Neutralizing Antibody Assay Research Articles

Development and comparison of immunologic assays to detect primary RSV infections in infants.

Effective respiratory syncytial virus (RSV) vaccines have been developed and licensed for elderly adults and pregnant women but not yet for infants and young children. The RSV immune state of the young child, i.e., previously RSV infected or not, is important to the conduct and interpretation of epidemiology studies and vaccine clinical trials. To address the need for sensitive assays to detect immunologic evidence of past infection, we developed, characterized, and evaluated 7 assays including 4 IgG antibody enzyme immunoassays (EIAs), two neutralizing antibody assays, and an IFN-γ EliSpot (EliSpot) assay. The four IgG EIAs used a subgroup A plus subgroup B RSV-infected Hep-2 cell lysate antigen (Lysate), an expressed RSV F protein antigen (F), an expressed subgroup A G protein antigen (Ga), or an expressed subgroup B G protein (Gb) antigen. The two neutralizing antibody assays used either a subgroup A or a subgroup B RSV strain. The EliSpot assay used a sucrose cushion purified combination of subgroup A and subgroup B infected cell lysate. All seven assays had acceptable repeatability, signal against control antigen, lower limit of detection, and, for the antibody assays, effect of red cell lysis, lipemia and anticoagulation of sample on results. In 44 sera collected from children >6 months after an RSV positive illness, the lysate, F, Ga and Gb IgG EIAs, and the subgroup A and B neutralizing antibody assays, and the EliSpot assays were positive in 100%, 100%, 86%, 95%, 43%, and 57%, respectively. The Lysate and F EIAs were most sensitive for detecting RSV antibody in young children with a documented RSV infection. Unexpectedly, the EliSpot assay was positive in 9/15 (60%) of PBMC specimens from infants not exposed to an RSV season, possibly from maternal microchimerism. The Lysate and F EIAs provide good options to reliably detect RSV antibodies in young children for epidemiologic studies and vaccine trials.

Intra-serotypic antigenic diversity of dengue virus serotype 3 in Thailand during 2004-2015.

In addition to the well-known differences among the four dengue serotypes, intra-serotypic antigenic diversity has been proposed to play a role in viral evolution and epidemic fluctuation. A replacement of genotype II by genotype III of dengue virus serotype 3 (DENV3) occurred in Thailand during 2007-2014, raising questions about the role of intra-serotypic antigenic differences in this genotype shift. We characterized the antigenic difference of DENV3 of genotypes II and III in Thailand, utilizing a neutralizing antibody assay with DENV3 vaccine sera and monotypic DENV3 sera. Although there was significant antigenic diversity among the DENV3, it did not clearly associate with the genotype. Our data therefore do not support the role of intra-serotypic antigenic difference in the genotype replacement. Amino acid alignment showed that eight positions are potentially associated with diversity between distinct antigenic subgroups. Most of these amino acids were found in envelope domain II. Some positions (aa81, aa124, and aa172) were located on the surface of virus particles, probably involving the neutralization sensitivity. Notably, the strains of both genotypes II and III showed clear antigenic differences from the vaccine genotype I strain. Whether this differencewill affect vaccine efficacy requires further studies.

Compatible co-administration of BioThrax® vaccine and ciprofloxacin—Results of a randomized open-label drug-vaccine interaction trial

