Design of universal Ebola virus vaccine candidates via immunofocusing.

The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Vaccine design via antigen reorientation.

A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.

Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine-Lysine Relay.

Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments.

Engineering a SARS-CoV-2 Vaccine Targeting the Receptor-Binding Domain Cryptic-Face via Immunofocusing.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response toward the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response toward the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential.

Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines.

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due in part to scalability, adaptability, and potency. Yet there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel depot technology which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available SARS-CoV-2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

Design of universal Ebola virus vaccine candidates via immunofocusing.

Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV) - one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here we describe a universal Ebola virus vaccine approach using structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Designing epitope-focused vaccines via antigen reorientation.

A major challenge in vaccine development, especially against rapidly evolving viruses, is the ability to focus the immune response toward evolutionarily conserved antigenic regions to confer broad protection. For example, while many broadly neutralizing antibodies against influenza have been found to target the highly conserved stem region of hemagglutinin (HA-stem), the immune response to seasonal influenza vaccines is predominantly directed to the immunodominant but variable head region (HA-head), leading to narrow-spectrum efficacy. Here, we first introduce an approach to controlling antigen orientation based on the site-specific insertion of short stretches of aspartate residues (oligoD) that facilitates antigen-binding to alum adjuvants. We demonstrate the generalizability of this approach to antigens from the Ebola virus, SARS-CoV-2, and influenza and observe enhanced antibody responses following immunization in all cases. Next, we use this approach to reorient HA in an "upside down" configuration, which we envision increases HA-stem exposure, therefore also improving its immunogenicity compared to HA-head. When applied to HA of H2N2 A/Japan/305/1957, the reoriented H2 HA (reoH2HA) on alum induced a stem-directed antibody response that cross-reacted with both group 1 and 2 influenza A HAs. Our results demonstrate the possibility and benefits of antigen reorientation via oligoD insertion, which represents a generalizable immunofocusing approach readily applicable for designing epitope-focused vaccine candidates.

Toward Point-of-Care Detection of Mycobacterium tuberculosis: A Brighter Solvatochromic Probe Detects Mycobacteria within Minutes.

There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes. 3-Hydroxychromone (3HC) dyes, known for having high fluorescence quantum yields, exhibit shifts in fluorescence intensity in response to changes in environmental polarity. We synthesized two analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3-Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial cells displayed a 10-fold increase in fluorescence intensity compared to our previous reports on the dye 4,4-N,N-dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb cells within 10 min of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-resource settings.