Antigenic Domains Research Articles

Detection and prognostic relevance of PLA2R epitopes in idiopathic membranous nephropathy: a simultaneous quantitative multiplex suspension array detection method

Abstract Objective This study aimed to develop a multiplex suspension assay for the simultaneous quantitative detection of anti-cysteine-rich domain (anti-CysR)/C-type lectin domain 1 (CTLD1)/C-type lectin domain 6–8 (CTLD678)-IgG4 antibodies of M-type phospholipase A2 receptor (PLA2R) in the serum samples of patients to evaluate the clinical application value of PLA2R epitope spreading in disease prognosis. Methods CysR, CTLD1, and CTLD678 antigen domains were coupled to three types of microspheres. The optimal dilution ratio of biotinylated anti-human IgG4 was identified, and a multiplex suspension assay was evaluated in terms of linearity, sensitivity, precision, specificity, and recovery rate. Lastly, the relationship between epitope spreading and the severity of idiopathic membranous nephropathy (IMN) was evaluated. Results The content of all three epitopes could be detected simultaneously within two hours. The intra-assay precision ranged from 4.74% to 9.48% , and the inter-assay precision was between 5.13% and 13.92% . Specific experiments showed that the human IgA antibody did not cause a cross-reaction. The recovery rates ranged between 90% and 100%. The cut-off values of epitopes CysR, CTLD1 and CTLD678 between healthy individuals and patients were 6.30, 12.38, and 10.06 Ru/mL, respectively. Among them, the positive rates of epitopes CysR, CTLD1 and CTLD678 were 100%, 34%, and 75%, respectively. In addition, group 3 (CTLD1 response) accounted for 73% of the twelve patients who were not in remission. Meanwhile, When the concentration of CTLD1 exceeds 42.76 Ru/mL, the patient's prognosis may be poorer. Conclusion A multiplex suspension assay was developed for the simultaneous quantitative detection of anti-CysR/CTLD1/CTLD678-IgG4 antibodies. The epitope migration sequence of PLA2R molecules during disease progression may not follow a simple linear rule. Among them, the epitope CTLD1 is likely to exert the most significant influence on patient remission.

Structures of the Varicella Zoster Virus Glycoprotein E and Epitope Mapping of Vaccine-Elicited Antibodies.

Background: Varicella zoster virus (VZV) is the causative agent for chickenpox and herpes zoster (HZ, shingles). HZ is a debilitating disease affecting elderly and immunocompromised populations. Glycoprotein E (gE) is indispensable for viral replication and cell-to-cell spread and is the primary target for anti-VZV antibodies. Importantly, gE is the sole antigen in Shingrix, a highly efficacious, AS01B-adjuvanted vaccine approved in multiple countries for the prevention of HZ, yet the three-dimensional (3D) structure of gE remains elusive. Objectives: We sought to determine the structure of VZV gE and to understand in detail its interactions with neutralizing antibodies. Methods: We used X-ray crystallography and cryo-electron microscopy to elucidate structures of gE bound by recombinant Fabs of antibodies previously elicited through vaccination with Zostavax, a live, attenuated vaccine. Results: The 3D structures resolve distinct central and C-terminal antigenic domains, presenting an array of diverse conformational epitopes. The central domain has two beta-sheets and two alpha helices, including an IgG-like fold. The C-terminal domain exhibits 3 beta-sheets and an Ig-like fold and high structural similarity to HSV1 gE. Conclusions: gE from VZV-infected cells elicits a human antibody response with a preference for the gI binding domain of gE. These results yield insights to VZV gE structure and immunogenicity, provide a framework for future studies, and may guide the design of additional herpesvirus vaccine antigens. Teaser: Structures of varicella zoster virus glycoprotein E reveal distinct antigenic domains and define epitopes for vaccine-elicited human antibodies.

The Autonomous Fusion Activity of Human Cytomegalovirus Glycoprotein B Is Regulated by Its Carboxy-Terminal Domain.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Hepatitis C Virus E1E2 Structure, Diversity, and Implications for Vaccine Development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Improving Access to Anti-HDV Testing: Development and Validation of an Affordable In-House ELISA Assay.

