Antibodies In Human Sera Research Articles

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

We describe a new approach that can rapidly measure how the antibodies in human serum inhibit infection by many different influenza strains. This new approach is useful for understanding how viral evolution affects antibody immunity. We apply the approach to study the effect of repeated influenza vaccination.

Diagnosis of viral infectious diseases through sensitive detection of human serum antibodies using a modified label-free electrochemical biosensor.

Rapid virus identification is crucial for preventing outbreaks. The COVID-19 pandemic has highlighted the critical nature of rapid virus detection. Here, we designed a label-free electrochemical biosensor modified with gold nanoparticles (AuNPs) to detect IgG antibodies from human serum, enabling rapid point-of-care diagnostics. AuNPs were synthesized and characterized. A multivariate optimization was carried out to determine the optimal condition for functionalizing AuNPs with anti-IgG. Subsequently, using a glassy carbon electrode (GCE), a modified AuNPs/GCE electrochemical biosensor was developed for IgG detection. The results indicated thatAuNPs displayed a spherical morphology with a size distribution of 19.54nm. Additionally, the zeta potential was recorded at -7.84mV. Central composite design (CCD) analysis determined the optimal conditions for functionalizing AuNPs to be an anti-IgG concentration of 320µgmL-1, atemperature of 25°C, and pH of 7.4. The characterization study confirmed the successful synthesis and functionalization of AuNPs. Through electrochemical impedance spectroscopy measurement, the biosensor demonstrated a limit of detection (LOD) of 0.2ngmL-1 and limit of quantification (LOQ) of 0.8ngmL-1. Furthermore, tests in real samples showed the interaction between IgG antibodies in serum samples and AuNPs/GCE, confirming the biosensor's ability to detect and quantify IgG in clinical samples.

Nanoparticle-Supported, Rapid, and Electronic Detection of SARS-CoV-2 Antibodies and Antigens at Attomolar Level.

A gold nanoparticle (AuNP)-supported rapid, electronic detection (NasRED) platform is demonstrated with high specificity to differentiate SARS-CoV-2 from human coronaviruses while achieving high sensitivity. Uniquely, active fluidic force is exerted on AuNPs in a microcentrifuge tube, through engineered centrifugation and vortex agitation, to accelerate signal transduction from biochemical protein binding to physical AuNP aggregation and precipitation and finally to electronic signals in a customized, stabilized circuitry. The limits of detection are found <80 aM (12 fg/mL) and ∼700 aM (105 fg/mL) to detect antibodies in human pooled serum and 20% diluted whole blood, and <900 aM (45 fg/mL) and 4.1 fM (200 fg/mL) to detect nucleocapsid proteins in human saliva and nasal fluid, respectively. With a small test volume (6 µL), a rapid turnaround time (as fast as <15 min), low reagent cost (estimated ∼$2), and a small system footprint, this novel digital platform can be utilized for accurate diagnosis and screening of a wide range of infectious and chronic diseases.

Broad-Spectrum Legionaminic Acid-Specific Antibodies in Pooled Human IgGs Revealed by Glycan Microarrays with Chemoenzymatically Synthesized Nonulosonosides.

The presence and the level of antibodies in human sera against bacterial glycans are indications of prior encounters with similar antigens and/or the bacteria that express them by the immune system. An increasing number of pathogenic bacteria that cause human diseases have been shown to express polysaccharides containing a bacterial nonulosonic acid called 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac2). To investigate the immune recognition of Leg5,7Ac2, which is critical for the fight against bacterial infections, a highly effective chemoenzymatic synthon strategy was applied to construct a library of α2-3/6-linked Leg5,7Ac2-glycans via their diazido-derivatives (Leg5,7diN3-glycans) formed by efficient one-pot three-enzyme (OP3E) synthetic systems from a diazido-derivative of a six-carbon monosaccharide precursor. Glycan microarray studies using this synthetic library of a Leg5,7Ac2-capped collection of diverse underlying glycan carriers and their matched sialoside counterparts revealed specific recognition of Leg5,7Ac2 by human IgG antibodies pooled from thousands of healthy donors (IVIG), suggesting prior human encounters with Leg5,7Ac2-expressing pathogenic bacteria at the population level. These biologically relevant Leg5,7Ac2-glycans and their immune recognition assays are important tools to begin elucidating their biological roles, particularly in the context of infection and host-pathogen interactions.

