Conventional Neutralization Research Articles

Bison, Elk, and Other Captive Wildlife Species Humoral Immune Responses against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has been found to infect various domestic and wild animal species. In this study, convenience serum samples from 575 bison, 180 elk, and 147 samples from various wildlife species collected between 2020 and 2023 from several regions in the United States were analyzed for the presence of SARS-CoV-2-specific antibodies. Two commercial ELISA assays based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) or the nucleocapsid protein (N-ELISA) of SARS-CoV-2 were used. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT). Our results indicated that 1.2% of bison, 2.2% of elk, and 4.1% of the other wildlife species serum samples were seropositive in the sVNT, whereas 4.2% of bison, 3.3% of elk, and 1.4% of the other captive wildlife species serum samples tested positive by the N-ELISA. Among the sVNT serum samples, two samples from bison, one sample from elk, and five serum samples from other wildlife species (one cheetah, one gorilla, two lions, and one hippopotamus) had neutralizing antibody titers in the VNT, indicating these species are susceptible to SARS-CoV-2 infection. These findings highlight the importance of broad surveillance efforts for the effective monitoring of SARS-CoV-2 in non-human hosts.

Facile quantitative diagnostic testing for neutralizing antibodies against Chikungunya virus

BackgroundViral neutralization (NT) assays can be used to determine the immune status of patients or assess the potency of candidate vaccines or therapeutic monoclonal antibodies (mAbs). Focus reduction neutralization test (FRNT) is a conventional neutralization test (cVNT) with superior specificity for measurement of neutralizing antibodies against a specific virus. Unfortunately, the application of FRNT to the chikungunya virus (CHIKV) involves a highly pathogenic bio-agent requiring biosafety level 3 (BSL3) facilities, which inevitably imposes high costs and limits accessibility. In this study, we evaluated a safe surrogate virus neutralization test (sVNT) that uses novel CHIKV replicon particles (VRPs) expressing eGFP and luciferase (Luc) to enable the rapid detection and quantification of neutralizing activity in clinical human serum samples.MethodsThis unmatched case-control validation study used serum samples from laboratory-confirmed cases of CHIKV (n = 19), dengue virus (DENV; n = 9), Japanese encephalitis virus (JEV; n = 5), and normal individuals (n = 20). We evaluated the effectiveness of sVNT, based on mosquito cell-derived CHIK VRPs (mos-CHIK VRPs), in detecting (eGFP) and quantifying (Luc) neutralizing activity, considering specificity, sensitivity, and reproducibility. We conducted correlation analysis between the proposed rapid method (20 h) versus FRNT assay (72 h). We also investigated the correlation between sVNT and FRNT in NT titrations in terms of Pearson’s correlation coefficient (r) and sigmoidal curve fitting.ResultsIn NT screening assays, sVNT-eGFP screening achieved sensitivity and specificity of 100%. In quantitative neutralization assays, we observed a Pearson’s correlation coefficient of 0.83 for NT50 values between sVNT-Luc and FRNT.ConclusionsFacile VRP-based sVNT within 24 h proved highly reliable in the identification and quantification of neutralizing activity against CHIKV in clinical serum samples.

Post-Hoc Analysis of Potential Correlates of Protection of a Recombinant SARS-CoV-2 Spike Protein Extracellular Domain Vaccine Formulated with Advax-CpG55.2-Adjuvant.

