Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA “breathing” on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Download Full-text

Conserved epitope on influenza-virus hemagglutinin head defined by a vaccine-induced antibody

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715471115 ◽

2017 ◽

Vol 115 (1) ◽

pp. 168-173 ◽

Cited By ~ 59

Author(s):

Donald D. Raymond ◽

Goran Bajic ◽

Jack Ferdman ◽

Pirada Suphaphiphat ◽

Ethan C. Settembre ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Selective Advantage ◽

Influenza Viruses ◽

Human Antibody ◽

Receptor Binding Site ◽

B Cell Memory ◽

Influenza Virus Hemagglutinin ◽

Viral Hemagglutinin ◽

Rapid Spread ◽

Conserved Epitope

Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA “head” and a region near the fusion peptide on the HA “stem” are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site (“lateral patch”) on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.

Download Full-text

Structure-guided molecular grafting of a complex broadly neutralizing viral epitope

10.1101/860825 ◽

2019 ◽

Author(s):

Goran Bajic ◽

Max J. Maron ◽

Ming Tian ◽

Garnett Kelsoe ◽

Masayuki Kuraoka ◽

...

Keyword(s):

Immune Responses ◽

Antigenic Variation ◽

Viral Evolution ◽

Molecular Scaffolds ◽

Receptor Binding Site ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Viral Epitope ◽

Viral Hemagglutinin ◽

Universal Influenza Vaccine

ABSTRACTAntigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a “universal” influenza vaccine will come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use non-circulating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These “resurfaced” HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can enrich for RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.

Download Full-text

Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Responses to the Induced Co-Receptor Binding Site

PLoS Pathogens ◽

10.1371/journal.ppat.1000445 ◽

2009 ◽

Vol 5 (5) ◽

pp. e1000445 ◽

Cited By ~ 98

Author(s):

Barna Dey ◽

Krisha Svehla ◽

Ling Xu ◽

Dianne Wycuff ◽

Tongqing Zhou ◽

...

Keyword(s):

Immune Responses ◽

Binding Site ◽

Receptor Binding ◽

Receptor Binding Site ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Hiv 1

Download Full-text

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

10.1101/2021.11.02.466200 ◽

2021 ◽

Author(s):

Zhi Yang ◽

Kim-Marie A. Dam ◽

Michael D. Bridges ◽

Magnus A.G. Hoffmann ◽

Andrew T. DeLaitsch ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibodies ◽

Resonance Spectroscopy ◽

Broadly Neutralizing Antibodies ◽

Receptor Binding Site ◽

Double Electron ◽

Immunogen Design ◽

Cd4 Binding Site ◽

Double Electron Electron Resonance ◽

Hiv 1

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterized Ab1303 and Ab1573, neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding was observed only when Env trimers were not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures showed that both antibodies recognized the CD4bs on Env trimer with an occluded-open conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation included outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, did not exhibit V1V2 displacement, co-receptor binding site exposure, or a 4-stranded gp120 bridging sheet. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggested an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Download Full-text

Biological Activity of the Carrier as a Factor in Immunogen Design for Haptens

Molecules ◽

10.3390/molecules23112977 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2977 ◽

Cited By ~ 2

Author(s):

Özlem Ertekin ◽

Esin Akçael ◽

Harun Kocaağa ◽

Selma Öztürk

Keyword(s):

Biological Activity ◽

Immune Responses ◽

Antibody Production ◽

Transferrin Receptor ◽

Carrier Protein ◽

Small Molecular Weight ◽

Humoral Immune ◽

Immunogen Design ◽

Antibody Titers ◽

Stimulate Antibody

Immunoanalytical methods are frequently employed in the detection of hazardous small molecular weight compounds. However, antibody development for these molecules is a challenge, because they are haptens and cannot induce a humoral immune response in experimental animals. Immunogenic forms of haptens are usually prepared by conjugating them to a protein carrier which serves as an immune stimulator. However, the carrier is usually considered merely as a bulk mass, and its biological activity is ignored. Here, we induced an endocytic receptor, transferrin receptor, by selecting its ligand as a carrier protein to enhance antibody production. We conjugated aflatoxin, a potent carcinogenic food contaminant, to transferrin and evaluated its potential to stimulate antibody production with respect to ovalbumin conjugates. Transferrin conjugates induced aflatoxin-specific immune responses in the second immunization, while ovalbumin conjugates reached similar antibody titers after 5 injections. Monoclonal antibodies were successfully developed with mice immunized with either of the conjugates.

Download Full-text

Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors

10.1101/2020.04.29.068742 ◽

2020 ◽

Author(s):

Iwona Wojcik ◽

Thomas Sénard ◽

Erik L de Graaf ◽

George MC Janssen ◽

Arnoud H de Ru ◽

...

Keyword(s):

Immune Responses ◽

Healthy Donor ◽

Cell Types ◽

Antigen Recognition ◽

Analytical Methodology ◽

Fc Gamma Receptor ◽

Site Specific ◽

Gamma Receptor ◽

Glycosylation Sites ◽

Human Material

ABSTRACTFc gamma receptors (FcγR) translate antigen-recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. In this study, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation from primary human material. In contrast to recently published alternatives, we assessed all glycosylation sites in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of FcγRIII glycosylation differences between individuals, cell types, subcellular locations and pathophysiological conditions.

