Non-pollinator selection for a floral homeotic mutant conferring loss of nectar reward in Aquilegia coerulea.

Here, we describe a polymorphic population of Aquilegia coerulea with a naturally occurring floral homeotic mutant, A. coerulea var. daileyae, where the characteristic petals with nectar spurs are replaced with a second set of sepals. Although it would be expected that this loss of pollinator reward would be disadvantageous to the mutant, we find that it has reached relatively high frequency (∼25%) and is under strong, positive selection across multiple seasons (s = 0.17-0.3) primarily due to reduced floral herbivory. We identify the underlying locus (APETALA3-3) and multiple causal loss-of-function mutations indicating an ongoing soft sweep. Elevated linkage disequilibrium around the two most common causal alleles indicates that positive selection has been occurring for many generations. Lastly, genotypic frequencies at AqAP3-3 indicate a degree of positive assortative mating by morphology. Together, these data provide both a compelling example that large-scale discontinuous morphological changes differentiating taxa can occur due to single mutations and a particularly clear example of linking genotype, phenotype, and fitness.

Semi-Supervised Pipeline for Autonomous Annotation of SARS-CoV-2 Genomes.

SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. However, autonomous genome annotation of SARS-CoV-2 genes, proteins, and domains is not readily accomplished by existing methods and results in missing or incorrect sequences. To overcome this limitation, we developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on the use of a single reference genome and by overcoming atypical genomic traits that challenge traditional bioinformatic methods. We analyzed an initial corpus of 66,000 SARS-CoV-2 genome sequences collected from labs across the world using our method and identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction, compared to proteome references, including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools, such as Prokka (base) and VAPiD, we yielded a 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 gene, protein, and domain sequences-some conserved across time and geography and others representing emerging variants. We observed 3362 non-redundant sequences per protein on average within this corpus and described key D614G and N501Y variants spatiotemporally in the initial genome corpus. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized receptor binding domain variants. We further demonstrated the robustness and extensibility of our method on an additional 4000 variant diverse genomes containing all named variants of concern and interest as of August 2021. In this cohort, we successfully identified all keystone spike glycoprotein mutations in our predicted protein sequences with greater than 99% accuracy as well as demonstrating high accuracy of the protein and domain annotations. This work comprehensively presents the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable, high-accuracy method to analyze newly sequenced infections as they arise.

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants.

Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2GΔHR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with comparable potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2GΔHR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in the mouse model. Compared with the soluble spike, the I3-01v9 SApNP showed sixfold longer retention, fourfold greater presentation on follicular dendritic cell dendrites, and fivefold stronger germinal center reactions in lymph node follicles.

Neutralizing Antibodies Induced by First-Generation gp41-Stabilized HIV-1 Envelope Trimers and Nanoparticles.

The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display. IMPORTANCE Self-assembling protein nanoparticles (NPs) presenting BG505 envelope (Env) trimers can elicit tier 2 HIV-1-neutralizing antibody (NAb) responses more effectively than soluble trimers. In the present study, monoclonal NAbs were isolated from previously immunized mice and rabbits for structural and functional analyses, which revealed that potent mouse NAbs recognize the C3/V4 region and small NP-elicited rabbit NAbs primarily target known glycan holes on BG505 Env. This study validates the gp41 stabilization strategy for HIV-1 Env vaccine design and highlights the challenge in eliciting a broad NAb response.

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines.

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants.

Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2GΔHR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with the same potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2GΔHR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in mice. Compared with the soluble spike, the I3-01v9 SApNP showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold stronger germinal center reactions in lymph node follicles.

Single-component, self-assembling, protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates.

Vaccination against SARS-CoV-2 provides an effective tool to combat the COVID-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2GΔHR2 elicited twofold higher NAb titers than S2P, while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T cell immunity, thereby providing a promising vaccine candidate.

Single-component, self-assembling, protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates.

Vaccination against SARS-CoV-2 provides an effective tool to combat the COIVD-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2GΔHR2 elicited two-fold-higher NAb titers than S2P, while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T-cell immunity, thereby providing a promising vaccine candidate.