SpikoGen® vaccine is a subunit COVID-19 vaccine composed of an insect cell expressed recombinant spike protein extracellular domain formulated with Advax-CpG55.2™ adjuvant. A randomized double-blind, placebo-controlled Phase II clinical trial was conducted in 400 adult subjects who were randomized 3:1 to receive two intramuscular doses three weeks apart of either SpikoGen® vaccine 25 μg or saline placebo, as previously reported. This study reports a post hoc analysis of the trial data to explore potential immune correlates of SpikoGen® vaccine protection. A range of humoral markers collected pre- and post-vaccination, including spike- and RBD-binding IgG and IgA, surrogate (sVNT), and conventional (cVNT) virus neutralization tests were compared between participants who remained infection-free or got infected over three months of follow-up. From 2 weeks after the second vaccine dose, 21 participants were diagnosed with SARS-CoV-2 infection, 13 (4.2%) in the SpikoGen® group and 8 (9%) in the placebo group. Those in the vaccinated group who experienced breakthrough infections had significantly lower sVNT titers (GMT 5.75 μg/mL, 95% CI; 3.72-8.91) two weeks after the second dose (day 35) than those who did not get infected (GMT 21.06 μg/mL, 95% CI; 16.57-26.76). Conversely, those who did not develop SARS-CoV-2 infection during follow-up had significantly higher baseline sVNT, cVNT, spike-binding IgG and IgA, and RBD-binding IgG, consistent with a past SARS-CoV-2 infection. SpikoGen® further reduced the risk of re-infection (OR 0.29) in baseline seropositive (previously infected) as well as baseline seronegative participants. This indicates that while SpikoGen vaccine is protective in seronegative individuals, those with hybrid immunity have the most robust protection.

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.

Geochemical Evaluation of Manganese Neutralization Sludge for Mn-Containing Mine Drainage Treatment

Conventional neutralization treatment for manganese (Mn)-bearing mine drainage provides a challenge of cost-efficiency, and new approaches should be explored for efficient removal of Mn. We focused on Mn neutralization sludge, a by-product of the Mn-bearing drainage treatment process, which was assumed to be useful as a water purification material. Mn and zinc (Zn) removal tests in simulated drainage were performed using Mn neutralization sludge, and the reaction mechanism was elucidated using geochemical modeling and X-ray absorption fine structure (XAFS) analysis. The results showed that the addition of sludge enabled to lower the Mn and Zn concentration below Japanese effluent standard (10 mg dm−3 for Mn; 2 mg dm−3 for Zn) within 1 h. Furthermore, heavy metal immobilization was achieved at neutral pH (7–8) with the sludge addition, while the conventional neutralization process without adding the sludge requires higher pH (>10) to lower Mn concentration. These removal behaviors were not explainable by considering only well-known phenomena: hydroxide precipitation, surface complexation reaction onto δ-MnO2, and autocatalytic Mn oxidation. Hence, we advanced the geochemical model for simulation, suggesting that a surface complexation reaction onto γ-MnOOH greatly contributed to the removal of Mn. Besides, Zn was calculated to be predominantly precipitated as ZnMn2O4. Solid residue analysis by XAFS measurement supported the result of above calculation, validating the reliability of the constructed geochemical model. Overall, we concluded that the advanced geochemical model would be useful in predicting the Mn and Zn behavior during mine drainage treatment with Mn neutralization sludge.Graphical abstract

A bipolar SMPS network for measuring atmospheric aerosols using natural air ions

To study atmospheric aerosols, it is essential to build networks that are continuously monitoring particle number size distribution (PNSD). PNSDs are often measured using electrical mobility spectrometers such as scanning mobility particle sizer (SMPS) which includes a neutralizer that conditions the charge state of particles to the pre-assumed distributions. During long-term atmospheric measurement, the charge state can deviate from these assumed values. Measuring both positively and negatively charged particles using a bipolar SMPS helps to capture the variations in aerosol charge state. In this study, we set up a network of bipolar SMPSs at three urban sites and three suburban sites in six Chinese cities. Removing the conventional neutralizer, the bipolar SMPS utilizes the natural aerosol charging process by atmospheric ions. Newly developed aerosol classifiers and detectors were used. Harmonized quality assurance procedures were adopted to assure the performance and the data quality, including laboratory calibrations and intercomparisons, maintenance and monitoring during field measurements, and data inversion and validation. PNSDs are effectively measured by bipolar SMPSs using natural charging process at all six sites. During six months of field measurement in these sites, ion mobility ratios fluctuate significantly while the daily averaged values are stable around 1.0. Particle number concentrations integrated from PNSDs are consistent with those directly measured by the detectors (within a variation of 10%).

