Detection Of Neutralizing Antibodies Research Articles

Development of a vesicular stomatitis virus pseudotyped with herpes B virus glycoproteins and its application in a neutralizing antibody detection assay.

Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.

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.

Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2.

Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r=0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.

SARS-CoV-2 Neutralization Assays Used in Clinical Trials: A Narrative Review.

Since the emergence of COVID-19, extensive research efforts have been undertaken to accelerate the development of multiple types of vaccines to combat the pandemic. These include inactivated, recombinant subunit, viral vector, and nucleic acid vaccines. In the development of these diverse vaccines, appropriate methods to assess vaccine immunogenicity are essential in both preclinical and clinical studies. Among the biomarkers used in vaccine evaluation, the neutralizing antibody level serves as a pivotal indicator for assessing vaccine efficacy. Neutralizing antibody detection methods can mainly be classified into three types: the conventional virus neutralization test, pseudovirus neutralization test, and surrogate virus neutralization test. Importantly, standardization of these assays is critical for their application to yield results that are comparable across different laboratories. The development and use of international or regional standards would facilitate assay standardization and facilitate comparisons of the immune responses induced by different vaccines. In this comprehensive review, we discuss the principles, advantages, limitations, and application of different SARS-CoV-2 neutralization assays in vaccine clinical trials. This will provide guidance for the development and evaluation of COVID-19 vaccines.

Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein.

The Crimean-Congo hemorrhagic fever virus (CCHFV), the most geographically widespread tick-borne virus, is endemic in Africa, Eastern Europe and Asia, with infection resulting in mortality in up to 30% of cases. Currently, there are no approved vaccines or effective therapies available for CCHF. The CCHFV should only be manipulated in the BSL-4 laboratory, which has severely hampered basic seroprevalence studies. In the present study, two antibody detection methods in the forms of an enzyme-linked immunosorbent assay (ELISA) and a surrogate virus neutralization test (sPVNT) were developed using a recombinant glycoprotein (rGP) and a vesicular stomatitis virus (VSV)-based virus bearing the CCHFV recombinant glycoprotein (rVSV/CCHFV) in a biosafety level 2 (BSL-2) laboratory, respectively. The rGP-based ELISA and rVSV/CCHFV-based sVNT were established by using the anti-CCHFV pre-GC mAb 11E7, known as a broadly cross-reactive, potently neutralizing antibody, and their applications as diagnostic antigens were validated for the specific detection of CCHFV IgG and neutralizing antibodies in experimental animals. In two tests, mAb clone 11E7 (diluted at 1:163840 or 512) still displayed positive binding and neutralization, and the presence of antibodies (IgG and neutralizing) against the rGP and rVSV/CCHFV was also determined in the sera from the experimental animals. Both mAb 11E7 and animal sera showed a high reactivity to both antigens, indicating that bacterially expressed rGP and rVSV/CCHFV have good immunoreactivity. Apart from establishing two serological testing methods, their results also demonstrated an imperfect correlation between IgG and neutralizing antibodies. Within this limited number of samples, the rGP and rVSV/CCHFV could be safe and convenient tools with significant potential for research on specific antibodies and serological samples.

Abstract 3249: Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum

Abstract Adeno-associated viruses (AAV) are prevalent gene therapy vectors, delivering therapeutic transgenes to treat various diseases including cancer and rare monogenic diseases. Individuals with pre-existing immunity to AAVs are less likely to benefit from AAV-based therapies due to both, a reduction in cellular AAV uptake and activation of a cytotoxic T-cell response to transduced cells, decreasing efficacy or changing the safety profile. Measurement of serum neutralizing antibodies (NAbs) against AAV is essential to determine treatment eligibility and to identify clinical trial candidates. Here we introduce a cell-based assay using NanoLuc® luciferase to detect NAbs in serum samples. The superior brightness of NanoLuc® luciferase in our AAV reporter system results in the need of low concentrations of AAV and a short 24h assay time. The minimal AAV concentration in our assay aids in detecting low NAb titers in serum samples, reducing false negatives and enhancing decision-making accuracy. Testing 60 human serum samples against a range of AAV serotypes, we found that a large fraction of the population had pre-existing NAbs to at least one serotype, and of the pre-exposed individuals a majority displayed NAbs against multiple AAV serotypes. Interestingly, fewer samples were seropositive for AAV-DJ than for either parental serotype (AAV2, AAV8 or AAV9), highlighting the assays utility in the development of engineered capsids. Furthermore, the broad dynamic range of our assay facilitates serum sample categorization into four NAb tiers: negative, low, medium, and high using a single serum dilution. Neutralizing titers (ND50) which was determined by serial dilution of the serum samples strongly agreed with the assigned NAb tiers. Our study with 40 naïve mice further validated the assay's robustness, showing a clear correlation between NAb levels and injected AAV viral loads. Taken together, this rapid, highly sensitive, and reliable assay enabled by NanoLuc® AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum. Further validation of the assay is required for future use in a clinical setting. Citation Format: Morten Seirup, Kirsten Wingate, Jeff Nelson, Kara Machleidt, Vanessa Ott, Trish Hoang, Marjeta Urh. Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum [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 3249.

