Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease

Abstract

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children and elicited nonprotective, pathogenic antibody. Immunized infants experienced increased morbidity after subsequent RSV exposure. No vaccine has been licensed since that time. A widely accepted hypothesis attributed the vaccine failure to formalin disruption of protective antigens. Here we show that the lack of protection was not due to alterations caused by formalin but instead to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor (TLR) stimulation. This study explains why the inactivated RSV vaccine did not protect the children and consequently led to severe disease, hampering vaccine development for 42 years. It also suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR agonists in their formulation, and it identifies affinity maturation as a key factor for the safe immunization of infants.

Figure 1. Non-replicating vaccines against RSV prime for ERD

(a) AHR 7 d after RSV challenge in previously immunized BALB/c mice. AHR to acetylcholine challenge is defined by the time-integrated rise in peak airway pressure. Results are means ± SEM (error bars) of 8–11 animals per group and are representative of two independent experiments. For FIRSV, P<0.05 vs. placebo or RSV; and P=NS vs. UVRSV or PFP. (b–d) Pulmonary histopathology 7 days after RSV challenge in mice that had received the indicated preimmunization: Hematoxylin and eosin (b) showing peribronchiolar pneumonia, Hematoxylin and Congo Red (c) showing pulmonary eosinophlia and periacid Schiff (d) showing enhanced bronchiolar mucus production in FIRSV, UVRSV and PFP recipients. Scale bar for b and d: 100 µm; scale bar for c: 25 µm (e) Pulmonary eosinophils per 40X field are increased in recipients of FIRSV, UVRSV and PFP. Results are means ± SEM (error bars) of 6–10 animals per group and are representative of two independent experiments. For FIRSV, P<0.05 vs. RSV, placebo and PFP; P=NS vs. UVRSV. (f) Lung viral titers 4 days after RSV challenge in recipients of inactivated vaccines, RSV or placebo. Results are means ± SEM (error bars) of 6 animals per group. For FIRSV, P<0.05 vs. RSV; P=NS vs. placebo, UVRSV and PFP.

Figure 2. Non-replicating vaccine elicit non-protective, low avidity antibody

(a) IgG antibody responses against the RSV F protein determined by immunoassay from sera of FIRSV, UVRSV and RSV recipients. For FIRSV, P=NS compared with UVRSV, and P<0.05 compared with RSV. (b) RSV-specific neutralization as measured by 50% plaque reduction (PRNT₅₀) on day 45 after immunization. Results are means ± SEM (error bars) of 5–8 animals per group and are representative of three independent experiments. For FIRSV, P=NS compared to UVRSV, and P=0.003 compared to RSV. For UVRSV, P=0.002 compared to RSV. (c) Determination of IgG avidity against RSV F after 7M urea wash in sera from pre-immunized BALB/c mice. For FIRSV, P=NS compared to UVRSV and P<0.05 compared to RSV. Results are means ± SEM (error bars) of 6–10 animals per group and are representative of three independent experiments. (d) Determination of IgG avidity against RSV F in sera from pre-immunized mice on day 45 after immunization. 50% avidity for FIRSV, P=NS vs. UVRSV and P<0.001 vs. RSV. For UVRSV, P<0.001 vs. RSV. (e) IgG_2a/IgG₁ ratio against RSV F in sera from pre-immunized mice.

Figure 3. Affinity is critical for protection against RSV

(a) Anti-F IgG avidity in sera. 50% avidity for FIRSV, P=NS vs.UVRSV and P<0.001 vs. RSV, UVRSV₅ and Rpn7. For UVRSV₅, P=NS vs. RSV and P<0.05 vs.UVRSV. (b) Correlation between RSV-specific PRNT₅₀ and 50% avidity in sera (pre-standardized for anti-F IgG levels). Black squares=RSV, half-shaded circles=UVRSV₅, white circles=UVRSV, black circles=FIRSV, half-shaded squares=Rpn7. (c) Lung viral titers in recipients of passively transferred sera (pre-standardized for anti-F IgG levels) from immunized mice. Results are means ± SEM of 3 animals/group. For UVRSV₅, P<0.01 vs. placebo and P=NS vs. RSV. (d) IgG antibody and (e) determination of IgG avidity after 7M urea wash against RSV F_422–438peptide from pooled sera of immunized mice. (f) Binding competition between sera (pre-standardized for anti-F IgG levels) from immunized mice and palivizumab for RSV F. For FIRSV, P<0.05 vs. naÏve, UVRSV₅ and RSV. FIRSV vs. UVRSV, P=NS. For UVRSV, P<0.05 vs. naÏve, UVRSV₅ and RSV. (g) IgG avidity against F after 7M urea wash in mice pre-immunized with FIRSV, FIRSV₅ or RSV. (h) Binding of anti-F IgG mAb panel (24,48) to FIRSV (white bars) or RSV (black bars). (i) IgG avidity against FIRSV or RSV after 7M urea wash in sera from mice pre-immunized with FIRSV₅. Results are means ± SEM of 7 animals/group. P=0.001. All data is representative of two-three independent experiments.