Comparative Immunogenicity and Neutralization Potency of Four Approved COVID-19 Vaccines in BALB/c Mice.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccine candidates have been developed within a short period of time. Although the potency of these vaccines was evaluated individually, their comparative potency was not comprehensively evaluated. To compare the immunogenicity and neutralization efficacy of four approved COVID-19 vaccines in Iran, including: PastoCovac Plus, Sinopharm, SpikoGen, and Noora in BALB/c mice. Different groups of female BALB/c mice were vaccinated with three doses of each vaccine. The serum levels of antibodies against the viral receptor binding domain (anti-RBD) and spike (anti-spike) protein as well as the vaccine formulation (anti-vaccine) were evaluated using enzyme-linked immunosorbent assay (ELISA). The neutralization efficacy of these four vaccines was assessed through four neutralization assays: conventional virus neutralization test (cVNT), pseudotype virus neutralization test (pVNT), surrogate virus neutralization test (sVNT), and inhibition flow cytometry. All four vaccines induced seroconversion in vaccinated animals. All vaccines successfully induced high levels of anti-vaccine antibody; however, PastoCovac Plus and Sinopharm vaccines induced significantly higher levels of anti-RBD antibody titer compared to Noora and SpikoGen. Moreover, the results of the antibody response were corroborated by the virus neutralization tests, which revealed very weak neutralization potency by Noora and SpikoGen in all tests. Our results indicate significant immunogenicity and neutralization efficacy induced by PastoCovac Plus and Sinopharm, but not by Noora and SpikoGen. This suggests the need for additional comparative assessment of the potency and efficacy of these four vaccines in vaccinated subjects.

Alkaline Chemical Neutralization to Treat Acid Mine Drainage with High Concentrations of Iron and Manganese

Due to its high acidity and toxic metal content, acid mine drainage (AMD) needs to be properly treated before being discharged into the environment. This study took the AMD collected from one specific mine in China as a sample and investigated the treatment methodology for AMD. The water quality of the AMD was measured, and the sample was treated with caustic soda (NaOH) and shell powder (one kind of conventional neutralizer, mainly composed of CaCO3) by the neutralization method. The results show that the AMD has a relatively low pH (2.16) and contains high concentrations of Fe (77.54 g/L), Mn (621.29 mg/L), Cu (6.54 mg/L), Ca (12.39 mg/L), and Mg (55.04 mg/L). NaOH was an effective neutralizer to treat the AMD and performed much better than shell powder. Various metals were precipitated, in the order of Fe(III), Cu, Fe(II), Mn, Ca, and Mg. The metal removal mechanisms included precipitation, adsorption, and co-precipitation. The optimal reaction conditions were the reaction duration was selected as 5 min and the mass ratio of NaOH to AMD was 0.16:1 (w:v). By this stage, the pH rapidly increased from 2.16 to 8.53 during AMD-NaOH interactions and various metals were efficiently removed (from 86.71% to 99.99%) by NaOH. The residual mass concentrations of Fe, Mn, Cu, Ca, and Mg after the treatment were 1.52, 1.77, 0.10, 1.65, and 2.17 mg/L, respectively. These data revealed that NaOH was a good treatment regent for this kind of AMD, based on the discharge criteria of China (GB28661 2012). Also, the shell powder was a helpful neutralizer for pH adjustment and copper removal. This neutralization method has the advantages of convenient operation, high speed, good effect, simple equipment, and low infrastructure cost. In addition, the resulting neutralized residue is a valuable and high-quality raw material, which can be used in metal smelting and separation.

Immunogenicity and safety of RAZI recombinant spike protein vaccine (RCP) as a booster dose after priming with BBIBP-CorV: a parallel two groups, randomized, double blind trial

BackgroundThe immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV).MethodsWe conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions.ResultsWe recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26–3.39) and 21 (GMFI = 21.51, 95% CI 16.35–28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32–8.72), 1.77 (1.15–2.72), and 2.37 (1.62–3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths.ConclusionsBBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses.Trial registrationThis study was registered with the Iranian Registry of Clinical Trial at www.irct.ir, IRCT20201214049709N4. Registered 29 November 2021.

Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Fluorescence-Quenching Lateral Flow Immunoassay for "Turn-On" and Sensitive Detection of Anti-SARS-Cov-2 Neutralizing Antibodies in Human Serum.

The titer of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs) in the human body is an essential reference for evaluating the acquired protective immunity and resistance to SARS-CoV-2 infection. In this study, a fluorescence-quenching lateral flow immunoassay (FQ-LFIA) is established for quantitative detection of anti-SARS-CoV-2 NAbs in the sera of individuals who are vaccinated or infected within 10min. The ultrabright aggregation-induced emission properties encapsulated in nanoparticles, AIE490 NP, are applied in the established FQ-LFIA with gold nanoparticles to achieve a fluorescence "turn-on" competitive immunoassay. Under optimized conditions, the FQ-LFIA quantitatively detected 103 positive and 50 negative human serum samples with a limit of detection (LoD) of 1.29IUmL-1 . A strong correlation is present with the conventional pseudovirus-based virus neutralization test (R2 =0.9796, P<0.0001). In contrast, the traditional LFIA with a "turn-off" mode can only achieve a LoD of 11.06IUmL-1 . The FQ-LFIA showed excellent sensitivity to anti-SARS-CoV-2 NAbs. The intra- and inter-assay precisions of the established method are below 15%. The established FQ-LFIA has promising potential as a rapid and quantitative method for detecting anti-SARS-CoV-2 NAbs. FQ-LFIA can also be used to detect various biomarkers.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Immunogenicity and safety of heterologous boost immunization with PastoCovac Plus against COVID-19 in ChAdOx1-S or BBIBP-CorV primed individuals.

This study aimed at evaluation and comparison of PastoCovac Plus protein-subunit vaccine in parallel with ChAdOx1-S (AstraZeneca) and BBIBP-CorV (Sinopharm) in primarily vaccinated volunteers with two doses of ChAdOx1-S or BBIBP-CorV. 194 volunteers enrolled the study who were previously primed with 2 doses of ChAdOx1-S or BBIBP-CorV vaccines. They were divided into two heterologous regimens receiving a third dose of PastoCovac Plus, and two parallel homologous groups receiving the third dose of BBIBP-CorV or ChAdOx1-S. Serum samples were obtained just before and 4 weeks after booster dose. Anti-spike IgG and neutralizing antibodies were quantified and the conventional live-virus neutralization titer, (cVNT50) assay was done against Omicron BA.5 variant. Moreover, the adverse events data were recorded after receiving booster doses. ChAdOx1-S/PastoCovac Plus group reached 73.0 units increase in anti-Spike IgG rise compared to the ChAdOx1-S/ ChAdOx1-S (P: 0.016). No significant difference was observed between the two groups regarding neutralizing antibody rise (P: 0.256), indicating equivalency of both booster types. Adjusting for baseline titers, the BBIBP-CorV/PastoCovac Plus group showed 135.2 units increase (P<0.0001) in anti-Spike IgG, and 3.1 (P: 0.008) unit increase in mean rise of neutralizing antibodies compared to the homologous group. Adjustment for COVID-19 history, age, underlying diseases, and baseline antibody titers increased the odds of anti-Spike IgG fourfold rise both in the ChAdOx1-S (OR: 1.9; P: 0.199) and BBIBP CorV (OR: 37.3; P< 0.0001) heterologous groups compared to their corresponding homologous arms. The odds of neutralizing antibody fourfold rise, after adjustment for the same variables, was 2.4 (P: 0.610) for the ChAdOx1-S heterologous group and 5.4 (P: 0.286) for the BBIBP CorV heterologous groups compared to their corresponding homologous groups. All the booster types had the potency to neutralize BA.5 variant with no significant difference. The highest rate of adverse event incidence was recorded for ChAdOx1-S homologous group. PastoCovac Plus booster application in primed individuals with BBIBP-CorV or ChAdOx1-S successfully increased specific antibodies' levels without any serious adverse events. This vaccine could be administrated in the heterologous regimen to effectively boost humoral immune responses.