PET/CT Targeted Tissue Sampling Reveals Intravenously Administered HGN194 IgG1 Affects HIV Distribution after Rectal Exposure.

Neutralizing monoclonal antibodies hold great potential for prevention of human immunodeficiency virus (HIV) acquisition. IgG is the most abundant antibody in human serum, has a long half-life, and potent effector functions, making it a prime candidate for an HIV prevention therapeutic. We combined Positron Emission Tomography imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-green fluorescent protein HIV (PA-GFP-BaL) and fluorescently labeled HGN194 IgG1 to determine whether intravenously instilled IgG influences viral interaction with mucosal barriers and viral penetration in colorectal tissue 2 h after rectal viral challenge. Our results show that IgG1 did not alter the number of virions found throughout the colon or viral penetration into the epithelium of the rectum or descending colon. A minor increase in virions was observed in the transverse colon of IgG1 treated animals. Overall, the number of viral particles found in the mesenteric lymph nodes was low. However, IgG1 administration resulted in a significant reduction of virions found in mesenteric lymph nodes. Taken together, our results show that HGN194 IgG1 does not prevent virions from penetrating into the colorectal mucosa but may perturb HIV virion access to the lymphatic system.

Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Reduction in Xenogeneic Epitopes on Porcine Endothelial Cells by Periodate Oxidation.

Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO4) oxidation. Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO4 for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding. Treatment of porcine endothelial cells and tissues with NaIO4 led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO4 was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO4 for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions. Glycan epitopes on living cells can be oxidized with NaIO4 while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides

ABSTRACT Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer’s investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Development and validation of bioanalytical methods to support clinical study of disitamab vedotin.

Disitamab vedotin (RC48), a humanized anti-HER2 antibody conjugated with monomethyl auristatin E (MMAE), is the first antibody-drug conjugate in China with an approved biological license application. A bioanalytical method was established for three analytes (total antibody, conjugate antibody and free payload) to help characterize their pharmacokinetic behavior in clinical settings. The bioanalytical methods were validated according to M10 guidance. Electrochemiluminescence assaymethods were used for thequantitative measurement of total antibody and conjugated antibody in human serum. A LC-MS/MSmethod was used to quantify the concentration of MMAE in human serum. The method had high specificity and sensitivity with a quantitative range of 19.531-1250.000ng/ml (total antibody), 39.063-5000.000ng/ml (conjugated antibody)and 0.04-10.0ng/ml (MMAE), respectively.

Detection of Toxoplasmosis in Pregnant Women’s In Sabratha City of Using VIDS

Toxoplasmosis is usually asymptomatic, but can have severe consequences if It occurs in immunodeficient subject or fetuses. The diagnosis of toxoplasmosis during pregnancy is often based on maternal serological testing for IgM and IgG Anti- Toxoplasma antibodies. Persistence of IgM for long periods, posses' problems in distinguishing acute from chronic infection. The evaluation of specific IgG avidity enables more accurate dating, since avidity rises progressively during the course of infection. Test that measures immunoglobulin G (IgG) is used to determine if a person has been infected. If it is necessary to try to estimate the time of infection, which is of particular importance for pregnant women, a test which measures immunoglobulin M (IgM) is also used along with other tests such as an avidity test. Toxoplasma-specific IgG antibodies are detectable 1-3 weeks after infection and remain detectable for the life of the individual. Toxoplasma-specific IgM antibodies are also detectable 1-3 weeks after infection but generally decline to nil by one year after infection. The VIDAS® TOXO IgM (TXM) assay is intended for use on the instruments of the VIDAS family (VITEK® Immunodiagnostic Assay System) as an automated enzyme-linked fluorescent immunoassay (ELFA) for the presumptive qualitative detection of anti-Toxoplasma gondii IgM antibodies in human serum. Seventy-six (76) women in the first 16 weeks of pregnancy were tested for VIDAS IgM, IgG antibodies and VIDAS Toxo-IgG avidity. Low avidity antibodies were demonstrated in 2 (33.3%) of 6 sera positive with IgM assay and 4 (12.12%) of sera positive with IgG assay. Low avidity was also detected in 2 (3.27%) of 61 sera negative with IgM. The low avidity suggesting a recent infection, while high avidity in 3 (50%) of the 6 positive IgM and 24 (72.72%) of 33 positive IgG indicating that the Infection acquired in the distant past. These findings highlight the value of VIDAS IgG avidity when used in combination with the VIDAS IgM and IgG assays to provide a confirmatory evidence of an acute infection with a single serum specimen for pregnant women.

