Exposure Of Epitopes Research Articles

Quantifying allergenic proteins using antibody-based methods or liquid chromatography-mass spectrometry/mass spectrometry: A review about the influence of food matrix, extraction, and sample preparation.

Accurate quantification of allergens in food is crucial for ensuring consumer safety. Pretreatment steps directly affect accuracy and efficiency of allergen quantification. We systematically reviewed the latest advances in pretreatment steps for antibody-based methods and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) protein quantification methods in food. For antibody-based methods, the effects induced by food matrix like decreased allergen solubility, epitope masking, and nonspecific binding are of the upmost importance. To mitigate interference from the matrix, effective and proper extraction can be used to obtain the target allergens with a high protein concentration and necessary epitope exposure. Removal of interfering substances, extraction systems (buffers and additives), assistive technologies, and commercial kits were discussed. About LC-MS/MS quantification, the preparation of the target peptides is the crucial step that significantly affects the efficiency and results obtained from the MS detector. The advantages and limitations of each method for pre-purification, enzymatic digestion, and peptide desalting were compared. Additionally, the application characteristics of microfluidic-based pretreatment devices were illustrated to improve the convenience and efficiency of quantification. A promising research direction is the targeted development of pretreatment methods for complex food matrices, such as lipid-based and carbohydrate-based matrices.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

An inactivated SARS-CoV-2 vaccine based on a Vero cell culture-adapted high-titer virus confers cross-protection in small animals

Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production.

Anti-SARS-CoV-2 Omicron BA.5 RBD antibody titers generated by the protein subunit vaccine Abdala in breast milk

ObjectiveIn this work, we investigate the presence of antibodies against OmicronBA.5 RBD in breast milk from puerperal women, vaccinated during pregnancy with the protein subunit vaccine Abdala, based on the Wuhan-Hu-1 RBD. Materials and methodsBreast milk samples were collected between September 30 and November 25, 2021, from 2 groups of participants: 5- and 9-weeks after the third dose of the vaccine. Antibody titers against OmicronBA.5 RBD and Wuhan-Hu-1 RBD were evaluated simultaneously by a homemade ELISA. The relationship of antibody titers with the parity at the beginning of vaccination and with the participants health status was assessed. ResultsBinding of OmicronBA.5 RBD, such as Wuhan-Hu-1, to IgA and IgG antibodies was detected in all breast milk samples, although the anti-RBD-Wuhan-Hu-1 titers were higher. The immune response to OmicronBA.5 could be related to the glycosylation of the Abdala RBD, expressed in Pichia pastoris, which contributes to the immunogenicity and to the exposure of epitopes that are conserved in pandemic variants, such as Omicron. Antibody titers against both RBDs were lower in participants with chronic diseases and in multiparous. ConclusionsThis is the first report of simultaneous detection of antibody titers, against the Wuhan-Hu-1 RBD and OmicronBA.5 RBD, in breast milk from puerperal women vaccinated with 3 doses a COVID-19 protein subunit vaccine. There is a tendency to decrease the antibody response after vaccination, due to chronic diseases and multiparity.

Investigation into Potential Allergenicity and Digestion-Resistant Linear Epitopes of Fish Skin Gelatin in Cell-Cultured Meat Scaffolds.

As a key component of cell-cultured fish, fish skin gelatin (FSG)-based cell scaffold provides support structures for cell growth, proliferation, and differentiation. However, there are potential allergenicity risks contained in FSG-based scaffolds. In this study, 3D edible scaffolds were prepared by phase separation method and showed a contact angle of less than 90°, which indicated that the scaffolds were favorable for cell adhesion. Besides, the swelling ratio was greater than 200%, implying a great potential to support cell growth. The sequence homology analysis indicated that FSG was prone to cross-reaction with collagen analogues. Additionally, a food allergic model was constructed and represented that mice gavaged with cod FSG exhibited higher levels of specific antibodies, mast cell degranulation, vascular permeability, and intestinal barrier impairment than those gavaged with pangasius and tilapias FSG. Its higher allergenicity might be attributed to a higher number of digestion-resistant linear epitopes. Moreover, the higher hydrolysis degree linked to the exposure of linear epitopes to promote the combination with IgE, which was also responsible for maintaining the higher allergenicity of cod FSG. This study clarifies allergenic risks in cell-cultured fish and further study will focus on the allergenicity reduction of FSG-based cell scaffolds.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.

Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection

Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.

Abstract 4006: IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells

Abstract Introduction: EGFRvIII is a commonly studied tumor-specific antigen in glioblastoma (GBM) and poses an enticing target for immunotherapy. Its varied expression, however, complicates its effectiveness as a Chimeric Antigen Receptor (CAR) T cell target. Given the increasing prominence of Interleukin-12 (IL12) in treating solid tumors, its synergy with EGFRvIII specific CARs in treating preclinical models of GBM is explored. Methods: To simulate tumor heterogeneity, mice were implanted with equal ratios of CT2A and CT2A EGFRvIII+ cells. We developed EGFRvIII-specific third-generation CAR T cells, both with and without IL12 using a MSGV retrovirus backbone and transduced them into murine T cells. Their antitumor effectiveness was gauged by evaluating survival rates, and their capability to enhance and modify the tumor environment was determined using single-cell sequencing. Results: Standalone EGFRvIII CARs were unsuccessful in curing mice with heterogeneous tumors. However, 60% of mice treated with EGFRvIII CARs combined with IL12 were long term survivors. IL12 secreting CARs exhibited prolonged persistence in vivo, and there was no need for prior lymphodepletion before ALT introduction. Our studies reveal that EGFRvIII CARs secreting IL12 can eliminate EGFRvIII negative tumor cells by drawing on the inherent CD8 T cell compartment. Moreover, IL12 prominently impacted microglia among all Antigen Presenting Cells in the tumor environment, amplifying antigen presentation processes and broadening epitope exposure. Conclusion: By leveraging IL12, EGFRvIII CARs can counteract antigen heterogeneity in tumors by recruiting endogenous CD8 T cells and polarizing microglia. These IL12-secreting CARs outlast their traditional counterparts without requiring lymphodepletion. Harnessing CAR-T cells that secrete IL12 represents a novel strategy for overcoming tumor heterogeneity and treating GBM in a clinical context. Citation Format: Aditya Mohan, Steven Shen, Kelly Hotchkiss, Sarah Quackenbush, John Sampson, Peter Fecci, Anoop Patel. IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4006.

Effect of transglutaminase in conjunction with glucono‐δ‐lactone treatment on the antigenicity and conformational structure of β‐conglycinin

SummaryIn this study, β‐conglycinin was treated with transglutaminase (TGase) and glucono‐δ‐lactone (GDL) to meticulously examine the resulting changes in both antigenicity and structural attributes. The findings elucidated a significant reduction (P < 0.05) in the antigenicity of β‐conglycinin to 72.1% and 77.5% after the modified treatment employing TGase alone or in conjunction with GDL, respectively, in contrast to the untreated samples. Electrophoretic analysis showed a decrease in the density of 7S α, 7S α′, and 7S β, indicating the formation of high molecular weight protein aggregates because of the catalytic effect of TGase. Additionally, the simultaneous application of TGase and GDL induced modifications in the structural configuration of β‐conglycinin, as evidenced by a decrease in intrinsic fluorescence intensity, an augmentation in surface hydrophobicity, and a transition from β‐sheet to β‐turn and random coil structures. These observed alterations suggest that the decline in antigenicity is intricately linked to conformational changes, potentially influencing the exposure of antigenic epitopes. Overall, this study holds significant theoretical and practical implications for the development of hypoallergenic soy products and the enhancement of food safety.

A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 μg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 μg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura

BackgroundPatients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti–ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer–CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. ObjectivesTo explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. MethodsWe developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. ResultsIn 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. ConclusionADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

Defining a de novo non-RBM antibody as RBD-8 and its synergistic rescue of immune-evaded antibodies to neutralize Omicron SARS-CoV-2.

