SARS-CoV-2 evolution has reduced the efficacy of clinical monoclonal antibodies, underscoring the need for therapeutics targeting conserved viral regions. The Spike (S) heptad repeat 2 (HR2) stem helix is highly conserved across SARS-CoV-2 variants and related betacoronaviruses. Although antibodies to this region can neutralize infection, their natural occurrence and evolution remain poorly understood. We previously identified human neutralizing antibodies to a conserved peptide within this region (HR2 coldspot). Here, we show that plasma IgG reactivity to this region remains rare, even after repeated antigen exposure. Longitudinal analysis over 30 months revealed continued somatic hypermutation of HR2-specific antibodies, yet none surpassed the potency or breadth of hr2.016, which emerged shortly after primary infection. Crystal structures of four HR2 stem helix antibodies revealed convergent recognition across distinct antibody lineages. Comparison of hr2.016 with its non-neutralizing clonal relative hr2.086 showed that structural convergence masks distinct binding kinetics. Surface plasmon resonance and molecular dynamics simulations revealed a more stable interaction network for hr2.016, with slower dissociation and prolonged S residence time. Neutralization required the IgG format, supporting an avidity-driven mechanism. Together, these findings define kinetic and avidity constraints governing neutralization at the HR2 stem helix and position hr2.016 as a resilient therapeutic candidate.

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2′ site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence Summary:Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2’ site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Two novel types of hydrogel microspheres (MS) are presented. First, one-component microspheres (1-comMS) were produced from sodium alginate (Na-alg) equipped with thiol-functionalized hydroxyl groups. The functionalization pathway included the conversion of Na-alg into tetrabutylammonium alginate, insertion of new carboxyl groups, grafting of alpha-amine-omega-thiol poly(ethylene glycol), and restoration of the sodium salt. This modification conserves all original carboxyl groups of Na-alg and allows for covalent disulfide bond formation in addition to ionic cross-linking. Second, two-component microspheres (2-comMS) were obtained from a mixture of Na-alg and Na-alg functionalized with cysteamine. This functionalization was achieved by grafting cystamine dihydrochloride on some carboxyl groups followed by the reduction to cysteamine. Using the one-step MS formation process developed for both MS types, very fast ionic gelation with calcium ions conserves the spherical shape of the polymer solution droplets upon extrusion into the gelation bath, while simultaneously occurring slow covalent cross-linking reinforces the hydrogels. The physical properties of both MS types are adjustable by varying the polymer concentration, the degree of grafting, and the mixing ratio. In vitro cell microencapsulation studies confirmed the cytocompatibility of 1-comMS and 2-comMS. [GRAPHICS] .