The recommended treatment for post-exposure prophylaxis (PEP) following known/suspected exposure to Bacillus anthracis involves immunization with anthrax vaccine adsorbed (AVA, i.e., BioThrax® vaccine) and a course of antimicrobial therapy. A drug-vaccine interaction clinical trial was conducted to determine whether this combined treatment might modify antimicrobial exposure or vaccine immunogenicity.A Phase 2, randomized, open-label, multi-center trial involving 154 healthy adult participants was completed to evaluate the effect of AVA immunization (three doses administered subcutaneously (SC) at weeks 0, 2 and 4) on the pharmacokinetics (PK) of ciprofloxacin, as well as the effect of ciprofloxacin administration (500 mg po bid) on the immunogenicity of AVA. PK parameters were derived using noncompartmental analysis of ciprofloxacin serum concentrations. Immunogenicity was assessed using a toxin neutralizing antibody (TNA) assay resulting in 50 % neutralization factor (NF50) values. Safety was assessed via reports of adverse events (AEs), clinically significant changes in laboratory parameters and vital signs, and collection of solicited local and systemic reactogenicity reactions.Statistical analyses of the steady state (SS) and single dose PK parameters Cmax and AUC0-–12h indicated that the AVA PEP regimen did not significantly modify ciprofloxacin exposure. Comparison of the geometric mean TNA NF50 values between participants receiving AVA + ciprofloxacin and those receiving AVA alone showed that the combined treatment was non-inferior to AVA alone. The trial met all prospectively defined success criteria for the primary PK endpoint and for the secondary PK and immunogenicity endpoints. There were no deaths, SAEs or AEs leading to drug discontinuation or study withdrawal during the trial.Overall, concomitant administration of ciprofloxacin and AVA produced no significant changes in the PK profile of ciprofloxacin nor in the immunogenicity of AVA. Furthermore, this trial demonstrated that the co-administration of ciprofloxacin and AVA was well tolerated in healthy adult participants.

Comparative assessment of immunogenicity of recombinant insulin Aspart from BioGenomics and its originator NovoRapid® in adult patients with type 2 diabetes mellitus.

To assess and compare the immunogenicity of recombinant Insulin Aspart [manufactured by BioGenomics Limited (BGL-ASP)] with its originator NovoRapid® (manufactured by Novo Nordisk) in adult patients with type 2 diabetes mellitus. BGL-IA-CTP301 study was a randomized, open label, parallel group, multicenter phase-III clinical study to compare the efficacy and safety of recombinant Insulin Aspart 100U/mL [manufactured by BioGenomics Limited (BGL-ASP)] with its reference medicinal product (RMP); NovoRapid® [manufactured by Novo Nordisk], in adult patients with Type 2 diabetes mellitus(T2DM). The primary objective of the study was to compare the immunogenicity of BGL-ASP and RMP; NovoRapid® in patient serum samples collected from phase-III clinical study. Immunogenicity was studied as the incidence of patients positive for anti-insulin Aspart (AIA) antibodies, developed against BGL-ASP/RMP at baseline, end of 12week and end of 24week of the treatment period. The changes in incidence of patients positive for AIA antibodies post-baseline were also studied to assess and compare the treatment-emergent antibody response (TEAR) between the treatment groups (BGL-ASP and RMP). Statistical evaluation was done by Fisher's exact test to compare the overall incidence of patients positive for AIA antibodies and the TEAR positives observed post-baseline in both the treated groups. An in-vitro neutralizing antibody assay (Nab assay) was also performed to study the effect of AIA antibodies in neutralizing the biological activity/metabolic function of the insulin. The neutralizing potential of AIA was studied by its effect on %glucose uptake. We also evaluated the association between AIA antibody levels and its impact on biological activity by studying the correlation between them. Analysis of immunogenicity data suggested that the percentage of patients positive for AIA antibodies until week 24 was similar and comparable in both the treatment groups, BGL-ASP and RMP; NovoRapid®. The changes in incidence of patients positive for AIA post-baseline in terms of TEAR positives were also similar and comparable between the treatment groups. The results of the Nab assay with confirmed positive AIA samples from BGL-ASP- and RMP-treated groups did not have any negative impact on %glucose uptake by the cells in Nab assay, confirming the absence of neutralizing antibodies in both the treatment groups. The correlation studies also showed absence of association between AIA antibody levels and percentage glucose uptake in both BGL-ASP and RMP-NovoRapid® treatment groups. CONCLUSIONS: The immunogenicity assessment based on the overall incidence of patients positive for AIA, changes in incidence of patients positive for AIA post-baseline, TEAR rates and absence of neutralizing antibodies, were found to be apparently similar and comparable in both the treatment groups (BGL-ASP and RMP). We conclude from our studies that the immunogenicity of BGL-ASP is similar and comparable to RMP and the observed immunogenicity in terms of anti-insulin Aspart antibody levels had no impact on the biological activity of insulin.