Currently, monoclonal antibodies (MAbs) targeting the SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein are classified into seven classes based on their binding epitopes. However, most of these antibodies are seriously impaired by SARS-CoV-2 Omicron and its subvariants, especially the recent BQ.1.1, XBB and its derivatives. Identification of broadly neutralizing MAbs against currently circulating variants is imperative. In this study, we identified a "breathing" cryptic epitope in the S protein, named as RBD-8. Two human MAbs, BIOLS56 and IMCAS74, were isolated recognizing this epitope with broad neutralization abilities against tested sarbecoviruses, including SARS-CoV, pangolin-origin coronaviruses, and all the SARS-CoV-2 variants tested (Omicron BA.4/BA.5, BQ.1.1, and XBB subvariants). Searching through the literature, some more RBD-8 MAbs were defined. More importantly, BIOLS56 rescues the immune-evaded antibody, RBD-5 MAb IMCAS-L4.65, by making a bispecific MAb, to neutralize BQ.1 and BQ.1.1, thereby producing an MAb to cover all the currently circulating Omicron subvariants. Structural analysis reveals that the neutralization effect of RBD-8 antibodies depends on the extent of epitope exposure, which is affected by the angle of antibody binding and the number of up-RBDs induced by angiotensin-converting enzyme 2 binding. This cryptic epitope which recognizes non- receptor binding motif (non-RBM) provides guidance for the development of universal therapeutic antibodies and vaccines against COVID-19.

Construction of hypoallergenic microgel by soy major allergen β-conglycinin through enzymatic hydrolysis and lactic acid bacteria fermentation

Soy allergenicity is a public concern, and the combination of multiple processing methods may be a promising strategy for reducing soy allergenicity. In this study, a novel two-step enzymatic hydrolysis followed by lactic acid bacteria fermentation was proposed for the construction of hypoallergenic soybean protein microgel. β-Conglycinin was used as the main soy allergen. The effects of different enzymatic hydrolysis (Alcalase, Neutrase, and Protamex) and LAB fermentation on β-conglycinin microgel formation and its immunoreactivity were investigated. Results showed that the use of different enzymes and the attainment of different degrees of hydrolysis affected the particle distribution and zeta potential in the microgels and leads to differences in microstructure and immunoreactivity. All hydrolysates compared with intact protein accelerated the formation of gel during LAB fermentation. Among the three assayed enzymes, fermented Protamex hydrolysates at 60 min (PF-60) demonstrated a microgel with an overall reduced average particle size (741.20±7.18 nm), lower absolute values of zeta potential (10.43±0.65 mV), and regular gel network. The antigenicity and IgE-binding capacity decreased to the lowest value of 0.30 % and 6.93 %, respectively. Peptidomics and immunoinformatic analysis suggested that PF-60 disrupted 17/30, 16/44, and 23/75 epitopes in the α, α′, and β subunits, respectively. Unlike the LAB-fermented unhydrolyzed β-conglycinin disrupted epitopes mostly located at the loop domain, PF-60 primarily promoted the exposure and disruption of allergen epitopes with β-sheet structure located at the core barrel domain. These findings can provide new perspectives on the preparation of hypoallergenic soybean-gel products on edible particulate systems.

Exposure of Junctional Claudin-3 Epitopes in the Thick Ascending Limb of Cldn19-Deficient Mice

Exposure of Junctional Claudin-3 Epitopes in the Thick Ascending Limb of Cldn19-Deficient Mice

Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity, and neutralization sensitivity.

