Increased Antibody Binding Research Articles

Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this “affinity profiling” approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Abstract A057: Microscale analysis of histone modifications in rare cell populations in prostate cancer

Abstract Pre-clinical models have identified diverse epigenetic alterations in prostate cancer that associate with treatment resistance and may also be therapeutic targets. However, these assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input primary tumor samples. Exclusive Liquid Repellant (ELR) platforms pass magnetic bead bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of prostate cancer cell lines with LNCaP, V16D, 49F, 42D, and H660s with serial dilutions was performed to evaluate the sensitivity and specificity of this integrated assay. The CUT&Tag assay was performed in standard fashion on a serial dilution of 50-100,000 cells from LNCaP and V16D calls and showed dropout of recoverable DNA at 50 cells. Sequencing of DNA at these dilutions confirmed identification of differential histone modifications consistent with previously published data. CUT&Tag was adapted to ELR through the serial patterning of each buffer in distinct wells connected by an oil interface. In order to optimize the assay for reproducibility and increased sensitivity, we optimized antibody binding/washing to minimize the protein A-Transposase inhibition. This optimization showed increased antibody binding by 36% compared to protocol defined concentrations. We have now successfully performed this assay with a sensitivity of ~100 cells with recoverable DNA for sequencing to identify heterogenous histone patterns in each cell line. CUT&Tag has improved sensitivity to detect epigenetic alterations that associate with aggressive prostate cancer. The integration of CUT&Tag with ELR improves both sensitivity and reproducibility for epigenetic analysis at low input cell numbers. This assay is now being utilized to analyze patient tumor samples and prostate cancer heterogeneity. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Duane S. Juang, Cole S. Gilsdorf, Shuang G. Zhao, David J. Beebe, Joshua M. Lang. Microscale analysis of histone modifications in rare cell populations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A057.

Invasive Streptococcal Infection Can Lead to the Generation of Cross-Strain Opsonic Antibodies.

The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain specific or more broadly protective. Here, we quantified the opsonic-antibody response following invasive S. pyogenes infection. Four patients with S. pyogenes bacteremia between 2018 and 2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute- and convalescent-phase sera were obtained, and the S. pyogenes isolates were genome sequenced (emm118, emm85, and two emm1 isolates). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Antibody binding increased modestly against the infecting isolate and across emm types in convalescent- compared to acute-phase sera for all patients. For two patients, phagocytosis increased in convalescent-phase serum both for the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both nonfunctional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections. IMPORTANCE The bacterium Streptococcus pyogenes is a common cause of both mild and severe human diseases resulting in substantial morbidity and mortality each year. No vaccines are available, and our understanding of the antibody response to this human pathogen is still incomplete. Here, we carefully analyzed the opsonic antibody response following invasive infection in four patients. Unexpectedly, the patients did not always generate opsonic antibodies against the specific infecting strain. Instead, we found that some patients could generate cross-opsonic antibodies, leading to phagocytosis of bacteria across strains. The emergence of cross-opsonic antibodies is likely important for long-term immunity against S. pyogenes. Our findings question the dogma that mostly strain-specific immunity is developed after infection and add to our overall understanding of how immunity to S. pyogenes can evolve.

Electrochemical immunosensor based on hybrid MoS2/Pt@Au-nanoprism/PDA for simultaneous detection of free and total prostate specific antigen in serum

As important biomarkers for prostate cancer, total prostate specific antigen (tPSA) and free prostate specific antigen (fPSA) levels in serum play essential roles in early diagnosis. In this study, hybrid molybdenum disulfide (MoS2)/Pt@Au-nanoprism/polydopamine (PDA)-modified screen-printed electrodes were utilized to detect both fPSA and tPSA in serum simultaneously. MoS2 generated by the hydrothermal method assembled on the electrode to provide binding sites for further modification. Au nanoprisms were obtained by reducing chloroauric acid with sodium thiosulfate to achieve uniformity. Pt nanoparticles covered on Au nanoprisms by the displacement method, and the generated Pt@Au-nanoprism structure was then deposited onto the electrode to enhance the electrochemical signals. An electropolymerization-synthesized PDA film further modified the electrode to increase antibody binding sites. The results demonstrated that the proposed electrochemical immunosensor for fPSA and tPSA analysis in serum using differential pulse voltammetry (DPV) had a wide linear range from 0.0001 ng/mL to 100 ng/mL, with limits of detection (3 SD/slope) at 0.1 pg/mL and 1.1 fg/mL, respectively. For clinical patient samples, the proposed biosensor demonstrated good accuracy according to the clinical electrochemiluminescence immunoassay and improved the diagnostic specificity of tPSA concentrations in the “gray zone", suggesting promising prospects for clinical applications of our multiplex biosensors.