Figure 4. Adaptive immunity after inactivated vaccines

Determination of CD40 (a), CD80 (b) and CD86 (c) surface expression in dendritic cells (CD11c⁺) from regional popliteal lymph nodes (CCR7⁺) 24 h. after footpad immunization with FIRSV, UVRSV or RSV (mean fold-increase over immunization with placebo of 9–12 animals/group). (d) Lymphoproliferative responses (by BrdU immunoassay) in CD4⁺ T lymphocytes from regional popliteal nodes after footpad immunization with FIRSV (black circles), UVRSV (white circles), RSV (black squares) or placebo (black triangles). CD4⁺ T lymphocytes were incubated with PHA (1µg) for 48 h. Representative of three independent experiments. CD71(e) and CD40 ligand (f) surface expression in CD4⁺ T lymphocytes from regional popliteal nodes by flow cytometry 10 days after FIRSV, UVRSV or RSV footpad immunization (mean fold-increase over immunization with placebo of 6 animals/group). (g) IFN-γ and (h) IL-4 spots/10⁴ mononuclear cells from regional popliteal nodes after footpad immunization as determined by ELISPOT. Representative of two independent experiments. (i) Determination of B cell centrocytes (B220⁺ and GL7⁺) from regional popliteal nodes 14 days after footpad immunization. Representative of four independent experiments. (j) Germinal center detection (PNA⁺) in slides from regional popliteal nodes after footpad immunization. Scale bar: 100 µm. Representative of three independent experiments.

Figure 5. Deficient activation of TLRs by inactivated vaccines

(a) Iκ-Bα expression by Western Blot in dendritic cells (CD11c⁺) isolated from regional popliteal lymph nodes 24 h. after footpad inoculation of UVRSV or RSV. Representative of two independent experiments. (b) Determination of 50% avidity against RSV F and (c) RSV-specific neutralization (PRNT₅₀) in sera from wt B6 and Myd88^+/− mice inoculated intranasally with RSV. Results are means ± SEM (error bars) of 6–8 animals per group and representative of two independent experiments. P < 0.05 for both comparisons. (d) Determination of 50% avidity against RSV F and (e) RSV-specific neutralization (PRNT₅₀) in sera from pre-immunized BALB/c mice with RSV, UVRSV, UVRSV+LPS, and UVRSV+LPS+Poly(I:C)+PolyU. For UVRSV vs. UVRSV+LPS+Poly(I:C)+PolyU P<0.05, vs. RSV P<0.01. For RSV vs. UVRSV+LPS+Poly(I:C)+PolyU P =NS. Representative of two independent experiments. (f-h) Anti-F IgG responses, IgG avidity and RSV-specific neutralization in Myd88^−/− mice recipients of wt B cells and (i,j) anti-F IgG responses and neutralization in µMT mice recipients of Myd88^−/− B cells. Mice were inoculated with wtRSV (black squares), UVRSV+TLR (gray squares) or UVRSV (white circles). In F and H, P<0.05 for UVRSV vs. wtRSV and vs. UVRSV+TLR.

Figure 6. UVRSV plus TLR agonists protects against ERD

(a) Airways resistance 7 d after RSV challenge in previously immunized BALB/c mice. Results are means ± SEM (error bars) of 4–6 animals per group and are representative of two independent experiments. Mice were inoculated with wtRSV (black squares), UVRSV+TLR (gray squares), UVRSV (white circles) or placebo (white squares). For UVRSV+TLR, P<0.05 vs. UVRSV; and P=NS vs. wtRSV or placebo. (b–d) Pulmonary histopathology 7 days after RSV challenge in mice that had received UVRSV+TLR preimmunization: Hematoxylin and eosin (b), Hematoxylin and Congo Red (c) and periacid Schiff (d) showing absence of pneumonia, eosinophilia and mucus production. Scale bar for b and d: 100 µm; scale bar for c: 25 µm (e) Lung viral titers 4 days after RSV challenge in recipients of UVRSV+TLR, UVRSV, RSV or placebo. Results are means ± SEM (error bars) of 4–5 animals per group. For UVRSV+TLR, P<0.05 vs. UVRSV and placebo; P=NS vs. wtRSV. (f,g) Pulmonary histopathology in µMT mice immunized with FIRSV and passively transfused with sera obtained from UVRSV+TLR, UVRSV or wtRSV inoculated mice. µMT mice were subsequently challenged with wtRSV (day 7 post-challenge): Hematoxylin and eosin (f), Hematoxylin and Congo Red (g). Scale bar for f: 100 µm; scale bar for g: 25 µm Yellow arrows showing eosinophils.