The Omicron subvariants have substantially evaded host-neutralizing antibodies and adopted an endosomal route of entry. The virus has acquired several mutations in the receptor binding domain and N-terminal domain of S1 subunit, but remarkably, also incorporated mutations in S2 which are fixed in Omicron sub-lineage. Here, we found that the mutations in the S2 subunit affect the structural and biological properties such as neutralization escape, entry route, fusogenicity, and protease requirement. In vivo, these mutations may have significant roles in tropism and replication. A detailed understanding of the effects of S2 mutations on Spike function, immune evasion, and viral entry would inform the vaccine design, as well as therapeutic interventions aiming to block the essential proteases for virus entry. Thus, our study has identified the crucial role of S2 mutations in stabilizing the Omicron spike and modulating neutralization resistance to antibodies targeting the S1 subunit.

Ex-chitin-g news on drug-induced fungal epitope unmasking.

The microbial cell wall is an essential cellular organelle commonly targeted by antimicrobials. It is also a battleground of innate immune recognition where microbes can evade immune recognition by masking essential cell wall components. A recent study (A. S. Wagner, S. W. Lumsdaine, M. M. Mangrum, and T. B. Reynolds, mBio https://doi.org/10.1128/mbio.00074-23, 2023) provides insight into how echinocandin antifungals cause exposure of proinflammatory β(1,3)-glucan by driving excess chitin production in the weakened cell wall. Although many environmental and biological activities perturb cell wall integrity and regulate β(1,3)-glucan exposure, we still know little about which intracellular signaling components regulate the cell wall changes that result in disrupted cell wall architecture. Wagner et al. showed that calcineurin and the Mkc1p kinase regulate chitin deposition and β(1,3)-glucan unmasking. They further identified chitin synthesis as a key driving force in cell wall structure disruption leading to epitope exposure. Their findings highlight how fungal cell wall dynamics have important implications for antifungal immunity and future drug development.

Broad protective RBD heterotrimer vaccines neutralize SARS-CoV-2 including Omicron sub-variants XBB/BQ.1.1/BF.7.

SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.

Piperidine CD4-Mimetic Compounds Expose Vulnerable Env Epitopes Sensitizing HIV-1-Infected Cells to ADCC.

The ability of the HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1-infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies that are abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engages the gp120 within the Phe43 cavity by targeting the highly conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit the infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination of HIV-1-infected cells.

Autoreactivity against Denatured Type III Collagen Is Significantly Decreased in Serum from Patients with Cancer Compared to Healthy Controls.

Autoantibodies have the potential as cancer biomarkers as they may associate with the outcome and immune-related adverse events (irAEs) following immunotherapy. Cancer and other fibroinflammatory diseases, such as rheumatoid arthritis (RA), are associated with excessive collagen turnover leading to collagen triple helix unfolding and denaturation with exposure of immunodominant epitopes. In this study, we aimed to investigate the role of autoreactivity against denatured collagen in cancer. A technically robust assay to quantify autoantibodies against denatured type III collagen products (anti-dCol3) was developed and then measured in pretreatment serum from 223 cancer patients and 33 age-matched controls. Moreover, the association between anti-dCol3 levels and type III collagen degradation (C3M) and formation (PRO-C3) was investigated. Anti-dCol3 levels were significantly lower in patients with bladder (p = 0.0007), breast (p = 0.0002), colorectal (p < 0.0001), head and neck (p = 0.0005), kidney (p = 0.005), liver (p = 0.030), lung (p = 0.0004), melanoma (p < 0.0001), ovarian (p < 0.0001), pancreatic (p < 0.0001), prostate (p < 0.0001), and stomach cancers (p < 0.0001) compared to controls. High anti-dCol3 levels were associated with type III collagen degradation (C3M, p = 0.0002) but not type III collagen formation (PRO-C3, p = 0.26). Cancer patients with different solid tumor types have downregulated levels of circulating autoantibodies against denatured type III collagen compared to controls, suggesting that autoreactivity against unhealthy type III collagen may be important for tumor control and eradication. This autoimmunity biomarker may have the potential for studying the close relationship between autoimmunity and cancer.

Mechano-Redox Control of Macrophage-1 Antigen De-Adhesion From ICAM-1 (Intercellular Adhesion Molecule 1) by Protein Disulfide Isomerase Promotes Directional Movement Under Flow.

Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.