Performance Evaluation of Three Antibody Binding Assays, a Neutralizing Antibody Assay, and an Interferon-Gamma Release Assay for SARS-CoV-2 According to Vaccine Type in Vaccinated Group.

We evaluated the performance of SARS-CoV-2 assays in the vaccinated group using receptor-binding domain antibody assays (RBD Ab assay), neutralizing antibody assay (nAb assay), and interferon-gamma release assay (IGR assay). We also compared the performance of the SARS-CoV-2 assays based on vaccine type in a large population. We collected 1851 samples from vaccinated individuals with vector, mix-and-match (MM), and mRNA vaccines. The performance of the RBD Ab assays was assessed by SARS-CoV-2 IgG II Quant (Abbott Laboratories, Sligo, Ireland), SARS-CoV-2 IgG (Beckman Coulter, CA, USA), and anti-SARS-CoV-2 S (Roche Diagnostics GmbH, Mannheim, Germany). The nAb assay was assessed by cPass SARS-CoV-2 neutralization antibody detection kits (GenScript, NJ, USA). The IGR assay was assessed by QuantiFERON (Qiagen, Venlo, The Netherlands). Median values of the RBD Ab assays and nAb assay sequentially increased after the first and second vaccinations. RBD Ab assays and nAb assay showed very strong correlations. The median values of the RBD Ab, nAb, and IGR were higher in the mRNA vaccine group than in the vector and MM vaccine groups. The agreement and correlation among the RBD Ab assays, nAb assay, and IGR assay were higher in the mRNA vaccine group than in the vector and MM vaccine groups. We compared the performance of the RBD Ab assay, nAb assay, and IGR assay based on the vaccine types using the RBD Ab, nAb, and IGR assays. This study provides a better understanding of the assessment of humoral and cellular immune responses after vaccination.

Clinical Utility of SARS-CoV-2 Antibody Testing

SARS-CoV-2 antibody assay is a test that checks whether an antibody against the SARS-CoV-2 virus has been formed in the blood after SARS-CoV-2 infection or vaccination. SARS-CoV-2 antibody is detected 1–2 weeks after infection, and antibodies are produced in more than 90% of infected patients. The duration for the formation of antibodies differs by individual and by type of antibody. In the case of IgG, it is at least several months or longer, and the relationship between antibodies and immunity is being studied. As test methods,enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), immunochromatographic assay, and neutralizing antibody assay have been developed and used. The target antibody to be detected differs depending on the type of recombinant antigen and the type of secondary antibody in reagents. Many kinds of commercialized SARS-CoV-2 antibody assays are currently being developed, and the S (spike) protein, N (nucleocapsid) protein, S1 or RBD (receptor binding domain) part of the S protein, and a mixture of these antigens are used as recombinant antigens of reagents. IgG, IgM, IgA, or total immunoglobulin antibodies in patients’ blood that react with these reagent antigens are detected. In this review, the types and performance of SARS-CoV-2 antibody tests and the guidelines for COVID-19 antibody tests published domestically and abroad were investigated.

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743ng/mL and can detect 1500ng/mL of a surrogate positive control antibody in the presence of 1500ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

Validation of Anti-Adeno Associated Virus Serotype rh10 (AAVrh.10) Total and Neutralizing Antibody Immunogenicity Assays