A novel method for identifying SARS-CoV-2 infection mutants via an epitope-specific CD8+ T cell test

Since the outbreak of the coronavirus disease 2019 (COVID-19) epidemic in 2019, the public health system has faced enormous challenges. Tracking the individuals who test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key step for interrupting chains of transmission of SARS-CoV-2 and reducing COVID-19-associated mortality. With the increasing of asymptomatic infections, it is difficult to track asymptomatic infections through epidemiological surveys and virus whole-genome sequencing. However, due to the cross-reactivity of neutralizing antibodies produced by multiple virus subtypes, neutralizing antibody detection cannot be used to determine whether an individual has a history of infection with a specific subtype of SARS-CoV-2. We recruited 4 human leukocyte antigen A2 (HLA-A2) infections, 15 individuals who received three doses of inactivated vaccines, and 30 breakthrough infections after vaccination and discussed a case-tracking approach to detect epitope-specific CD8+ T cells in the peripheral blood of close contacts, including accurate HLA typing based on ribonucleic acid (RNA)-sequencing and flow cytometry data and the comparison and characterization of SARS-CoV-2 HLA-A2 and HLA-A24 epitope-specific CD8+ T cells. From individuals who received three doses of inactivated vaccine, we observed that the CD8+ T cell specificity for ancestral epitopes was significantly higher than for mutated epitopes, and the fold change of CD8+ T cells corresponding to mutated epitopes relative to ancestral epitopes was less than 1. The enzyme-linked immunospot (ELISpot) results further validate this result. This study forms a “method for understanding the infection history of SARS-CoV-2 subtypes based on the proportion of epitope-specific CD8+ T cells in the peripheral blood of subjects”, covering up to 46 % of the population, including HLA-A2+ and HLA-A24+ donors, providing a novel method for SARS-CoV-2 infected case tracing.

Prevalence of neutralizing antibodies against SARS-CoV-2 using a rapid serological test in health workers of a Spanish Department of Health in Alicante (Spain) before the booster dose of the vaccine

AimTo study the prevalence of neutralizing antibodies in healthcare workers and healthcare support personnel after the administration of the second dose of the BNT162b2 vaccine (Pfizer-BioNTech). Materials and MethodsIn December 2021, we undertook a study in the Health Department in Orihuela, Alicante (Spain), which consists of 1500 workers. We collected demographic variables about the study participants, and we performed a “point-of-care” immunochromatography test to measure the presence of neutralizing antibodies (OJABIO® SARS-CoV-2 Neutralizing Antibody Detection Kit, manufactured by Wenzhou OJA Biotechnology Co., Ltd. Wenzhou, Zhejiang, China) before the administration of the third dose of the vaccine. ResultsWe obtained complete information about 964 (64%) workers, which consisted of 290 men and 674 women. The average age was 45,8 years (min. 18, max. 68) and the average time since the last dose of the vaccine was 40,5 weeks (min. 1,71, max. 47,71). A total of 131 participants (13,5%) had suffered infection by SARS-CoV-2 confirmed using RT-PCR. The proportion of participants who showed presence of neutralizing antibodies was 38,5%. In the multivariable analysis, the time since the last dose of the vaccine (aOR week: 1,07; 95%CI: 1,04; 1,09) and previous infection by SARS-CoV-2 (aOR: 3,7; 95CI: 2,39; 5,63) showed a statistically significant association with the presence of neutralizing antibodies. ConclusionsThe time since the administration of the last dose of the vaccine and the previous infection by SARS-CoV-2 determined the presence of neutralizing antibodies in 38,5% of the healthcare workers and support workers.