In certain low-income nations, the hepatitis Delta virus and hepatitis B virus (HBV) pose a serious medical burden, where the prevalence of hepatitis B surface antigen (HBsAg) is greater than 8%. Especially in rural places, irregular diagnostic exams are the main restriction and reason for underestimation. Utilizing serum samples from a Pakistani isolate, an internal ELISA for the quick identification of anti-HDV was created, and the effectiveness of the test was compared to a commercial diagnostic kit. HDV-positive serum samples were collected, and a highly antigenic domain of HDAg antigen was derived from them. This antigenic HDAg was expressed in a bacterial expression system, purified by Ni-chromatography, and confirmed by SDS-PAGE and Western blot analysis. The purified antigen was utilized to develop an in-house ELISA assay for anti-HDV antibody detection of the patient's serum samples at very low cost. Purified antigens and positive and negative controls can detect anti-HDV (antibodies) in ELISA plates. The in-house developed kit's efficiency was compared with that of a commercial kit (Witech Inc., USA) by the mean optical density values of both kits. No significant difference was observed (a P value of 0.576) by applying statistical analysis. The newly developed in-house ELISA is equally efficient compared to commercial kits, and these may be useful in regular diagnostic laboratories, especially for analyzing local isolates.

Engineering Antigen-Specific Tolerance to an Artificial Protein Hydrogel.

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due to their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "nonself" artificial protein hydrogels, which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the nonself gels raised significantly higher antigel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the nonself hydrogel epitopes to DEC-205, an endocytic receptor involved in Treg induction, fully suppressed the elevated antibody titer against the nonself gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

More than a decade of research on Schmallenberg virus-Knowns and unknowns.

Schmallenberg virus, an arbovirus of the Orthobunyavirus genus that primarily infects ruminants, emerged in 2011 near the Dutch-German border region and subsequently caused a large number of abortions and the births of severely malformed newborns in the European livestock population. Immediate intensive research led to the development of reliable diagnostic tests, the identification of competent Culicoides vector species, and the elucidation of the pathogenesis in infected vertebrate hosts. In addition, the structure of the major antigenic domain has been elucidated in great detail, leading to the development of effective marker vaccine candidates. The knowledge gained over the last decade on the biology and pathogenesis of SBV and the experience acquired in its control will be of great value in the future for the control of any similar emerging pathogen of veterinary or public health importance such as Shuni or Oropouche virus. However, some important knowledge gaps remain, for example, the factors contributing to the highly variable transmission rate from dam to fetus or the viral factors responsible for the vector competence of Culicoides midges are largely unknown. Thus, questions still remain for the next decade of research on SBV and related viruses.

Evaluation of vaccine efficacy with 2B/T epitope conjugated porcine IgG-Fc recombinants against foot-and-mouth disease virus.

The inactivated vaccine is effective in controlling foot-and-mouth disease (FMD), but it has drawbacks such as the need for a biosafety level 3 laboratory facility to handle live foot-and-mouth disease virus (FMDV), high production costs, and biological safety risks. In response to these challenges, we developed a new recombinant protein vaccine (2BT-pIgG-Fc) containing porcine IgG-Fc to enhance protein stability in the body. This vaccine incorporates two-repeat B-cell and one-single T-cell epitope derived from O/Jincheon/SKR/2014. Our study confirmed that 2BT-pIgG-Fc and a commercial FMDV vaccine induced FMDV-specific antibodies in guinea pigs at 28 days post-vaccination. The percentage inhibition (PI) value of 2BT-pIgG-Fc was 90.43%, and the commercial FMDV vaccine was 81.75%. The PI value of 2BT-pIgG-Fc was 8.68% higher than that of commercial FMDV vaccine. In pigs, the primary target animals for FMDV, all five individuals produced FMDV-specific antibodies 42 days after vaccination with 2BT-pIgG-Fc. Furthermore, serum from 2BT-pIgG-Fc-vaccinated pigs exhibited neutralizing ability against FMDV infection. Intriguingly, the 2BT-pIgG-Fc recombinant demonstrated FMDV-specific antibody production rates and neutralization efficiency similar to commercial inactivated vaccines. This study illustrates the potential to enhance vaccine efficacy by strategically combining well-known antigenic domains in the development of recombinant protein-based vaccines.

Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

The structure of HSV-1 gB bound to a potent neutralizing antibody reveals a conservative antigenic domain across herpesviruses

This study presents the pioneering co-structure of herpes simplex virus-1 (HSV-1) glycoproteins B (gB) and its neutralizing antibody D48, offering not only a structural foundation for D48 neutralizing HSV-1 infection but also revealing a potentially conserved antigenic domain gB DII, across the herpesvirus family, spanning pre- and post-fusion configurations. This lends support to the proposition that DII domain holds a pivotal and shared role during viral infection, presenting an auspicious target for the development of broad-spectrum drugs across the herpesvirus family.

Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections.Key points• The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs.• Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs.• Inoculation of mutated SVPs induces antibodies with low cross-reactivity.