Immunogenicity assessment of Adeno-Associated Virus (AAV) vectors is a critical part of gene therapy drug development. Whether the assays are used for inclusion/exclusion criteria or to monitor the safety and efficacy of the gene therapy, they are critical bioanalytical assessments. While total anti-AAV assays are perceived as easier to develop and implement than neutralizing anti-AAV assays, the gene therapy field is still nascent, and it is not yet clear which of the assays should be implemented at what stage of drug development. Recently AAVrh.10 has gained interest for use in gene therapies targeting cardiac, neurological, and other diseases due to its enhanced transduction efficiency. There is limited information on anti-AAVrh.10 antibodies and their clinical impact; thus, the information presented herein documents the validation of both a total antibody assay (TAb) and a neutralizing antibody (NAb) assay for anti-AAVrh.10 antibodies. In this manuscript, the validation was performed in accordance with the 2019 FDA immunogenicity guidance with additional evaluations to comply with CLIA where applicable. The AAVrh.10 TAb and NAb assays were compared in terms of sensitivity, drug tolerance, and precision, along with a concordance analysis using the same individual serum samples. This comparison gave insight into the utility of each format as a screening assay for inclusion into clinical studies.

Control of maternal Zika virus infection during pregnancy is associated with lower antibody titers in a macaque model.

Zika virus (ZIKV) infection during pregnancy results in a spectrum of birth defects and neurodevelopmental deficits in prenatally exposed infants, with no clear understanding of why some pregnancies are more severely affected. Differential control of maternal ZIKV infection may explain the spectrum of adverse outcomes. Here, we investigated whether the magnitude and breadth of the maternal ZIKV-specific antibody response is associated with better virologic control using a rhesus macaque model of prenatal ZIKV infection. We inoculated 18 dams with an Asian-lineage ZIKV isolate (PRVABC59) at 30-45 gestational days. Plasma vRNA and infectious virus kinetics were determined over the course of pregnancy, as well as vRNA burden in the maternal-fetal interface (MFI) at delivery. Binding and neutralizing antibody assays were performed to determine the magnitude of the ZIKV-specific IgM and IgG antibody responses throughout pregnancy, along with peptide microarray assays to define the breadth of linear ZIKV epitopes recognized. Dams with better virologic control (n= 9) cleared detectable infectious virus and vRNA from the plasma by 7 days post-infection (DPI) and had a lower vRNA burden in the MFI at delivery. In comparison, dams with worse virologic control (n= 9) still cleared detectable infectious virus from the plasma by 7 DPI but had vRNA that persisted longer, and had higher vRNA burden in the MFI at delivery. The magnitudes of the ZIKV-specific antibody responses were significantly lower in the dams with better virologic control, suggesting that higher antibody titers are not associated with better control of ZIKV infection. Additionally, the breadth of the ZIKV linear epitopes recognized did not differ between the dams with better and worse control of ZIKV infection. Thus, the magnitude and breadth of the maternal antibody responses do not seem to impact maternal virologic control. This may be because control of maternal infection is determined in the first 7 DPI, when detectable infectious virus is present and before robust antibody responses are generated. However, the presence of higher ZIKV-specific antibody titers in dams with worse virologic control suggests that these could be used as a biomarker of poor maternal control of infection and should be explored further.

Addressing Domain Specificity in the Development of a Cell-Based Binding Assay for the Detection of Neutralizing Antibodies Against a CD47xPD-L1 Bispecific Antibody

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.Graphical

Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice.

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

Safety, tolerability, and immunogenicity of a CpG/Alum adjuvanted SARS-CoV-2 recombinant protein vaccine (ZR202-CoV) in healthy adults: Preliminary report of a phase 1, randomized, double-blind, placebo-controlled, dose-escalation trial

ABSTRACT This was a phase 1 dose-escalation study of ZR202-CoV, a recombinant protein vaccine candidate containing a pre-fusion format of the spike (S)-protein (S-trimer) combined with the dual-adjuvant system of Alum/CpG. A total of 230 participants were screened and 72 healthy adults aged 18–59 years were enrolled and randomized to receive two doses at a 28-day interval of three different ZR202-CoV formulations or normal saline. We assessed the safety for 28 days after each vaccination and collected blood samples for immunogenicity evaluation. All formulations of ZR202-CoV were well-tolerated, with no observed solicited adverse events ≥ Grade 3 within 7 days after vaccination. No unsolicited adverse events ≥ Grade 3, or serious adverse events related to vaccination occurred as determined by the investigator. After the first dose, detectable immune responses were observed in all subjects. All subjects that received ZR202-CoV seroconverted at 14 days after the second dose by S-binding IgG antibody, pseudovirus and live-virus based neutralizing antibody assays. S-binding response (GMCs: 2708.7 ~ 4050.0 BAU/mL) and neutralizing activity by pseudovirus (GMCs: 363.1 ~ 627.0 IU/mL) and live virus SARS-CoV-2 (GMT: 101.7 ~ 175.0) peaked at 14 days after the second dose of ZR202-CoV. The magnitudes of immune responses compared favorably with COVID-19 vaccines with reported protective efficacy.