Rapid, Multispecies Detection of SARS-CoV-2 Antibodies via a Meta-Surface Plasmon Resonance Biosensor

Public health concerns have been raised by numerous reports of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and its variations infecting a range of animals. Wildlife reservoirs may facilitate the evolution of viral types capable of causing human infection in the future. Therefore, epidemiological monitoring of animals in close contact with humans is necessary. Yet, infection symptoms are not obvious in most animals, which leads to a short nucleic acid test-detection period and limits the application of this method in animals. The use of virus- and pseudovirus-based neutralizing antibody detection techniques is restricted to establishments with elevated biosafety standards. Traditional enzyme-linked immunosorbent assays (ELISA) do not offer multispecies detection and are time-consuming and labor-intensive. This work developed a polyethyleneimine-gold nanoparticle meta-surface plasmon resonance biosensor system-based multispecies SARS-CoV-2 antibody detection platform that is fast, sensitive, has a high throughput, and is fully automated. The test can be done in 30 min and specificity is up to 100% for detection in cats, dogs, and minks. Moreover, the coincidence rate was up to 99.36% (313/315) for the detection of pseudovirus in clinical and immune sera. Additionally, this method’s detection sensitivity in cat, dog, and mink serum is 2,048, 1,024, and 4,096 times, which is much better than indirect ELISA and comparable to indirect immunofluorescence assays. An efficient method for COVID-19 epidemiology screening in animal serum will be made available by this platform.

Development of a quantum dot-based lateral flow immunoassay strip for rapid and sensitive detection of SARS-CoV-2 neutralizing antibodies

To a certain extent, the development and vaccination of COVID-19 vaccine have reduced the alarming rate of transmission speed and mortality rate. At present, vaccine coverage is quite high in countries around the world. Since individual differences are unavoidable, it is necessary to assess the efficacy of the vaccine in each vaccinated person in order to reflect the protective effect of the vaccine in different populations. In this study, we developed a novel COVID-19 neutralizing antibody detection kit combining lateral flow immunochromatography and novel quantum dot technology with 85.23% sensitivity, 92.50% specificity. The novel QD-ICA could achieve an accurate detection of SARS-CoV-2 neutralizing antibodies with 10 minutes, two steps, small equipment size, and broad testing application, suggesting its capability to assess vaccine effectiveness on a large scale in areas of world that currently affected by the pandemic.

A community study of neutralizing antibodies against SARS-CoV-2 in China.

The immune background of the overall population before and after the outbreak of SARS-CoV-2 in China remains unexplored. And the level of neutralizing antibodies is a reliable indicator of individual immunity. This study aimed to assess the immune levels of different population groups during a viral outbreak and identify the factors influencing these levels. We measured the levels of neutralizing antibodies in 12,137 participants using the COVID19 Neutralizing Antibody Detection kit. The dynamics of neutralizing antibodies were analyzed using a generalized additive model, while a generalized linear model and multi-factor analysis of variance were employed to investigate the influencing factors. Additionally, statistical methods were used to compare neutralizing antibody levels among subgroups of the real-world population. Participants who received booster doses exhibited significantly higher levels of neutralizing antibodies compared to those who received only one or two doses (p<0.001). Both elderly [22.55 (5.12, 62.03) IU/mL, 55%] and minors [21.41 (8.15, 45.06) IU/mL, 56%] showed lower positivity rates and neutralizing antibody levels compared to young adults [29.30 (9.82, 188.08) IU/mL, 62%] (p<0.001). Furthermore, the HIV-positive group demonstrated a slightly lower seropositivity rate compared to the healthy group across the three vaccination time points. Notably, three months after the large-scale infection, both the neutralizing antibody level and positivity rate in real-world populations were higher than the previous record [300 (300, 300) IU/mL, 89%; 27.10 (8.77, 139.28) IU/mL, 60%], and this difference was statistically significant. Increasing vaccine dosage enhances neutralizing antibody levels, resulting in greater and longer-lasting immunity. Monitoring immune levels in older individuals and those with AIDS is crucial. Additionally, the neutralizing antibodies generated from vaccination have not yet reached the threshold for achieving herd immunity, while individuals exhibit higher immune levels following a large-scale infection. These findings provide valuable insights for guiding new strategies in vaccine administration.