Association Study between SNPs in MST1 and MST2 and H. pylori Infection as well as Noncardia Gastric Carcinogenesis

Introduction: Gastric cancer (GC) remains a global health challenge, and H. pylori infection is a main risk factor for noncardia GC. The present study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in mammalian sterile 20-like kinase 1 (MST1) and MST2, H. pylori (H. pylori) infection, and the risk of noncardia gastric cancer (GC). Methods: A case-control study was conducted using enzyme-linked immunosorbent assay (ELISA) and TaqMan method to detect the titer of anti-H. pylori antibody in normal human serum and genotype 9 SNPs of MST1 and MST2 genes among 808 samples. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between SNPs and H. pylori infection, as well as the risk of noncardia gastric cancer in codominant, dominant, overdominant, recessive, and log-additive genetic models. Haplotypes were constructed using the Haploview 4.2 software. Results: The CC genotype of MST2 SNP rs10955176 was associated with a reduced risk of H. pylori infection compared to the TT + CT genotype. None of other SNPs were associated with H. pylori infection. The TT genotype of MST2 SNP rs7827435 was associated with a reduced risk of noncardia gastric cancer compared to the AA + AT genotype. None of the SNPs were associated with noncardia gastric cancer. There were no associations between haplotypes and H. pylori infection or the risk of noncardia gastric cancer. Conclusions: The CC genotype of rs10955176 and the TT genotype of rs7827435 may serve as protective factors against H. pylori infection and noncardia gastric cancer risk, respectively.

Highly heterogenous humoral immune response in Lyme disease patients revealed by broad machine learning-assisted antibody binding profiling with random peptide arrays.

Lyme disease (LD), a rapidly growing public health problem in the US, represents a formidable challenge due to the lack of detailed understanding about how the human immune system responds to its pathogen, the Borrelia burgdorferi bacterium. Despite significant advances in gaining deeper insight into mechanisms the pathogen uses to evade immune response, substantial gaps remain. As a result, molecular tools for the disease diagnosis are lacking with the currently available tests showing poor performance. High interpersonal variability in immune response combined with the ability of the pathogen to use a number of immune evasive tactics have been implicated as underlying factors for the limited test performance. This study was designed to perform a broad profiling of the entire repertoire of circulating antibodies in human sera at the single-individual level using planar arrays of short linear peptides with random sequences. The peptides sample sparsely, but uniformly the entire combinatorial sequence space of the same length peptides for profiling the humoral immune response to a B.burg. infection and compare them with other diseases with etiology similar to LD and healthy controls. The study revealed substantial variability in antibody binding profiles between individual LD patients even to the same antigen (VlsE protein) and strong similarity between individuals diagnosed with Lyme disease and healthy controls from the areas endemic to LD suggesting a high prevalence of seropositivity in endemic healthy control. This work demonstrates the utility of the approach as a valuable analytical tool for agnostic profiling of humoral immune response to a pathogen.

A robust and validated LC–MS/MS method for the quantification of ramucirumab in rat and human serum using direct enzymatic digestion without immunoassay

A new liquid chromatography tandem mass spectrometry (LC–MS/MS) method was established to quantify the anti-gastric cancer fully human monoclonal antibody (ramucirumab) in rat and human serum. The surrogate peptide (GPSVLPLAPSSK) for ramucirumab was generated by trypsin hydrolysis and quantified using the isotopically labeled peptide GPSVLPLAPSSK[13C6, 15N2]ST containing two more amino acids at the carboxyl end as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry changes. Additionally, the oxidation and deamidation of unstable peptides (VVSVLTVLHQDWLNGK and NSLYLQMNSLR) were detected. The quantitative range of the proposed method was 1–1000 μg/mL, and complete methodological validation was performed. The precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference of the measurements met the required standards. The validated LC-MS/MS method was applied to pharmacokinetic studies in rats administered ramucirumab at 15 mg/kg intravenously. Overall, a robust, efficient, and cost-effective LC–MS/MS method was successfully developed for quantifying ramucirumab in rat and human serum.