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.

Development of a Cell-based Neutralizing Antibody Assay for Zinpentraxin Alfa: Challenges and Mitigation Strategies.

Therapeutic protein drugs can potentially induce immune responses in patients and result in the production of anti-drug antibodies (ADAs), including a subset of ADAs called neutralizing antibodies (NAbs) that might cause loss of efficacy by inhibiting clinical activities of the drug. Herein, we describe the unique challenges encountered during the development of a fit-for-purpose cell-based NAb assay for a new protein modality, zinpentraxin alfa, including our strategies for assay design to overcome various matrix interferences and improve assay drug tolerance. We demonstrated that a typical biotin-drug extraction with acid dissociation (BEAD) approach alone was not sufficient to eliminate matrix interferences in this assay. Instead, the combination of the BEAD and ZebaTM spin size exclusion plate (SEP) was required to achieve the desirable assay performance. We also demonstrated that appropriate acidic buffers were critical in sample pretreatment to improve assay drug tolerance, which not only dissociated the drug/NAb immune complex but also effectively and irreversibly denatured the free drug. The final assay performed well with confirmed assay robustness and suitability for the clinical applications.

ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood–cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Neutralizing Antibody Validation Testing and Reporting Harmonization

Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists’ Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.Graphical

Safety and immunogenicity against ancestral, Delta and Omicron virus variants following a booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001): Interim analysis of an open-label extension of the randomized, controlled, phase 3 COV-COMPARE trial