Dual-Function Janus Nanozymes for Performance Evaluation and Application in a Surrogate Virus Neutralization Test with Vaccinated Samples.

The need exists for biosensing technologies capable of sensitively and accurately detecting various biomarkers. In response, the development of nanozymes is actively underway; they have advantages in stability, cost, performance, and functionalization over natural enzymes commonly used for signal amplification in sensing technologies. However, the performance of nanozymes is interdependent with factors such as shape, size, and surface functional moiety, making it challenging to perform quantitative performance comparisons based on the nanozyme material. In this study, we propose a physical synthetic approach to fabricate double-layered bimetallic nanozymes with identical shapes, sizes, and surfaces but different material compositions. These Janus nanozymes consist of a nanozymatic layer responsible for catalytic activity and a gold layer responsible for quantification and efficient surface modification. Based on their identical physicochemical properties, the synthesized double-layered bimetallic nanozymes allow, for the first time, a quantitative comparison of nanozymatic activities in terms of various kinetic parameters. We compared several candidates and found that the Ir-Au nanozyme exhibited the best performance. Subsequently, we applied this nanozyme to detect neutralizing antibodies against SARS-CoV-2 based on a surrogate virus neutralization test. The results demonstrated a limit of detection as low as 2 pg/mL and selectivity specifically toward MERS-CoV. The performance of this assay was further validated using vaccinated samples, demonstrating the potential of our approach as a cost-effective, rapid, and sensitive diagnostic tool for neutralizing antibody detection against viruses such as SARS-CoV-2.

Naked-Eye Readable Microarray for Rapid Profiling of Antibodies against Multiple SARS-CoV-2 Variants.

Novel high-throughput protein detection technologies are critically needed for population-based large-scale SARS-CoV-2 antibody detection as well as for monitoring quality and duration of immunity against virus variants. Current protein microarray techniques rely heavily on labeled transduction methods that require sophisticated instruments and complex operations, limiting their clinical potential, particularly for point-of-care (POC) applications. Here, we developed a label-free and naked-eye readable microarray (NRM) based on a thickness-sensing plasmon ruler, enabling antibody profiling within 30 min. The NRM chips provide 100% accuracy for neutralizing antibody detection by efficiently screening antigen types and experimental conditions and allow for the profiling of antibodies against multiple SARS-CoV-2 variants in clinical samples. We further established a flexible "barcode" NRM assay with a simple tape-based operation, enabling an effective smartphone-based readout and analysis. These results demonstrate new strategies for high-throughput protein detection and highlight the potential of novel protein microarray techniques for realistic clinical applications.

Evaluation of immune response to SARS-CoV-2 Omicron sublineages six months after different vaccination regimens in Italy

The Omicron variant is the most divergent, displaying more mutations than previous SARS-CoV-2 variants, particularly in the gene that encodes the spike protein. This study aimed to assess the persistence of neutralizing antibodies towards the SARS-CoV-2 Omicron sublineages (BA.2, BA.5, BQ.1, XBB and XBB1.5) six months after the third dose in different vaccination regimens. Subjects who received 3 doses of mRNA vaccine retained their neutralization activity against BA.2 and BA.5, even though 56.3% and 66.7% showed a ≥ 2-fold reduction in the neutralizing antibody titre, respectively. Subjects who had received the adenovirus-based vaccine plus a booster dose of mRNA vaccine retained their neutralization activity especially against BA.2. With regard to BQ.1, XBB and XBB.1.5, the majority of the subjects showed a ≥ 2-fold reduction in neutralizing antibody titre, with the greatest evasion being observed in the case of XBB. Overall, our results provide further evidence that triple homologous/heterologous vaccination and hybrid immunity result in detectable neutralizing antibodies against the ancestral virus; however, emerging Omicron sublineages, such as XBB and XBB.1.5, show a great evasive capacity, which compromises the effectiveness of current COVID-19 vaccines.

