Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine.

Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-γ levels 1 d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-γ. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.

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.

Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors.

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

Targeted glycan degradation potentiates the anticancer immune response in vivo.

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.

Protect, modify, deprotect (PMD): A strategy for creating vaccines to elicit antibodies targeting a specific epitope.

In creating vaccines against infectious agents, there is often a desire to direct an immune response toward a particular conformational epitope on an antigen. We present a method, called protect, modify, deprotect (PMD), to generate immunogenic proteins aimed to direct a vaccine-induced antibody (Ab) response toward an epitope defined by a specific monoclonal Ab (mAb). The mAb is used to protect the target epitope on the protein. Then the remaining exposed surfaces of the protein are modified to render them nonimmunogenic. Finally, the epitope is deprotected by removal of the mAb. The resultant protein is modified at surfaces other than the target epitope. We validate PMD using a well-characterized antigen, hen egg white lysozyme, then demonstrate the utility of PMD using influenza virus hemagglutinin (HA). We use an mAb to protect a highly conserved epitope on the stem domain of HA. Exposed surface amines are then modified with short polyethylene glycol chains. The resultant antigen shows markedly reduced binding to mAbs that target the head region of HA, while maintaining binding to mAbs at the epitope of interest. This antigenic preference is also observed with yeast cells displaying Ab fragments. Antisera from guinea pigs immunized with the PMD-modified HA show increased cross-reactivity with HAs from other influenza strains, compared with antisera obtained with unmodified HA trimers. PMD has the potential to direct an Ab response at high resolution and could be used in combination with other such strategies. There are many attractive targets for the application of PMD.

Bringing immunofocusing into focus.

Immunofocusing is a strategy to create immunogens that redirect humoral immune responses towards a targeted epitope and away from non-desirable epitopes. Immunofocusing methods often aim to develop "universal" vaccines that provide broad protection against highly variant viruses such as influenza virus, human immunodeficiency virus (HIV-1), and most recently, severe acute respiratory syndrome coronavirus (SARS-CoV-2). We use existing examples to illustrate five main immunofocusing strategies-cross-strain boosting, mosaic display, protein dissection, epitope scaffolding, and epitope masking. We also discuss obstacles for immunofocusing like immune imprinting. A thorough understanding, advancement, and application of the methods we outline here will enable the design of high-resolution vaccines that protect against future viral outbreaks.

Engineering a SARS-CoV-2 vaccine targeting the RBD 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 towards 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 towards 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.

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.

Efficient evolution of human antibodies from general protein language models.

Natural evolution must explore a vast landscape of possible sequences for desirable yet rare mutations, suggesting that learning from natural evolutionary strategies could guide artificial evolution. Here we report that general protein language models can efficiently evolve human antibodies by suggesting mutations that are evolutionarily plausible, despite providing the model with no information about the target antigen, binding specificity or protein structure. We performed language-model-guided affinity maturation of seven antibodies, screening 20 or fewer variants of each antibody across only two rounds of laboratory evolution, and improved the binding affinities of four clinically relevant, highly mature antibodies up to sevenfold and three unmatured antibodies up to 160-fold, with many designs also demonstrating favorable thermostability and viral neutralization activity against Ebola and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudoviruses. The same models that improve antibody binding also guide efficient evolution across diverse protein families and selection pressures, including antibiotic resistance and enzyme activity, suggesting that these results generalize to many settings.

Decreased efficacy of a COVID-19 vaccine due to mutations present in early SARS-CoV-2 variants of concern.

With the SARS-CoV-2 virus still circulating and evolving, there remains an outstanding question if variant-specific vaccines represent the optimal path forward, or if other strategies might be more efficacious towards providing broad protection against emerging variants. Here, we examine the efficacy of strain-specific variants of our previously reported, pan-sarbecovirus vaccine candidate, DCFHP-alum, a ferritin nanoparticle functionalized with an engineered form of the SARS-CoV-2 spike protein. In non-human primates, DCFHP-alum elicits neutralizing antibodies against all known VOCs that have emerged to date and SARS-CoV-1. During development of the DCFHP antigen, we investigated the incorporation of strain-specific mutations from the major VOCs that had emerged to date: D614G, Epsilon, Alpha, Beta, and Gamma. Here, we report the biochemical and immunological characterizations that led us to choose the ancestral Wuhan-1 sequence as the basis for the final DCFHP antigen design. Specifically, we show by size exclusion chromatography and differential scanning fluorimetry that mutations in the VOCs adversely alter the antigen's structure and stability. More importantly, we determined that DCFHP without strain-specific mutations elicits the most robust, cross-reactive response in both pseudovirus and live virus neutralization assays. Our data suggest potential limitations to the variant-chasing approach in the development of protein nanoparticle vaccines, but also have implications for other approaches including mRNA-based vaccines.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.

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.

Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold.

Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)─this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ∻one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly booster vaccine, and as a primary vaccine for pediatric use including in infants.

A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice.

The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

New-onset IgG autoantibodies in hospitalized patients with COVID-19.

COVID-19 is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. Here we develop three protein arrays to measure IgG autoantibodies associated with connective tissue diseases, anti-cytokine antibodies, and anti-viral antibody responses in serum from 147 hospitalized COVID-19 patients. Autoantibodies are identified in approximately 50% of patients but in less than 15% of healthy controls. When present, autoantibodies largely target autoantigens associated with rare disorders such as myositis, systemic sclerosis and overlap syndromes. A subset of autoantibodies targeting traditional autoantigens or cytokines develop de novo following SARS-CoV-2 infection. Autoantibodies track with longitudinal development of IgG antibodies recognizing SARS-CoV-2 structural proteins and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. We developed three different protein arrays to measure hallmark IgG autoantibodies associated with Connective Tissue Diseases (CTDs), Anti-Cytokine Antibodies (ACA), and anti-viral antibody responses in 147 hospitalized COVID-19 patients in three different centers. Autoantibodies were identified in approximately 50% of patients, but in <15% of healthy controls. When present, autoantibodies largely targeted autoantigens associated with rare disorders such as myositis, systemic sclerosis and CTD overlap syndromes. Anti-nuclear antibodies (ANA) were observed in ∼25% of patients. Patients with autoantibodies tended to demonstrate one or a few specificities whereas ACA were even more prevalent, and patients often had antibodies to multiple cytokines. Rare patients were identified with IgG antibodies against angiotensin converting enzyme-2 (ACE-2). A subset of autoantibodies and ACA developed de novo following SARS-CoV-2 infection while others were transient. Autoantibodies tracked with longitudinal development of IgG antibodies that recognized SARS-CoV-2 structural proteins such as S1, S2, M, N and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. COVID-19 patients with one or more autoantibodies tended to have higher levels of antibodies against SARS-CoV-2 Nonstructural Protein 1 (NSP1) and Methyltransferase (ME). We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice.

Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.