Pneumococcal capsule blocks protection by immunization with conserved surface proteins

Vaccines targeting Streptococcus pneumoniae (Spn) are limited by dependence on capsular polysaccharide and its serotype diversity. More broadly-based approaches using common protein antigens have not resulted in a licensed vaccine. Herein, we used an unbiased, genome-wide approach to find novel vaccine antigens to disrupt carriage modeled in mice. A Tn-Seq screen identified 198 genes required for colonization of which 16 are known to express conserved, immunogenic surface proteins. After testing defined mutants for impaired colonization of infant and adult mice, 5 validated candidates (StkP, PenA/Pbp2a, PgdA, HtrA, and LytD/Pce/CbpE) were used as immunogens. Despite induction of antibody recognizing the Spn cell surface, there was no protection against Spn colonization. There was, however, protection against an unencapsulated Spn mutant. This result correlated with increased antibody binding to the bacterial surface in the absence of capsule. Our findings demonstrate how the pneumococcal capsule interferes with mucosal protection by antibody to common protein targets.

Free Energy Landscapes from SARS-CoV-2 Spike Glycoprotein Simulations Suggest that RBD Opening Can Be Modulated via Interactions in an Allosteric Pocket.

The SARS-CoV-2 coronavirus is an enveloped, positive-sense single-stranded RNA virus that is responsible for the COVID-19 pandemic. The spike is a class I viral fusion glycoprotein that extends from the viral surface and is responsible for viral entry into the host cell and is the primary target of neutralizing antibodies. The receptor binding domain (RBD) of the spike samples multiple conformations in a compromise between evading immune recognition and searching for the host-cell surface receptor. Using atomistic simulations of the glycosylated wild-type spike in the closed and 1-up RBD conformations, we map the free energy landscape for RBD opening and identify interactions in an allosteric pocket that influence RBD dynamics. The results provide an explanation for experimental observation of increased antibody binding for a clinical variant with a substitution in this pocket. Our results also suggest the possibility of allosteric targeting of the RBD equilibrium to favor open states via binding of small molecules to the hinge pocket. In addition to potential value as experimental probes to quantify RBD conformational heterogeneity, small molecules that modulate the RBD equilibrium could help explore the relationship between RBD opening and S1 shedding.

Abstract 1032: Super-resolution imaging studies of zanidatamab: Providing insights into its bispecific mode of action

Abstract Zanidatamab (ZW25) is a biparatopic antibody that simultaneously binds two distinct epitopes of the oncogenic cell surface receptor HER2. Zanidatamab is currently being evaluated in global Phase 1, Phase 2 and registration-enabling clinical trials as a potential new treatment for patients with HER2-expressing cancers, including biliary tract, gastroesophageal adenocarcinomas, breast, and other tumor types. The extracellular domain of the HER2 receptor comprises four domains with zanidatamab binding to epitopes on domain 2 and the membrane proximal domain 4. Domain 2 and domain 4 of HER2 are also respectively targeted by the antibodies pertuzumab and trastuzumab, currently approved for clinical use in a subset of HER2 overexpressing cancers. The unique engineering and HER2-engagement of zanidatamab results in multiple mechanisms of action, including dual HER2 signal blockade, increased antibody binding, receptor clustering, and removal of HER2 from the cell surface, and potent effector function. Preclinical work shows that zanidatamab can exhibit differentiated activity over trastuzumab or combination of trastuzumab and pertuzumab but the precise molecular mechanism by which zanidatamab differentiates itself from these approved agents remains unclear. Structural modeling of zanidatamab in complex with HER2 suggests that unlike trastuzumab and pertuzumab, steric features of zanidatamab induce a complex of this antibody with HER2 comprised of alternating chains of the antibody and HER2 molecules, with each copy of zanidatamab bridging two HER2 molecules and vice-versa. In this study we report on the spatial distribution of cell surface HER2 molecules and the effect of antibody-induced receptor reorganization on prototypical cancer cells with high and low expression levels of this receptor. We employ super-resolution single-molecule microscopy to map the receptor distribution below the diffraction limit of traditional optical imaging techniques. The use of Direct Stochastic Optical Reconstruction Microscopy (dSTORM) allows us to quantitatively differentiate properties such as cluster size, frequency and receptor density induced by the antibodies. We demonstrate that the unique geometry of HER2 engagement achieved by zanidatamab results in the induction of strikingly aggregated HER2 receptor cluster we refer to as “capping” on the cell surface. This effect induced by zanidatamab is notably distinct relative to the receptor reorganization observed with trastuzumab or combination of trastuzumab and pertuzumab which typically induce multiple smaller microclusters on the cell surface. We observe differences in the lifetime and persistence of these capped configurations induced by zanidatamab, leading us to believe that these cell surface reorganization events are likely coupled to events such as internalization and receptor down regulation. Citation Format: Surjit Dixit, Libin Abraham, Nina Weiser, Michael R. Gold. Super-resolution imaging studies of zanidatamab: Providing insights into its bispecific mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1032.