A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies

Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.

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.

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

Comprehensive Cross-Sectional Evaluation of Human Sandfly-Borne Phlebovirus Exposure in an Endemic Region.

Sandfly-borne phleboviruses are endemic in countries around the Mediterranean Basin and pose a significant health threat for populations, with symptoms spanning from febrile diseases to central nervous system involvement. We carried out a comprehensive cross-sectional screening via microneutralization (MN) assays for a quantitative assessment of neutralizing antibodies (NAs) to seven phleboviruses representing three distinct serocomplexes, using samples previously screened via immunofluorescence assays (IFAs) in Turkey, an endemic region with various phleboviruses in circulation. We detected NAs to three phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), and sandfly fever Sicilian virus (SFSV), while assays utilizing Adana virus, Punique virus, Massilia virus, and Zerdali virus remained negative. The most frequently observed virus exposure was due to TOSV, with a total prevalence of 22.6%, followed by SFNV (15.3%) and SFSV (12.1%). For each virus, IFA reactivity was significantly associated with NA detection, and further correlated with NA titers. TOSV and SFSV seroreactivities were co-detected, suggesting exposure to multiple pathogenic viruses presumably due to shared sandfly vectors. In 9.6% of the samples, multiple virus exposure was documented. In conclusion, our findings demonstrate widespread exposure to distinct pathogenic phleboviruses, for which diagnostic testing and serological screening efforts should be directed.

Refined semi-lethal aerosol H5N1 influenza model in cynomolgus macaques for evaluation of medical countermeasures

Highly pathogenic avian influenza A H5N1 viruses cause high mortality in humans and have pandemic potential. Effective vaccines and treatments against this threat are urgently needed. Here, we have refined our previously established model of lethal H5N1 infection in cynomolgus macaques. An inhaled aerosol virus dose of 5.1 log10 plaque-forming unit (pfu) induced a strong febrile response and acute respiratory disease, with four out of six macaques succumbing after challenge. Vaccination with three doses of adjuvanted seasonal quadrivalent influenza vaccine elicited low but detectable neutralizing antibody to H5N1. All six vaccinated macaques survived four times the 50% lethal dose of aerosolized H5N1, while four of six unvaccinated controls succumbed to disease. Although vaccination did not protect against severe influenza, vaccinees had reduced respiratory dysfunction and lower viral load in airways compared to controls. We anticipate that our macaque model will play a vital role in evaluating vaccines and antivirals against influenza pandemics.

The instantly blocking-based fluorescent immunochromatographic assay for the detection of SARS-CoV-2 neutralizing antibody.

At present, there is an urgent need for the rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs) to evaluate the ability of the human body to resist coronavirus disease 2019 (COVID-19) after infection or vaccination. The current gold standard for neutralizing antibody detection is the conventional virus neutralization test (cVNT), which requires live pathogens and biosafety level-3 (BSL-3) laboratories, making it difficult for this method to meet the requirements of large-scale routine detection. Therefore, this study established a time-resolved fluorescence-blocking lateral flow immunochromatographic assay (TRF-BLFIA) that enables accurate, rapid quantification of NAbs in subjects. This assay utilizes the characteristic that SARS-CoV-2 neutralizing antibody can specifically block the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2) to rapidly detect the content of neutralizing antibody in COVID-19-infected patients and vaccine recipients. When 356 samples of vaccine recipients were measured, the coincidence rate between this method and cVNT was 88.76%, which was higher than the coincidence rate of 76.97% between cVNT and a conventional chemiluminescence immunoassay detecting overall binding anti-Spike-IgG. More importantly, this assay does not need to be carried out in BSL-2 or 3 laboratories. Therefore, this product can detect NAbs in COVID-19 patients and provide a reference for the prognosis and outcome of patients. Simultaneously, it can also be applied to large-scale detection to better meet the needs of neutralizing antibody detection after vaccination, making it an effective tool to evaluate the immunoprotective effect of COVID-19 vaccines.