Electroneutralization with simultaneous highly-efficient desalination exemplified by acidic brackish wastewater

Chemical neutralization has commonly been employed for the treatment of acidic brackish wastewater through the addition of external alkaline chemicals. However, this approach can potentially compromise the health of aquatic environment and resource sustainability. In this study, an innovative electroneutralization stack incorporating highly-efficient desalination was developed as a green solution for acidic hot-spring discharge treatment. Two distinct stack configurations, i.e., AECE and AEBP, were designed to evaluate the performance of simultaneous neutralization and desalination under different operating conditions. The results demonstrated the effectiveness of neutralizing acidic discharges while concurrently achieving a significant reduction in salinity. The brine solution produced during the process also presented the possibility of being harnessed as a valuable resource. Notably, the design parameters for the process were determined using a first-order kinetic model integrated with the Nernst-Planck equation. The maximum rate constants for sulfate and chloride removal were found to be 0.129 min−1 and 0.125 min−1 for AECE, and 0.039 min−1 and 0.042 min−1 for AEBP, respectively. The corresponding chloride-to-sulfate selectivities were observed to range from 0.42 to 1.00 for AECE, and from 0.15 to 1.21 for AEBP. Furthermore, the optimal operational conditions were identified through a comprehensive multi-objective assessment, resulting in process energy consumption ranging from 1.5 to 2.2 kWh/m3, while maintaining a clean water productivity of ∼30 L/m2/h. Significantly, in comparison to conventional chemical neutralization methods, the developed electroneutralization process demonstrated a substantial reduction in operating costs by 2 to 5-fold, along with a remarkable decrease in CO2 emissions (by approximately 4-fold) and the avoidance of sludge generation.

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.

SARS-CoV-2 Neutralizing Antibodies in Free-Ranging Fallow Deer (Dama dama) and Red Deer (Cervus elaphus) in Suburban and Rural Areas in Spain

Effective control of zoonotic infectious diseases requires identifying the animal species involved in the infectious cycle as transmitters or reservoirs where the pathogen could persist and evolve, increasing the risk of reintroduction of new variants in humans and animals. Multiple surveillance studies have detected the presence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or specific antibodies in free-ranging white-tailed deer (Odocoileus virginianus) in North America, being the wild mammal species with the most reported cases of SARS-CoV-2 infection. However, so far, all attempts to detect the infection in European cervids have been unsuccessful. In this study, we demonstrated the presence of specific antibodies against SARS-CoV-2 in European fallow deer (Dama dama) and red deer (Cervus elaphus) in Spain. However, all samples of European roe deer (Capreolus capreolus), taxonomically related to the American white-tailed deer, were seronegative for the virus. We tested 215 serum samples from roe deer (n = 116), red deer (n = 63), and fallow deer (n = 36) collected in eight provinces of central-southern Spain between 2017 and 2022. We first screened sera by a surrogate virus neutralization test (sVNT) ELISA based on the binding of the receptor-binding domain of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor and then confirmed positive samples by a conventional virus neutralization test (cVNT) against the Alpha, Delta, and Omicron variants. Four fallow deer and two red deer samples were seropositive in both neutralization assays. Four samples of fallow deer and one of red deer, collected in a suburban park near Madrid in February 2022, had antibodies against the Alpha, Delta, and Omicron variants, while a seropositive sample of red deer, collected in a rural area in February 2021, was specific for the Delta variant. All samples collected before the start of the pandemic in Spain were seronegative for sVNT, which also indicates that there are not cross-reactive antibodies potentially elicited by other viruses antigenically related to SARS-CoV-2. The results indicate probable human-to-deer transmission of SARS-CoV-2, but do not clarify whether there was subsequent spread within herds.