Development and characterization of a hemolysis inhibition assay to determine functionality of anti-Streptolysin O antibodies in human sera

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies.

Application of the Peroxidase‒like Activity of Nanomaterials for the Detection of Pathogenic Bacteria and Viruses.

Infectious diseases caused by pathogenic bacteria and viruses pose a significant threat to human life and well-being. The prompt identification of these pathogens, characterized by speed, accuracy, and efficiency, not only aids in the timely screening of infected individuals and the prevention of further transmission, but also facilitates the precise diagnosis and treatment of patients. Direct smear microscopy, microbial culture, nucleic acid-based polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) based on microbial surface antigens or human serum antibodies, have made substantial contributions to the prevention and management of infectious diseases. Due to its shorter processing time, simple equipment requirements, and no need for professional and technical personnel, ELISA has inherent advantages over other methods for detecting pathogenic bacteria and viruses. Horseradish peroxidase mediated catalysis of substrate coloration is the key for the detection of target substances in ELISA. However, the variability, high cost, and environmental susceptibility of natural peroxidase greatly limit the application of ELISA in pathogen detection. Compared with natural enzymes, nanomaterials with enzyme-mimicking activity are inexpensive, highly environmentally stable, easy to store and mass producing, etc. Based on their peroxidase-like activities and unique physicochemical properties, nanomaterials can greatly improve the efficiency and ease of use of ELISA-like detection methods for pathogenic bacteria and viruses. This review introduces recent advances in the application of nanomaterials with peroxidase-like activity for the detection of pathogenic bacteria (both gram-negative bacteria and gram-positive bacteria) and viruses (both RNA viruses and DNA viruses). The emphasis is on the detection principle and the evaluation of effectiveness. The limitations and prospects for future translations are also discussed.

Cross-Reactivity of Human, Wild Boar, and Farm Animal Sera from Pre- and Post-Pandemic Periods with Alpha- and Βeta-Coronaviruses (CoV), including SARS-CoV-2.

Panels of pre- and post-pandemic farm animals, wild boar and human sera, including human sera able to neutralize SARS-CoV-2 in vitro, were tested in serological tests to determine their cross-reactivity with β- and α-CoV originating from farm animals. Sera were tested in neutralization assays with high ascending concentrations (up to 1 × 104 TCID50 units/well) of β-CoV Bovine coronavirus (BCV), SARS-CoV-2, and porcine α-CoV-transmissible gastroenteritis virus (TGEV). In addition, sera were tested for immunostaining of cells infected with β-CoV porcine hemagglutinating encephalomyelitis (PHEV). Testing revealed a significantly higher percentage of BCV neutralization (78%) for sera of humans that had experienced a SARS-CoV-2 infection (SARS-CoV-2 convalescent sera) than was observed for human pre-pandemic sera (37%). Also, 46% of these human SARS-CoV-2 convalescent sera neutralized the highest concentration of BCV (5 × 103 TCID50/well) tested, whereas only 9.6% of the pre-pandemic sera did. Largely similar percentages were observed for staining of PHEV-infected cells by these panels of human sera. Furthermore, post-pandemic sera collected from wild boars living near a densely populated area in The Netherlands also showed a higher percentage (43%) and stronger BCV neutralization than was observed for pre-pandemic sera from this area (21%) and for pre- (28%) and post-pandemic (20%) sera collected from wild boars living in a nature reserve park with limited access for the public. High percentages of BCV neutralization were observed for pre- and post-pandemic sera of cows (100%), pigs (up to 45%), sheep (36%) and rabbits (60%). However, this cross-neutralization was restricted to sera collected from specific herds or farms. TGEV was neutralized only by sera of pigs (68%) and a few wild boar sera (4.6%). None of the BCV and PHEV cross-reacting human pre-pandemic, wild boar and farm animal sera effectively neutralized SARS-CoV-2 in vitro. Preexisting antibodies in human sera effectively neutralized the animal β-CoV BCV in vitro. This cross-neutralization was boosted after humans had experienced a SARS-CoV-2 infection, indicating that SARS-CoV-2 activated a "memory" antibody response against structurally related epitopes expressed on the surface of a broad range of heterologous CoV, including β-CoV isolated from farm animals. Further research is needed to elucidate if a symptomless infection or environmental exposure to SARS-CoV-2 or another β-CoV also triggers such a "memory" antibody response in wild boars and other free-living animals.