Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses

Abstract

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

Keywords: RSV; antibody repertoire; cryo-EM structure; fusion glycoprotein; memory B cells; neutralizing antibodies; prefusion; public clonotypes; respiratory syncytial virus.

Figure 1.. RSV F memory B cells identified in DS-Cav1 vaccinees

(A) Serum neutralization titers for six individuals vaccinated with 150 μg DS-Cav1 + alum. Titers are shown before vaccination (week 0) and 4 weeks after vaccination. (B) Fold increase in serum neutralization titer 4 weeks after vaccination for all 15 subjects immunized with 150 μg DS-Cav1 + alum adjuvant. The six vaccinees studied in group 4 (4B, 4C, 4F, 4G, 4H, and 4K) are colored. Error bars indicate mean with standard deviation. (C) The percentage of CD19⁺/CD27⁺ memory B cells with probe phenotype specified as pre-F-specific, dual, or post-F-specific before vaccination (week 0) and 2 weeks after vaccination. Statistical significance was determined using the Wilcoxon test. (D) Fluorescence-activated cell sorting (FACS) index plots for six vaccinees (before and 2 weeks after immunization) showing RSV F memory B cells reactive with the indicated fluorescently labeled probes DS-Cav1 or post-F. The x and y axes show MFI. Gate regions in each plot show B cells as pre-F-specific, dual, or post-F-specific binders.

Figure 2.. DS-Cav1 boosts pre-existing B cell lineages that target prefusion sites

(A) Circle plots show RSV F-specific B cell lineages identified before and after vaccination with DS-Cav1. Lineages were identified from probe-sorted memory B cell heavy-chain sequences and augmented with data from plasmablasts and unpaired NGS. Each slice of the plot represents a lineage, with slice size proportional to the number of sequences for the lineage. Some lineages were found before and after vaccination, as indicated by the same color for that subject. The value in the circle indicates the number of lineages found before or after vaccination for each subject. Gray slices represent remaining lineages found only before or after DS-Cav1 vaccination. (B) Summary of RSV F-specific lineages. For each subject, the number of lineages identified before and after vaccination is shown. Shared lineages are those with sequences identified before and after vaccination. Total identified lineages are the number of distinct RSV F lineages per subject. (C) Violin plots showing all sequences from shared (boosted) lineages in six subjects before (n = 299) or after (n = 1,639) vaccination. Percent nucleotide divergence from the inferred germline heavy chain gene (% HV SHM) is shown on the y axis. Statistical significance was determined by unpaired Mann-Whitney nonparametric test with p < 0.0001. (D) Probe-sorted RSV F binding memory B cells from the top three clonally expanded lineages per subject are overlayed on a flow plot (18 of 125 shared lineages). Probe-sorted sequences are colored by subject. The x and y axes show MFI and the indicated antigen-specific probe used. Gate regions in each plot show B cells as pre-F- or post-F-specific or dual binding.

Figure 3.. Identification of RSV F-specific public clonotypes

Thirty-four public clonotypes were identified by augmenting probe-sorted memory B cell heavy-chain sequences with paired plasmablast and unpaired NGS data for all six subjects and by adding published RSV F-specific antibody heavy-chain sequences to the dataset. Public clonotypes are numbered 1–34. The second column shows subjects, each with a distinct color as shown in the key on the right, in which the clonotype was found. The third column shows the heavy- and light-chain germline gene pairing based on IMGT. The fourth column shows the CDR H3 and L3 amino acid length, respectively. The fifth column shows the total number of heavy-chain sequences identified for each clonotype. The sixth column shows the distribution of SHM within a clonotype, based on nucleotide divergence from the germline heavy chain gene. The rectangular box indicates SHM ranging from 0%–20% (bottom of the sixth column). The intensity of the blue hue corresponds to the number of heavy-chain sequences (as seen on the right in the bottom figure legend) at a specified SHM percentage. The last column indicates the antigenic site of RSV F recognized by the clonotype.

Figure 4.. The 3.2-Å cryo-EM structure of pre-F bound by site Ø Fab 32.4K

(A) The RSV F + Fab 32.4K (317.4K.L62.004) complex is shown as a molecular surface, with individual F protomers colored green, tan, or pink. Two 32.4K molecules are shown as molecular surfaces, and one is shown in ribbons, each with the heavy chain in orange and the light chain in white. The site V Fab 01.4B is not shown here for the sake of visual clarity (see Figure S4 for a full overview). (B) Fab 32.4K heavy-chain contacts. RSV F and Fab 32.4K are shown as ribbon diagrams, and the green and pink RSV F protomers are not shown. Residues that form key interactions are shown as sticks, with oxygen atoms colored red and nitrogen atoms colored blue. Hydrogen bonds are shown as black dots. (C) The view from (B), rotated by roughly 130° to show additional interactions.

Figure 5.. Structural basis of site V recognition by clonotype 1 antibodies isolated after DS-Cav1 vaccination or natural RSV infection

(A) Cryo-EM structure of 01.4B Fab in complex with pre-F. The three protomers of pre-F are shown as molecular surfaces colored tan, green, and pink. Two 01.4B molecules are shown as molecular surfaces, and one is shown in ribbons, each with the heavy chain in orange and the light chain in white. The constant domains of the Fabs were not modeled in the structure. (B) Magnified view of the interface between 01.4B and pre-F, colored as in (A), except the third protomer of pre-F is shown in ribbons, with regions within 5.5 Å of 01.4B colored blue. (C) The interface between ADI-14442 and pre-F, determined by cryo-EM, is shown with the same orientation and coloring as in (B), with the exception of the ADI-14442 heavy chain, which is colored gold. (D) Further magnified view of the interface between the heavy chain of ADI-14442 and pre-F with an approximately 180° rotation about the trimeric axis relative to the orientation shown in (C). (E) Magnified view of the interface between the light chain of ADI-14442 and pre-F. (F) Magnified view of the ADI-14442 CDR H3 interactions with CDR H2, CDR L1, and pre-F with an approximately 180° rotation about the trimeric axis relative to the orientation shown in (E). (G) Magnified view of the 01.4B CDR H3 interactions with CDR H2, CDR L1, and pre-F in the same orientation as in (F). (H) Pre-F is shown as green molecular surfaces. The CDR H3s of 01.4B and ADI-14442 are shown as sticks colored orange and gold, respectively. The CDR H3 sequences are shown in the inset and are colored to match the structures. Side chains of residues involved in hydrogen bonding or salt bridges are shown as sticks, with oxygen and nitrogen atoms colored red and blue, respectively. Hydrogen bonds and salt bridges are depicted as black dotted lines. Transparent molecular surfaces are shown for residues involved in hydrophobic interactions.