Lectin Fingerprinting Distinguishes Antibody Neutralization in SARS-CoV-2.

Enveloped viruses co-opt host glycosylation pathways to decorate their surface proteins. As viruses evolve, emerging strains can modify their glycosylation patterns to influence host interactions and subvert immune recognition. Still, changes in viral glycosylation or their impact on antibody protection cannot be predicted from genomic sequences alone. Using the highly glycosylated SARS-CoV-2 Spike protein as a model system, we present a lectin fingerprinting method that rapidly reports on changes in variant glycosylation state, which are linked to antibody neutralization. In the presence of antibodies or convalescent and vaccinated patient sera, unique lectin fingerprints emerge that distinguish neutralizing versus non-neutralizing antibodies. This information could not be inferred from direct binding interactions between antibodies and the Spike receptor-binding domain (RBD) binding data alone. Comparative glycoproteomics of the Spike RBD of wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylation differences as a key determinant of immune recognition differences. These data underscore the interplay between viral glycosylation and immune recognition and reveal lectin fingerprinting to be a rapid, sensitive, and high-throughput assay to distinguish the neutralization potential of antibodies that target critical viral glycoproteins.

Mechanisms that promote the evolution of cross-reactive antibodies upon vaccination with designed influenza immunogens.

Immunogens that elicit broadly neutralizing antibodies targeting the conserved receptor-binding site (RBS) on influenza hemagglutinin may serve as candidates for a universal influenza vaccine. Here, we develop a computational model to interrogate antibody evolution by affinity maturation after immunization with two types of immunogens: a heterotrimeric "chimera" hemagglutinin that is enriched for the RBS epitope relative to other B cell epitopes and a cocktail composed of three non-epitope-enriched homotrimers of the monomers that comprise the chimera. Experiments in mice find that the chimera outperforms the cocktail for eliciting RBS-directed antibodies. We show that this result follows from an interplay between how B cells engage these antigens and interact with diverse helper T cells and requires T cell-mediated selection of germinal center B cells to be a stringent constraint. Our results shed light on antibody evolution and highlight how immunogen design and T cells modulate vaccination outcomes.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

An epitope-enriched immunogen expands responses to a conserved viral site.

Pathogens evade host humoral responses by accumulating mutations in surface antigens. While variable, there are conserved regions that cannot mutate without compromising fitness. Antibodies targeting these conserved epitopes are often broadly protective but remain minor components of the repertoire. Rational immunogen design leverages a structural understanding of viral antigens to modulate humoral responses to favor these responses. Here, we report an epitope-enriched immunogen presenting a higher copy number of the influenza hemagglutinin (HA) receptor-binding site (RBS) epitope relative to other B cell epitopes. Immunization in a partially humanized murine model imprinted with an H1 influenza shows H1-specific serum and >99% H1-specific B cells being RBS-directed. Single B cell analyses show a genetically restricted response that structural analysis defines as RBS-directed antibodies engaging the RBS with germline-encoded contacts. These data show how epitope enrichment expands B cell responses toward conserved epitopes and advances immunogen design approaches for next-generation viral vaccines.

Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting.

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Protein engineering strategies for rational immunogen design.

Antibody immunodominance refers to the preferential and asymmetric elicitation of antibodies against specific epitopes on a complex protein antigen. Traditional vaccination approaches for rapidly evolving pathogens have had limited success in part because of this phenomenon, as elicited antibodies preferentially target highly variable regions of antigens, and thus do not confer long lasting protection. While antibodies targeting functionally conserved epitopes have the potential to be broadly protective, they often make up a minority of the overall repertoire. Here, we discuss recent protein engineering strategies used to favorably alter patterns of immunodominance, and selectively focus antibody responses toward broadly protective epitopes in the pursuit of next-generation vaccines for rapidly evolving pathogens.

CD209L/L-SIGN and CD209/DC-SIGN Act as Receptors for SARS-CoV-2.

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelia and endothelia. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor-binding domain (S-RBD) or S1 encompassing both N termal domain and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection, and interference with CD209L activity by a knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent and may have implications for antiviral drug development.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Rapid generation of potent antibodies by autonomous hypermutation in yeast.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates.

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2.

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.

Engineered receptor binding domain immunogens elicit pan-sarbecovirus neutralizing antibodies outside the receptor binding motif.

Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. In mice, we find that a cocktail of these homotrimeric sarbecovirus RBDs elicits antibodies to conserved viral epitopes outside of the ACE2 receptor binding motif (RBM). Importantly, these responses neutralize all sarbecovirus components even in context of prior SARS-CoV-2 imprinting. We further show that a substantial fraction of the neutralizing antibodies elicited after vaccination in humans also engages non-RBM epitopes on the RBD. Collectively, our results suggest a strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine. AUTHOR SUMMARY: Immunity to SARS-CoV-2 in the human population will be widespread due to natural infection and vaccination. However, another novel coronavirus will likely emerge in the future and may cause a subsequent pandemic. Humoral responses induced by SARS-CoV-2 infection and vaccination provide limited protection against even closely related coronaviruses. We show immunization with a cocktail of trimeric coronavirus receptor binding domains induces a neutralizing antibody response that is broadened to related coronaviruses with pandemic potential. Importantly, this broadening occurs in context of an initial imprinted SARS-CoV-2 spike immunization showing that preexisting immunity can be expanded to recognize other related coronaviruses. Our immunogens focused the serum antibody response to conserved epitopes on the receptor binding domain outside of the ACE2 receptor binding motif; this contrasts with current SARS-CoV-2 therapeutic antibodies, which predominantly target the receptor binding motif.

Assessment of Maternal and Neonatal SARS-CoV-2 Viral Load, Transplacental Antibody Transfer, and Placental Pathology in Pregnancies During the COVID-19 Pandemic.

Importance: Biological data are lacking with respect to risk of vertical transmission and mechanisms of fetoplacental protection in maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objective: To quantify SARS-CoV-2 viral load in maternal and neonatal biofluids, transplacental passage of anti-SARS-CoV-2 antibody, and incidence of fetoplacental infection. Design, Setting, and Participants: This cohort study was conducted among pregnant women presenting for care at 3 tertiary care centers in Boston, Massachusetts. Women with reverse transcription-polymerase chain reaction (RT-PCR) results positive for SARS-CoV-2 were recruited from April 2 to June 13, 2020, and follow-up occurred through July 10, 2020. Contemporaneous participants without SARS-CoV-2 infection were enrolled as a convenience sample from pregnant women with RT-PCR results negative for SARS-CoV-2. Exposures: SARS-CoV-2 infection in pregnancy, defined by nasopharyngeal swab RT-PCR. Main Outcomes and Measures: The main outcomes were SARS-CoV-2 viral load in maternal plasma or respiratory fluids and umbilical cord plasma, quantification of anti-SARS-CoV-2 antibodies in maternal and cord plasma, and presence of SARS-CoV-2 RNA in the placenta. Results: Among 127 pregnant women enrolled, 64 with RT-PCR results positive for SARS-CoV-2 (mean [SD] age, 31.6 [5.6] years) and 63 with RT-PCR results negative for SARS-CoV-2 (mean [SD] age, 33.9 [5.4] years) provided samples for analysis. Of women with SARS-CoV-2 infection, 23 (36%) were asymptomatic, 22 (34%) had mild disease, 7 (11%) had moderate disease, 10 (16%) had severe disease, and 2 (3%) had critical disease. In viral load analyses among 107 women, there was no detectable viremia in maternal or cord blood and no evidence of vertical transmission. Among 77 neonates tested in whom SARS-CoV-2 antibodies were quantified in cord blood, 1 had detectable immunoglobuilin M to nucleocapsid. Among 88 placentas tested, SARS-CoV-2 RNA was not detected in any. In antibody analyses among 37 women with SARS-CoV-2 infection, anti-receptor binding domain immunoglobin G was detected in 24 women (65%) and anti-nucleocapsid was detected in 26 women (70%). Mother-to-neonate transfer of anti-SARS-CoV-2 antibodies was significantly lower than transfer of anti-influenza hemagglutinin A antibodies (mean [SD] cord-to-maternal ratio: anti-receptor binding domain immunoglobin G, 0.72 [0.57]; anti-nucleocapsid, 0.74 [0.44]; anti-influenza, 1.44 [0.80]; P < .001). Nonoverlapping placental expression of SARS-CoV-2 receptors angiotensin-converting enzyme 2 and transmembrane serine protease 2 was noted. Conclusions and Relevance: In this cohort study, there was no evidence of placental infection or definitive vertical transmission of SARS-CoV-2. Transplacental transfer of anti-SARS-CoV-2 antibodies was inefficient. Lack of viremia and reduced coexpression and colocalization of placental angiotensin-converting enzyme 2 and transmembrane serine protease 2 may serve as protective mechanisms against vertical transmission.

Rapid generation of potent antibodies by autonomous hypermutation in yeast.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens ( e . g ., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe A utonomous H ypermutation y E ast surf A ce D isplay (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naïve nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw ∼580-fold and ∼925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.