assessing early immunity in DCs. Further research into vaccine efficacy assessment approaches on various types of immune cells could lead to a systemic regimen for vaccine development in the future.

Selection of viral variants with enhanced transmission and reduced neutralization susceptibility alongside lateral introductions may explain the persistence of porcine reproductive and respiratory syndrome virus in vaccinated breeding herds.

This study investigates the long-term evolutionary dynamics of porcine reproductive and respiratory syndrome virus (PRRSV-1) in an endemically infected and vaccinated pig herd. Over a one year and a half period, piglets from seven farrowing batches in a 300-sow PRRSV-vaccinated farm were monitored from birth to nine weeks of age by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Eighty-five PRRSV-positive samples were subjected to whole genome sequencing (Illumina Miseq), and 251 samples to open reading frame 5 (ORF5) sequencing. Farm-specific PRRSV variants' impact on anti-PRRSV antibodies was evaluated using enzyme-linked immunosorbent and neutralizing antibody assays. The replication kinetics and cytokine inhibition capabilities (IFN-α and TNF-α) of these variants were assessed in porcine alveolar macrophages. The study revealed fluctuating PRRSV-1 incidences in farrowing units and nurseries, attributed to two key evolutionary events: an escape variant emergence and a lateral introduction of a new strain. Initially, strain 1 variant α was swiftly replaced within weeks by variant 1β (99.5 per cent genomic similarity), with twenty-five amino acid mutations, primarily in nsp1α, GP2, GP3, and GP5, including an additional glycosylation site and a deletion downstream the neutralization epitope of GP5. This shift to 1β correlated with increased incidence in nurseries and higher viral loads, with sera from 1α-exposed animals showing reduced neutralization against 1β. Consistently for in vitro assays, variant 1β demonstrated enhanced replication in porcine alveolar macrophages but no difference regarding IFN-α or TNF-α responses. Later, a new strain (strain 2, 83.3 per cent similarity to strain 1) emerged and led to incidence resurgence because of the low cross reactivity with the previous antibodies. The study highlights PRRSV's rapid adaptability and challenges in controlling its spread, underscoring the necessity for more effective vaccines and eradication approaches.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays

BackgroundThe highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. MethodsTo generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. ResultsWe successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. SignificanceThis assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

Tailored different sizes of quantum dot nanobeads for sensitive and quantitative detection based on the competition fluorescence-linked immunosorbent assay platform

Highly stable and multicolor photoluminescent (PL) quantum dots (QDs) have attracted widespread attention as ideal probe materials in the field of in vitro diagnostics (IVD), especially the fluorescence-linked immunosorbent assay (FLISA), due to their advantages of high-throughput, high stability, and high sensitivity. However, the size of QDs as fluorescent probes have significant effects on antigen-antibody performance. Therefore, it is critical to design suitable QDs for obtain excellent quantitative detection-based biosensors. In this paper, we prepared different sizes of aqueous QDs (30 nm, 116 nm, 219 nm, and 320 nm) as fluorescent probes to optimize the competitive FLISA platform. The SARS-CoV-2 neutralizing antibody (NTAB) assay was used as an example, and it was found that the size of the QDs has a significant impact on the antigen-antibody binding efficiency and detection sensitivity in competitive FLISA platform. The results showed that these QD nanobeads (QBs, ∼219 nm) could be used as a labeled probe for competitive FLISA, with half-maximal inhibitory concentration (IC50) of 1.34 ng/mL and limit of detection (LOD) of 0.21 pg/mL for NTAB detection. More importantly, the results showed good specificity and accuracy, and the QB219 probe was able to efficiently bind NTAB without interference from other substances in the serum. Given the above advantages, the nanoprobe material (∼200 nm) offers considerable potential as a competitive FLISA platform in the field of IVD.

An inactivated PDCoV vaccine induces robust neutralizing antibodies and immune protection in pigs lasting for three months

Porcine deltacoronavirus (PDCoV), a novel enteropathogenic coronavirus, causes diarrhea mainly in suckling piglets and has the potential to infect humans. Whereas, there is no commercially available vaccine which can effectively prevent this disease. In this study, to ascertain the duration of immune protection of inactivated PDCoV vaccine, suckling piglets were injected subcutaneously with inactivated PDCoV vaccine using a prime/boost strategy at 3 and 17-day-old. Neutralizing antibody assay showed that the level of the inactivated PDCoV group was still ≥1:64 at three months after prime vaccination. The three-month-old pigs were orally challenged with PDCoV strain CZ2020. Two pigs in challenge control group showed mild to severe diarrhea at 10-11 day-post-challenge (DPC), while the inactivated PDCoV group had no diarrhea. High levels of viral shedding, substantial intestinal villus atrophy, and positive straining of viral antigens in ileum were detected in challenge control group, while the pigs in inactivated PDCoV group exhibited significantly reduced viral load, minor intestinal villi damage and negative straining of viral antigens. These results demonstrated that PDCoV was pathogenic against three-month-old pigs and inactivated PDCoV vaccine can provide effective protection in pigs lasting for three months.

