Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies

Abstract

Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.

Keywords: CX3CR1; G glycoprotein; G protein; RSV; antibodies; respiratory syncytial virus; vaccine.

Figure 1

Rational design and expressiom of RSV G protein immunogens. (A) RSV G protein central conserved domain (CCD) (cyan) with CX3C motif highlighted, and sites of anti-G protein mAb binding: 2D10 (orange), 3G12 (yellow), and 3D3 (magenta). Serine 177 mutations modeled with (B) glutamine and (C) arginine. (D) Coomassie-stained SDS-PAGE gel of RSV G protein immunogens at ~90kDa. Lane 1: wild-type, lane 2: CX4C, lane 3: 177R, lane 4: 177Q.

Figure 2

Outline of vaccination and challenge scheme. Mice were i.p. vaccinated with 10 μg/immunogen + 10 μg MPLA on days 0, 28, and 60. Mice were i.n. challenged (D72) with 10⁶ PFU RSV/A2 and sera were collected prior to and 7 days post-challenge (D79).

Figure 3

Immunogen vaccination induces anti-RSV IgG. Antibody levels were detected by indirect anti-RSV ELISA specific for total IgG (A,D); IgG1 (B,E); IgG2a (C,F) on days 0 (A–C) and day 7 (D–F) post-challenge. Graphs are representative of three independent experiments. Data represents the mean value of 3 experiments subtracted from the background. Bars represent the mean OD450 + SEM. n= 3–5 animals per group. Data were analyzed by one-way ANOVA tests where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to adjuvant-only group.

Figure 4

RSV G protein enriched sera. Antibody levels were detected by indirect anti-RSV IgG ELISA. Sera from n = 3–5 animals/group were panned against F protein to remove anti-F Abs prior to analysis by indirect ELISA. Bars represent the mean + SEM. Data were analyzed by one-way ANOVA tests where * p < 0.05, **** p < 0.0001 compared to adjuvant only group in three independent experiments.

Figure 5

CX3CR1.293 cells abundantly express CX3CR1. Representative histogram (A) and percentages (B) of CX3CR1 expression in 293 (black) and CX3CR1.293 (gray) cells after staining with anti-CX3CR1-Alexa647. 20,000 events were collected. Bars represent the mean of three independent experiments + SEM where * p < 0.001 by two-tailed T test.

Figure 6

FKN and G protein CX3C binding to CX3CR1 in the presence or absence of heparin. 20 nM FKN (CX3CL1) (A) or 500 nM G protein (B) was pre-incubated with or without 5 μg/mL heparin to block non-specific binding. FKN binding was observed using Streptavidin-PE. RSV G protein was observed using anti-G protein mAb (clone 130-5F) followed by secondary anti-mouse conjugated to AlexaFluor-488. 20,000 events were collected. Bars represent the mean of three independent experiments + SEM analyzed by one-way ANOVA where ** p < 0.01, **** p < 0.0001, ns (no significance).

Figure 7

RSV G protein CX3C-CX3CR1 binding is inhibited by serum IgG from G protein immunogen vaccinated mice. Purified serum IgG from G protein immunogen or adjuvant vaccinated mice was examined for the ability to inhibit (A) FKN or (B) G protein CX3C binding to CX3CR1. IgG was co-incubated with FKN or G protein and 5 μg/mL heparin to block non-specific binding. Ligand-specific binding and percent inhibition was determined by: [1 − (% Alexa Fluor-488+ CX3CR1.293⁺ cells treated with ligand + antibody mixture) / (% Alexa Fluor-488+ CX3CR1.293 cells treated with ligand + normal mouse IgG)] × 100, as previously described. Bars represent mean of at least three independent experiments + SEM. Analysis by one-way ANOVA compared to adjuvant-only control where, ** p < 0.01, *** p < 0.001 **** p < 0.00001.

Figure 8

Disease markers in challenged mice. Mice were vaccinated with G protein immunogens and challenged with 10⁶ PFU RSV/A2. (A) Mice were weighed daily from days 0–12 post-challenge. Each shape represents the mean weight change from starting weight in each group each day. (B) Pulmonary leuokcyte infiltrates were collected seven days post-challenge. Graph indicates the mean BAL cells/mL + SEM. * p < 0.05 by one-way ANOVA compared to adjuvant-only group. (C) BAL cells were pooled and analyzed by flow cytometry. Percent granulocytes were determined using SSC^hi singlets. Twenty-thousand events were collected on LSR-II. (D) Lungs of challenged mice were collected on days 3 and 7 post-challenge and evaluated for RSV M protein transcripts by qRT-PCR. Data represent the mean of triplicate experiments. Standard curve was generated with a known concentration of RSV/A2 and two-way ANOVA was performed.