Optimal treatment technology selection for acid mine drainage via spherical fuzzy analytic hierarchy process

The Philippines is endowed with large deposits of mineral resources, but mining operations have the potential to negatively impact the environment. For example, acid mine drainage (AMD) is a problem that occurs when sulfide minerals are exposed to air, water, and microbial activity, releasing hazardous elements. Many treatment options have been proposed to date to address these negative impacts of AMD. However, little effort has been made over the years to develop a generalized framework for the selection of the optimum technology for AMD treatment. This study thus proposes the spherical fuzzy analytic hierarchy process (SFAHP) as a multi-criteria decision analysis tool to select the best treatment technology for the treatment of AMD. The use of SFAHP is novel in this context as it addresses uncertainty due to indeterminacy by incorporating a “hesitancy parameter” in the decision-making process. Three treatment options were evaluated using a set of criteria such as technical, economical, and environmental aspects. The results showed that the combination of the two-step neutralization ferrite formation and sulfidogenic bioreactor (TSNFF + SB) was considered the most sustainable, with the environmental aspect being the most important factor. The second most preferred option is the high-density sludge (HDS) process while the least preferred is the combination of the conventional lime neutralization and sulfidogenic bioreactor (CLN + SB). A sensitivity analysis was also done, and a comparison with traditional AHP was conducted, which also found TSNFF + SB to be the best option. This illustrative case study demonstrates the robust framework built from SFAHP for selecting the best technology to manage AMD and addresses uncertainty in the decision-making process.

Mechanism of removal of toxic arsenic (As) from zinc sulfate solution by ultrasonic enhanced neutralization with zinc roasting dust

Arsenic in zinc sulfate solution greatly hinders the zinc hydrometallurgy process, and the treatment of arsenic residue with lime also generates large cost due to the large amount of residue. Herein, a new process of neutralization by zinc roasting dust (ZRD) and ultrasound (US) field enhancement of arsenic removal was proposed. The process includes the following steps:one-stage neutralization, KMnO4 oxidation and two-stage neutralization to remove arsenic. The process has the advantages of a good arsenic removal effect, no introduction of new impurities, and less residue. The effects of the ZRD dosage, temperature and US power on arsenic removal were investigated. The optimal conditions were 12 g/L, 70 ℃ and 300 W, respectively, and the arsenic removal rate reached 99.3%. The X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FE-SEM-EDS) were utilized to analyze the raw materials and residue. The results showed that, compared with conventional conditions, US strengthening treatment led to more complete reaction of ZRD. The arsenic removal residue after US treatment mainly existed in the form of ZnFe2O4, Fe3O4 and ZnS, which exhibited low reactivity. Ultrasound could effectively destroy the wrapper on the surface of ZRD, and enhance the liquid–solid reaction, which reduced the amount of residue and improved the arsenic removal efficiency. Interestingly, we found that ultrasound could enhance arsenic removal by increasing the ligand exchange between arsenate and sulfate. Compared with the lime and conventional ZRD neutralization, the wet residue weight after US treatment was reduced by 46.8% and 16.3%, respectively.

Reduced Binding between Omicron B.1.1.529 and the Human ACE2 Receptor in a Surrogate Virus Neutralization Test for SARS-CoV-2.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

Development of SARS-CoV-2 neutralizing antibody detection assay by using recombinant plant-produced proteins

Detecting immunity against SARS-CoV-2 is vital for evaluating vaccine response and natural infection, but conventional virus neutralization test (cVNT) requires BSL3 and live viruses, and pseudo-virus neutralization test (pVNT) needs specialized equipment and trained professionals. The surrogate virus neutralization test (sVNT) was developed to overcome these limitations. This study explored the use of angiotensin converting enzyme 2 (ACE2) produced from Nicotiana benthamiana for the development of an affordable neutralizing antibodies detection assay. The results showed that the plant-produced ACE2 can bind to the receptor binding domain (RBD) of the SARS-CoV-2, and was used to develop sVNT with plant-produced RBD protein. The sVNT developed using plant-produced proteins showed high sensitivity and specificity when validated with a group of 30 RBD vaccinated mice sera and the results were correlated with cVNT titer. This preliminary finding suggests that the plants could offer a cost-effective platform for producing diagnostic reagents.