Heterologous immunization with Covishield and Pfizer vaccines against SARS-CoV-2 elicits a robust humoral immune response.

Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford-AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

A phase IIb, open-label, single-arm study of zanidatamab (ZW25) monotherapy in subjects with advanced or metastatic HER2-amplified biliary tract cancers.

TPS352 Background: Advanced biliary tract cancers (BTCs), including gallbladder cancer (GBC) and cholangiocarcinoma (CC), have a poor prognosis. Zanidatamab (ZW25) is a novel bispecific antibody that targets HER2 domains ECD2 and ECD4, resulting in increased antibody binding density and improved receptor internalization and downregulation relative to trastuzumab. In an ongoing phase I trial (ZWI-ZW25-101; NCT02892123), single-agent zanidatamab was well tolerated and showed promising anti-tumor activity across HER2-expressing solid tumors, including BTCs. These results formed the basis for a phase IIb study of zanidatamab in patients with BTC. Methods: Study ZWI-ZW25-203 (NCT04466891) is a global, multicenter, open-label, single-arm, phase IIb trial designed to evaluate the anti-tumor activity of zanidatamab monotherapy in patients with HER2-amplified, inoperable and advanced or metastatic BTCs, including GBC and CC. Patients must have received at least 1 prior gemcitabine-containing systemic chemotherapy regimen for advanced disease and have experienced disease progression after (or developed intolerance to) their most recent prior therapy. New or archival tumor tissue is required from all patients for HER2 amplification and protein expression testing at a central lab using in situ hybridization (ISH) and immunohistochemistry (IHC) assays. Approximately 100 patients with HER2 amplification by ISH will be enrolled. Zanidatamab 20 mg/kg will be administered intravenously every 2 weeks until one of the treatment discontinuation criteria is met. The primary endpoint of the study is the confirmed objective response rate (ORR) by independent central review per the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Secondary endpoints include duration of response (DOR), proportion of patients with DOR ≥ 16 weeks, disease control rate, progression-free survival, overall survival, safety, quality of life, and disease-related pain. The safety and tolerability of zanidatamab will be assessed by recording the frequency and severity of adverse events, serious adverse events, and laboratory abnormalities, as well as the frequency of zanidatamab dose modifications. The study is currently open for enrollment. Clinical trial information: NCT04466891.

Simultaneously targeting nitrocellulose and antibody by a dual-headed protein

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix – a protein that has a strong affinity to nitrocellulose membrane and protein A – a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.

Abstract B001: Safety and efficacy of ZW25, a HER2-targeted bispecific antibody, in combination with chemotherapy in patients with locally advanced and/or metastatic HER2-expressing gastroesophageal cancer