Vaccination with a replication-defective cytomegalovirus vaccine elicits a glycoprotein B-specific monoclonal antibody repertoire distinct from natural infection

Human Cytomegalovirus (HCMV) is the leading infectious congenital infection globally and the most common viral infection in transplant recipients, therefore identifying a vaccine for HCMV is a top priority. Humoral immunity is a correlate of protection for HCMV infection. The most effective vaccine tested to date, which achieved 50% reduction in acquisition of HCMV, was comprised of the glycoprotein B protein given with an oil-in-water emulsion adjuvant MF59. We characterize gB-specific monoclonal antibodies isolated from individuals vaccinated with a disabled infectious single cycle (DISC) CMV vaccine, V160, and compare these to the gB-specific monoclonal antibody repertoire isolated from naturally-infected individuals. We find that vaccination with V160 resulted in gB-specific antibodies that bound homogenously to gB expressed on the surface of a cell in contrast to antibodies isolated from natural infection which variably bound to cell-associated gB. Vaccination resulted in a similar breadth of gB-specific antibodies, with binding profile to gB genotypes 1–5 comparable to that of natural infection. Few gB-specific neutralizing antibodies were isolated from V160 vaccinees and fewer antibodies had identifiable gB antigenic domain specificity compared to that of naturally-infected individuals. We also show that glycosylation of gB residue N73 may shield binding of gB-specific antibodies.

An Overview of Recent Developments in the Application of Antigen Displaying Vaccine Platforms: Hints for Future SARS-CoV-2 VLP Vaccines.

Recently, a great effort has been devoted to studying attenuated and subunit vaccine development against SARS-CoV-2 since its outbreak in December 2019. It is known that diverse virus-like particles (VLPs) are extensively employed as carriers to display various antigenic and immunostimulatory cargo modules for vaccine development. Single or multiple antigens or antigenic domains such as the spike or nucleocapsid protein or their variants from SARS-CoV-2 could also be incorporated into VLPs via either a genetic or chemical display approach. Such antigen display platforms would help screen safer and more effective vaccine candidates capable of generating a strong immune response with or without adjuvant. This review aims to provide valuable insights for the future development of SARS-CoV-2 VLP vaccines by summarizing the latest updates and perspectives on the vaccine development of VLP platforms for genetic and chemical displaying antigens from SARS-CoV-2.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTRN-S1NCOE to obtain vC/SM3'UTRN-tPAsS1NCOE and vC/SM3'UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-β induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-β induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1′‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SignificanceThis article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker - located on the mucin protein MUC16 - is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies - OC125, M11, "OC125-like," and "M11-like" - to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.

A fully human neutralizing monoclonal antibody targeting a highly conserved epitope of the human cytomegalovirus glycoprotein B.

Human cytomegalovirus causes severe diseases in children (by congenital infection) and immunocompromised patients. Treatment with antiviral agents, such as ganciclovir, is limited by their toxicity. In this study, we investigated the effectiveness of a fully human neutralizing monoclonal antibody to inhibit human cytomegalovirus infection and viral cell-to-cell spread. We isolated a potent neutralizing antibody, EV2038 (IgG1 lambda), targeting human cytomegalovirus glycoprotein B using Epstein-Barr virus transformation. This antibody inhibited human cytomegalovirus infection by all four laboratory strains and 42 Japanese clinical isolates, including ganciclovir-resistant isolates, with a 50% inhibitory concentration (IC50) ranging from 0.013 to 0.105 μg/mL, and 90% inhibitory concentration (IC90) ranging from 0.208 to 1.026 μg/mL, in both human embryonic lung fibroblasts (MRC-5) and human retinal pigment epithelial (ARPE-19) cells. Additionally, EV2038 prevented cell-to-cell spread of eight clinical viral isolates, with IC50 values ranging from 1.0 to 3.1 μg/mL, and IC90 values ranging from 13 to 19 μg/mL, in ARPE-19 cells. EV2038 recognized three discontinuous sequences on antigenic domain 1 of glycoprotein B (amino acids 549-560, 569-576, and 625-632), which were highly conserved among 71 clinical isolates from Japan and the United States. Pharmacokinetics study in cynomolgus monkeys suggested the potential efficacy of EV2038 in vivo, the concentration of which in serum remained higher than the IC90 values of cell-to-cell spread until 28 days after intravenous injection of 10 mg/kg EV2038. Our data strongly support EV2038 as a promising candidate and novel alternative for the treatment of human cytomegalovirus infection.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.