Download Full-text

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

10.1101/2021.08.02.454546 ◽

2021 ◽

Author(s):

Walther Mothes ◽

Wenwei Li ◽

Yaozong Chen ◽

Jeremie Prevost ◽

Irfan Ullah ◽

...

Keyword(s):

Immune Responses ◽

Mode Of Action ◽

Neutralizing Antibodies ◽

Structural Basis ◽

Receptor Binding Domain ◽

Broadly Neutralizing Antibodies ◽

Vaccine Immunogen ◽

Immunogen Design

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the RBD-up position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among beta-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Download Full-text

Persistent Immunogenicity of Integrase Defective Lentiviral Vectors delivering membrane tethered Native-Like HIV-1 Envelope Trimers

10.1101/2021.10.08.462761 ◽

2021 ◽

Author(s):

Alessandra Gallinaro ◽

Maria Franca Pirillo ◽

Yoann Aldon ◽

Serena Cecchetti ◽

Zuleika Michelini ◽

...

Keyword(s):

Immune Responses ◽

Lentiviral Vectors ◽

Igg Antibodies ◽

Vaccine Platform ◽

Neutralization Activity ◽

Cynomolgus Macaques ◽

Immunogen Design ◽

Humoral Responses ◽

Hiv 1 ◽

Vector Particles

Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.

Download Full-text

Design of a new effector recognition specificity in a plant NLR immune receptor by molecular engineering of its integrated decoy domain

10.1101/2021.04.24.441256 ◽

2021 ◽

Author(s):

Stella Cesari ◽

Yuxuan Xi ◽

Nathalie Declerck ◽

Véronique Chalvon ◽

Léa Mammri ◽

...

Keyword(s):

Immune Responses ◽

Crop Protection ◽

Molecular Engineering ◽

Future Research ◽

Pathogen Effectors ◽

Recognition Specificity ◽

Heterologous System ◽

Binding Surface ◽

Engineering Strategy ◽

Effector Recognition

SUMMARYPlant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that specifically recognize pathogen effectors and induce immune responses. Designing artificial NLRs with new effector recognition specificities is a promising prospect for sustainable, knowledge-driven crop protection. However, such strategies are hampered by the complexity of NLR function. Here, we tested whether molecular engineering of the integrated decoy domain (ID) of an NLR could extend its recognition spectrum to a new effector. To this aim, we relied on the detailed molecular knowledge of the recognition of distinct Magnaporthe oryzae MAX (Magnaporthe AVRs and ToxB-like) effectors by the rice NLRs RGA5 and Pikp-1. For both NLRs, effector recognition involves physical binding to their HMA (Heavy Metal-Associated) IDs. However, AVR-PikD, the effector recognized by Pikp-1, binds to a completely different surface of the HMA domain compared to AVR-Pia and AVR1-CO39, recognized by RGA5. By introducing into the HMA domain of RGA5 the residues of the Pikp-1 HMA domain involved in AVR-PikD binding, we created a high-affinity binding surface for this new effector. In the Nicotiana benthamiana heterologous system, RGA5 variants carrying this engineered binding surface still recognize AVR-Pia and AVR1-CO39, but also perceive the new ligand, AVR-PikD, resulting in the activation of immune responses. Therefore, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs. However, it pinpoints significant knowledge gaps that limit the full deployment of this NLR-ID engineering strategy and provides hypotheses for future research on this topic.

Download Full-text

Pathogenesis of SARS-CoV-2 and Immune Response in COVID-19

Türk Mikrobiyoloji Cemiyeti Dergisi ◽

10.5222/tmcd.2020.183 ◽

2020 ◽

Author(s):

Mehmet Demirci ◽

Özge Ünlü ◽

Akın Yiğin ◽

Fadime Yıldız Zeyrek

Keyword(s):

Immune Response ◽

Immune Responses ◽

Host Immune Response ◽

Amino Acid Sequences ◽

Receptor Binding Site ◽

The Third ◽

Protease Cleavage ◽

The World ◽

Short Time ◽

First Time

SARS-CoV-2, which emerged in Wuhan province of China for the first time in December 2019 and spread rapidly all over the world, is still causing an epidemic. SARS-CoV-2 is the third coronavirus outbreak we encountered after SARS-CoV and MERS-CoV infections. Due to SARS-CoV and MERS-CoV infections, we have gained experience about the pathogenesis and immune responses of coronaviruses. However, studies have shown that, unlike the information derived from our experience, SARS-CoV-2 is both very infectious and its effect on cells is different. Therefore, we aimed to compile the data of the published studies on the pathogenesis of SARS-CoV-2 and the resulting host immune response. In many studies, it has been reported that not only the presence of the host ACE2 receptor is sufficient for the infection of the host cell, but also the cleavage of the structural S protein by proteases should be materialized. It has been shown that, unlike SARS-CoV-2, SARS-CoV and MERS-CoV, it contains different protease cleavage systems and amino acid sequences in the ACE2 receptor binding site. In SARS-CoV-2 infection, as reported in studies conducted up to now, Th1 and Th2-mediated cytokine and chemokine levels in the host are different than SARS-CoV infection, and also different chemokines can be upregulated compared to other CoVs. Considering that effective vaccines have not been developed for the infectious RNA viruses despite the ongoing trials for many years, in order to reveal all these differences in the pathogenesis and immune response process and to develop effective antivirals against SARS-CoV-2 within a short time. the need for comprehensive studies on host immune response is evident.

Download Full-text