Abstract Background: Up to 30% of gastroesophageal adenocarcinoma (GEA) overexpress HER2. ZW25 is a novel bispecific antibody that targets HER2 domains ECD2 and ECD4, resulting in multiple differentiated and unique mechanisms of action, including increased antibody binding density and improved receptor internalization and downregulation relative to trastuzumab. In an ongoing phase 1 trial (ZWI-ZW25-101; NCT02892123), single-agent ZW25 was well tolerated and showed promising anti-tumor activity across HER2-expressing solid tumors. This abstract presents data for GEA patients treated with ZW25 in combination with chemotherapy in Study ZWI-ZW25-101. Methods Eligible patients had HER2-expressing (IHC 3+, IHC 2+/FISH+, or IHC 2+/FISH-) GEA and had disease progression after 1–3 lines of prior chemotherapy for advanced and/or metastatic disease. Fresh or archived tumor tissue was required for central review of HER2 status, although patients could be enrolled based on local results. Patients received ZW25 in combination with paclitaxel or capecitabine. Evaluations included standard safety assessments, tumor re-staging (Q8W by RECIST 1.1), and assessment of peak/trough pharmacokinetic (PK) levels during each cycle. Results To date, 14 patients have been enrolled, with safety and efficacy data available for 11. ZW25 was administered in combination with paclitaxel (n=5) or capecitabine (n=6). The median age was 62 yrs (range, 26-80). The median number of prior anti-cancer regimens was 3 and the median number of prior HER2-targeted therapies was 1. Six patients (55%) were HER2 IHC3+ or IHC2+ and FISH+ per central review. The most common adverse events (AEs) considered related to ZW25 or ZW25 and chemotherapy were diarrhea (n=5; 45%), fatigue (n=3; 27%), and nausea (n=3; 27%); the majority of events were Grade 1 or 2. Grade 3 or higher events considered related to ZW25 or ZW25 and chemotherapy occurred in 3 patients (27%), including fatigue, hypokalemia, and neutropenia (n=1 each). One additional patient experienced a Grade 5 serious adverse event of pneumonitis, reported as related to the combination of ZW25 and paclitaxel. This patient had been previously treated with trastuzumab, cisplatin, and capecitabine; pembrolizumab and margetuximab; and most recently DS8201, and had asymptomatic interstitial lung disease at study entry. A total of 9/11 patients (82%) had measurable disease and 8/9 (89%) were response-evaluable. The objective response rate was 63% (95% CI: 25, 92) (5/8 patients; all partial responses, with 4 confirmed to date) and the disease control rate was 75% (6/8 patients). Responses were seen in patients with FISH+ and FISH- disease, as well as in combination with either paclitaxel or capecitabine. Patients have received between 1 and 12 cycles of treatment and 4 patients remain on treatment. PK data will be presented at the meeting. Conclusions The combination of ZW25 with chemotherapy was generally well tolerated in heavily pre-treated patients with HER2 high- and low-expressing GEA and the preliminary anti-tumor activity is promising. Further evaluation of the safety and efficacy of ZW25 in combination with standard of care chemotherapy regimens has been initiated in frontline GEA (ZW25-201; NCT03929666). Citation Format: Funda Meric-Bernstam, Jorge Chaves, Do-Youn Oh, Jeeyun Lee, Yoon-Koo Kang, Erika Hamilton, Jose Mayordomo, Melody Cobleigh, Christos Vaklavas, Elena Elimova, Jaffer Ajani, Jordi Rodon, Gerry Rowse, Todd Gray, Rose Lai, Diana Hanna. Safety and efficacy of ZW25, a HER2-targeted bispecific antibody, in combination with chemotherapy in patients with locally advanced and/or metastatic HER2-expressing gastroesophageal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B001. doi:10.1158/1535-7163.TARG-19-B001

Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.

Impact of the variations in potential glycosylation sites of the hemagglutinin of H9N2 influenza virus

Variations in the potential glycosylation sites were observed in hemagglutinin (HA) sequences of H9N2 avian influenza virus isolated in China, deposited in the Influenza Virus Resource of NCBI before 2017, which showed a deleted glycosylation site at amino acid residue 218, and an introduced glycosylation site at amino acid residue 313. Based on the variations in the glycosylation sites at these amino acids, H9N2 avian influenza viruses could be divided into three categories. Firstly, most of the H9N2 influenza viruses were 218G+ viruses; less 313G+ viruses were isolated between 1997 and 2004. Secondly, the occurrence of the 218G+/313G+ viruses increased, while the 218G+/313G− viruses decreased from 2005 to 2012. Thirdly, from 2013 to 2016, the 218G−/313G+ viruses were predominant compared to the 218G+/313G+ viruses. Here, based on an F/98 virus backbone, a 218G+/313G− virus, two reassortment viruses were generated, and named rF/HA218G+/313G+ and rF/HA 218G+/313G−, respectively. HA protein migration demonstrated that the glycosylation sites at amino acid residues 313 and 218 were both functional. The absence of the glycosylation site at amino acid residue 218 and the presence of the glycosylation site at amino acid residue 313 increased antibody binding and moderately prevented the virus from escaping neutralization with homologous antisera. Additionally, compared to the F/98 virus (218G+/313G−), the viruses rF/HA218G+/313G+ or rF/HA218G−/313G+ showed significantly increased infectivity of MDCK cells, chicken embryo eggs, and trachea and lung tissue of SPF chickens, but did not display differences in airborne spread in chickens or infectivity of mice compared with its parental virus F/98.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3hour) and concentrations (5, 10, 20, and 40μL) was determined by flow cytometry. An increase in incubation time from 30minutes to 2hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5μL to 20μL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro.

Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy.

Female infertility induced by vanadium inhalation in a murine model

To characterize antibody specificities associated with pre-eclampsia (PE), bacterial displayed peptide library screening and evolution was applied to identify peptide epitopes recognized by plasma antibodies present in women with PE near the time of delivery. Pre-eclamptic women exhibited elevated IgG1 titers towards a peptide epitope KRPSCIGCK within the Epstein-Barr virus nuclear antigen 1 (EBNA-1). EBNA-1 epitope antibodies cross-reacted with a similar epitope within the extracellular N-terminus of the human G protein-coupled receptor, GPR50, expressed in human placental tissue and immortalized placental trophoblast cells. We observed increased antibody binding activity to epitopes from EBNA-1 and GPR50 among women with PE (n = 42) compared to healthy-outcome pregnancies (n = 43) and nulligravid samples (n = 21). The EBNA-1 peptide potently blocked binding of the PE-associated antibody to the GPR50 epitope (IC50 = 58–81 pM). These results reveal the existence of molecular mimicry between EBNA-1 and placental GPR50, supporting a mechanism for IgG1 deposition in the pre-eclamptic placenta.

A pre-eclampsia-associated Epstein-Barr virus antibody cross-reacts with placental GPR50

To characterize antibody specificities associated with pre-eclampsia (PE), bacterial displayed peptide library screening and evolution was applied to identify peptide epitopes recognized by plasma antibodies present in women with PE near the time of delivery. Pre-eclamptic women exhibited elevated IgG1 titers towards a peptide epitope KRPSCIGCK within the Epstein-Barr virus nuclear antigen 1 (EBNA-1). EBNA-1 epitope antibodies cross-reacted with a similar epitope within the extracellular N-terminus of the human G protein-coupled receptor, GPR50, expressed in human placental tissue and immortalized placental trophoblast cells. We observed increased antibody binding activity to epitopes from EBNA-1 and GPR50 among women with PE (n=42) compared to healthy-outcome pregnancies (n=43) and nulligravid samples (n=21). The EBNA-1 peptide potently blocked binding of the PE-associated antibody to the GPR50 epitope (IC50=58–81pM). These results reveal the existence of molecular mimicry between EBNA-1 and placental GPR50, supporting a mechanism for IgG1 deposition in the pre-eclamptic placenta.

Determination of the Dynamic Structures of Igg Antibody by Individual-Particle Electron Tomography

Antibody, also known as immunoglobulin, functions as one of the key parts in human immune response. Studying antibodies provides benefits in treating immune system diseases, medical diagnostics utilizing particular antibodies, and applications such as monoclonal antibody therapy. Antibodies perform functions through identifying and neutralizing various foreign objects based on a unique Y-shaped highly dynamic structure. In antibody structure, two antigen-binding domains (Fabs) are connected to effector domain (Fc) via a flexible hinge-loop region allowing two antigen-binding sites to vary. Dynamics of the loop region allow antibodies to undergo large conformational change to search binding sites on various biological macromolecule surfaces, fitting the paratope to a targeted antigen. Conventional techniques, such as X-ray crystallography, nuclear magnetic resonance technique, or single particle TEM reconstruction method, in studying antibody structure are faced with difficulty in revealing structural dynamic properties in 3D, due to their requirement of averaging vast numbers of particles.Here, we use our recently developed individual-particle electron tomography (IPET) to obtain nearly a hundred 3D density maps each from an individual antibody. Each 3D map provided the spacing and orientations of the three domains of each antibody. By comparison of the domain spatial relation among different antibody molecules, the 3D distributions of the two Fab domains uncovered the dynamics and fluctuations of this antibody type in solution. These dynamic characteristics provide us insight into antibody functional expression, and show potential approaches to increase antibody binding affinity.