Booster doses for COVID-19 vaccinations have been shown to amplify the waning immune response after primary vaccination and to enhance protection against emerging variants of concern (VoCs). Here, we aimed to assess the immunogenicity and safety of a booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) after primary vaccination with 2 doses of either VLA2001 or ChAdOx1-S (Oxford-Astra Zeneca), including the cross-neutralization capacity against the Delta and Omicron VoCs. This interim analysis of an open-label extension of a randomized, controlled phase 3 trial assessed a single booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) in healthy or medically stable adults aged 18 years and above, recruited in 21 clinical sites in the UK, who had previously received two doses of either VLA2001 or ChAdOx1-S. Safety outcomes were frequency and severity of solicited injection site and systemic reactions within 7 days after booster vaccination as well as frequency and severity of any unsolicited adverse events (AE) after up to 6 months. Immunogenicity outcomes were the immune response to ancestral SARS-CoV-2 assessed 14 days post booster expressed as geometric mean titres (GMT), GMT fold ratios and seroconversion of specific neutralizing antibodies and S-protein binding IgG antibodies. Immunogenicity against the Delta and Omicron VoCs was assessed as a post-hoc outcome with a pseudovirus neutralization antibody assay. This study is registered with ClinicalTrials.gov, NCT04864561, and is ongoing. A booster dose of VLA2001 was administered to 958 participants, of whom 712 had been primed with VLA2001, and 246 with ChAdOx1-S. Within 7 days following these booster doses, 607 (63.4%) participants reported solicited injection site reactions, and 487 (50.8%) reported solicited systemic reactions. Up to 14 days post booster, 751 (78.4%) participants reported at least one adverse event. The tolerability profile of a booster dose of VLA2001 was similar in VLA2001-primed and ChAdOx1-S-primed participants. In VLA2001-primed participants, the GMT (95% CI) of neutralizing antibodies increased from 32.5 (22.8, 46.3) immediately before to 521.5 (413.0, 658.6) 2 weeks after administration of the booster dose, this corresponds to a geometric mean fold rise (GMFR) of 27.7 (20.0, 38.5). Compared to 2 weeks after the second priming dose, the GMFR was 3.6 (2.8, 4.7). In the ChAdOx1-S primed group, the GMT (95% CI) of neutralizing antibodies increased from 65.8 (43.9, 98.4) immediately before to 188.3 (140.3, 252.8) 2 weeks after administration of the booster dose, a geometric mean fold rise (GMFR) of 3.0 (2.2, 4.0). Compared to 2 weeks after the second priming dose, the GMFR was 1.6 (1.1, 2.2). For S-protein binding IgG antibodies, the pre- versus post-booster GMT fold ratio (95% CI) was 34.6 (25.0, 48.0) in the VLA2001-primed group and 4.0 (3.0, 5.2) in the ChAdOx1-S-primed group. Compared to 2 weeks after the second priming dose, the GMT fold rise of IgG antibodies was 3.8 (3.2, 4.6) in the VLA2001-primed group and 1.2 (0.9, 1.6) in the ChAdOx1-S-primed group. The GMT against Delta (B.1.617.2) and Omicron (BA.4/5) increased from 4.2 to 260, and from 2.7 to 56.7, respectively, when boosting subjects previously primed with VLA2001. Following the boost, 97% of subjects primed with VLA2001 had detectable Delta- and 94% Omicron-neutralizing antibodies. In subjects primed with ChAdOx1-S, the GMT against Delta and Omicron titres increased from 9.1 to 92.5, and from 3.6 to 12.3, respectively. After boosting, 99% of subjects primed with ChAdOx1-S had detectable Delta- and 70% Omicron-neutralizing antibodies. In both VLA2001 and ChAdOx1-S primed subjects, the additional VLA2001 dose boosted T cell responses against SARS-CoV-2 antigens to levels above those observed before the booster dose. A booster dose of VLA2001 was safe and well tolerated after primary immunization with VLA2001 and ChAdOx1-S. The tolerability of a booster dose of VLA2001 was similar to the favourable profile observed after the first and second priming doses. Both in a homologous and a heterologous setting, boosting resulted in higher neutralizing antibody titres than after primary immunization and significant increases in cross-neutralization titres against Delta and Omicron were observed after the booster dose. These data support the use of VLA2001 in booster programmes in ChadOx1-S primed groups.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin.

Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT). The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT. We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed. The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.

Goats with low levels of AAV antibody may serve as candidates for large animal gene therapy

AAV vector-mediated gene therapy has been proposed as a feasible strategy for several eye diseases. However, AAV antibodies in the serum prior to treatment hinder the transduction efficiency and thus the therapeutic effect. Therefore, it is necessary to evaluate AAV antibodies in the serum before gene therapy. As large animals, goats are more closely related to humans than rodents and more economically available than nonhuman primates. Here, we first evaluated the AAV2 antibody serum level in rhesus monkeys before AAV injection. Then, we optimized a cell-based neutralizing antibody assay for detecting AAV antibodies in the serum of Saanen goats and evaluated the consistency of the cell-based neutralizing antibody assay and ELISA for goat serum antibody evaluation.The cell-based neutralizing antibody assay showed that the percentage of macaques with low antibody levels was 42.86%; however, there were no macaques with low antibody levels when the serum was evaluated by ELISA. The proportion of goats with low antibody levels was 56.67% according to the neutralizing antibody assay and 33. 33% according to the ELISA, and McNemar's test showed that the results of the two assays were not significantly different (P = 0.754), but that their consistency is poor (Kappa = 0.286, P = 0.114). Moreover, longitudinal evaluation of serum antibodies before and after intravitreal injection of AAV2 in goats revealed that the level of AAV antibodies increased and transduction inhibition subsequently increased, as reported in humans, indicating that transduction inhibition should be taken into account at different stages of gene therapy. In summary, starting with an evaluation of monkey serum antibodies, we optimized a detection method of goat serum antibodies, providing an alternative large animal model for gene therapy, and our serum antibody measurement method may be applied to other large animals.