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.

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.

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.

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.

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.

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.

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.