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

Defining and Manipulating B Cell Immunodominance Hierarchies to Elicit Broadly Neutralizing Antibody Responses against Influenza Virus

© 2020 Elsevier Inc. The antibody repertoire possesses near-limitless diversity, enabling the adaptive immune system to accommodate essentially any antigen. However, this diversity explores the antigenic space unequally, allowing some pathogens like influenza virus to impose complex immunodominance hierarchies that distract antibody responses away from key sites of virus vulnerability. We developed a computational model of affinity maturation to map the patterns of immunodominance that evolve upon immunization with natural and engineered displays of hemagglutinin (HA), the influenza vaccine antigen. Based on this knowledge, we designed immunization protocols that subvert immune distraction and focus serum antibody responses upon a functionally conserved, but immunologically recessive, target of human broadly neutralizing antibodies. We tested in silico predictions by vaccinating transgenic mice in which antibody diversity was humanized to mirror clinically relevant humoral output. Collectively, our results demonstrate that complex patterns in antibody immunogenicity can be rationally defined and then manipulated to elicit engineered immunity

The quantitative homologous recombination assay detected deletion strains with reduced recombination that was not detected with the patch assay.

<p>A. Quantitative recombination assays of deletion strains expressing wild-type Tf1-<i>nat</i>AI and the INfs. B. Quantitative recombination frequencies are shown in a histogram of strains sorted from highest to lowest. The numbers on the x-axis identify strains in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006775#pgen.1006775.s011" target="_blank">S3 Table</a>. The deletion strains here were shown by the yeast patch assays to have defects in transposition but not homologous recombination (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006775#pgen.1006775.s011" target="_blank">S3 Table</a>). The red line illustrates the homologous recombination activity of wild-type Tf1 in wild-type <i>S</i>. <i>pombe</i>. The green line shows the homologous recombination activity of the INfs in Wild-type <i>S</i>. <i>pombe</i>. C. Quantitative homologous recombination assays of cells with catalytically inactive mutants in the catalytic core (CC) of IN.</p

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes

<div><p>Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of <i>Schizosaccharomyces pombe</i> is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 <i>S</i>. <i>pombe</i> strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in <i>Saccharomyces cerevisiae</i>, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.</p></div

Host factors that function in Tf1 integration.

<p>Host factors that function in Tf1 integration.</p

Genes that promote Tf1 integration were identified by screening deletion strains with assays that measure transposition, homologous recombination, and expression of Tf1 protein.

<p>Out of 150 strains with low transposition, 109 supported high levels of homologous recombination. These candidates were further analyzed with a quantitative recombination assay to detect reduced cDNA in the nucleus and with immunoblots to detect reduced levels of Gag and IN.</p

Tf1 integration clustered upstream of ORFs in both wild-type and the strains with transposition defects.

<p>The x-axis is the distance upstream (-3000 bp to 0 bp) and downstream (0 bp to +3000 bp) from ORFs divided into bins of 100 bp. The y-axis is the amount of integrations within a bin as a percent of all integrations. Insertions closer to the 5’ end (-) of an ORF were plotted upstream of the ORF and insertions closer to the 3’ end (+) were plotted downstream of the ORF. The red vertical dashes delineate the body of ORFs, and insertions in ORFs are tabulated within 15 bins of equal proportion; total insertions in ORFs are labeled in percentages. (A) Wild-type (indicated with black bars); (B-H) deletion mutants (indicated with blue bars); (I) a matched random control of integrations in wild-type cells.</p

Assays that measure Tf1 transposition and homologous recombination of cDNA detect defects in integration.

<p>Transposition is detected by expressing Tf1-<i>nat</i>AI in cells on agar plates and replica printing patches of cells to medium containing FOA and Nat. The intron in <i>nat</i> is spliced out, the mRNA (red) is reverse transcribed, and IN inserts Tf1 cDNA with an active <i>nat</i> into <i>S</i>. <i>pombe</i> chromosomes. Frame shift mutations at the N termini of PR (PRfs) and IN (INfs) greatly reduce transposition (right panel). Tf1 cDNA is detected in the nucleus by replica printing cell patches to medium containing Nat (left panel).</p

Distribution of Tf1 integration within intergenic regions in transposition-defective mutants compared to wild-type.

<p>Density scatter plot and linear regression analysis are shown for each indicated deletion strain. The x-axis is the amount of insertions in WT cells per intergenic region normalized as a percent of all insertions and sorted by increasing amount of integrations. The y-axis is the corresponding normalized insertion number per intergenic region in the deletion (Δ) mutant. Data points are plotted such that color gradient indicates the density of overlapping points. The correlation coefficient (R²) from linear regression of each WT/Δ pair is indicated and a trend line is shown in red dash. A diagonal reference line (y = x) is shown in black. A. WT plotted against a biologically independent set of integration in wild-type cells, WT2. B-H, WT plotted against deletion mutants, I, WT plotted against MRC.</p

Interactions of IN with Rhp18 and Cwf3 as detected by two-hybrid assays.

<p>Interactions between IN and IN, Rhp18, and Cwf3 resulted in lacZ expression that was detected as blue CTY10-5d cells on nitrocellulose filters. The multimerization of IN produced by LexA-IN and Gal4-IN was our positive control. Technical replicates of this positive control produced the three blue patches on the top panel and the two blue patches on the bottom panel. The negative control was cells expressing LexA-IN and Gal4. Technical replicates of this negative control produced the three white patches on the top panel and the two white patches on the bottom panel. Another negative control was cells expressing LexA-IN and Gal4 fused to a non-interacting protein. Nine independent transformants expressing LexA-IN and Gal4-Rhp18 produced blue coloration indicating a significant interaction. Four independent transformants expressing LexA-IN and Gal4-Cwf3 also produced blue signal indicating interaction.</p