Altered transcriptomic immune responses of maintenance hemodialysis patients to the COVID-19 mRNA vaccine.

End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines. The immune response to the COVID-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and 7 d after the second dose (V2D7) using anti-spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified 6 mo after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response. Transcriptomic analyses demonstrated differing time courses of immune responses, with prolonged myeloid cell activity in HD at 1 wk after the first vaccination dose. HD also demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p<0.05). Anti-spike IgG remained elevated above baseline at 6 mo in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development. Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance HD subjects comparable to healthy controls and identify transcriptomic and clinical predictors of anti-spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of ESRD. F30HD102093, F30HL151182, T32HL144909, R01HL138628. This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.

Abstract 2030: N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is FDA-approved for the treatment of glioblastoma and unresectable pleural mesothelioma, and has recently demonstrated benefit (together with an immune checkpoint inhibitor) for the treatment of metastatic non-small cell lung carcinoma (NSCLC) progressing on or after platinum-based therapy. The current study aimed to uncover cellular response mechanisms to TTFields. Methods: A2780 ovarian cancer cells and H1299 NSCLC cells were treated with TTFields for 72 h (200 and 150 kHz, respectively; 1.7 V/cm RMS). Changes in signaling pathways were analyzed using the Luminex multiplex assay and validated by protein expression. Fluorescence microscopy, calcium switch assay, antibody neutralization assay, and immunoprecipitation were employed to investigate the mechanism of action. The efficacy of concomitant treatment with TTFields and a potential inhibitor was tested in cell lines (cytotoxic and clonogenic effects) and two orthotopic mouse models: MOSE-L-FFL ovarian cancer and LL/2 lung cancer. Immunohistochemistry was performed on tumor sections from both models. Results: Increased AKT phosphorylation was observed in the cancer cells treated with TTFields. N-cadherin, known to be involved in the activation of the PI3K/AKT pathway, was elevated in the membranes of cells treated with TTFields. Elimination of calcium ions required for N-cadherin homophilic ligation abrogated TTFields-induced AKT phosphorylation, whereas calcium supplementation restored AKT phosphorylation. Inhibition of N-cadherin-mediated cell-cell contacts by neutralizing antibody resulted in a significant reduction in TTFields-induced AKT phosphorylation. Pull-down assays with an anti-N-cadherin antibody demonstrated increased recruitment of the p85 regulatory subunit of PI3K to N-cadherin complexes following TTFields application. TTFields-induced activation of AKT could be mitigated in cell cultures as well as in animal models by application of the PI3K inhibitor alpelisib. Co-treatment with alpelisib also enhanced the effectiveness of TTFields in vitro and in vivo. Conclusions: The PI3K/AKT signaling pathway, activated via cell-cell N-cadherin interactions, plays a role in the cancer cell response to TTFields treatment. Inhibiting the PI3K/AKT pathway may potentially sensitize tumors to TTFields. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Kerem Wainer-Katsir, Alexandra Volodin, Bella Koltun, Boris Brant, Yiftah Barsheshet, Tal Kan, Cfir David, Tharwat Haj Khalil, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2030.

Real-world data on the Immunity Response to the COVID-19 Vaccine among Patients with Central Nervous System Immunological Diseases

Objective: The effects of immunotherapies on the immune response to various regimens of SARS-CoV-2 vaccines in patients with autoimmune neurological disease have been demonstrated in limited data. Thus, we evaluated the immune responses in each platform of COVID-19 vaccination between patients with autoimmune neurological disease and a healthy population. Materials and Methods: We conducted a prospective observational study. We collected serum from patients with autoimmune neurological diseases to perform serological methods using anti-RBD IgG assay, neutralizing antibodies assay, and interferon SARS-CoV-2 immunoassay. Serological response level was analyzed by platforms of vaccines and types of immune modifying therapy. Results: Fifty-eight patients had tested for an anti-RBD IgG response, and those receiving no immunotherapy/ healthy controls had the highest median anti-RBD IgG levels amongst immunotherapy statuses. Rituximab in those who received inactivated or mRNA vaccine regimens had the lowest antibody level compared with other immunotherapies. In vector-based vaccine regimens, significant reductions of anti-RBD IgG response were observed in all other immunotherapy groups except for azathioprine, with the greatest difference seen compared to rituximab. Thirty-five patients with positive anti-RBD responses were further tested for neutralizing antibodies. The mRNA vaccine regimen demonstrated the highest inhibition percentage among the Delta and Omicron variants. Twentytwo patients were tested for T cell responses, with no significant difference in T-cell activity across all groups. Conclusion: We have demonstrated a significant decrease in antibody response against SARS-CoV-2 in patients with autoimmune neurological diseases receiving immunotherapies compared to a healthy population, especially for patients taking rituximab.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.