A peptide mimic of a protective epitope of respiratory syncytial virus selected from a combinatorial library induces virus-neutralizing antibodies and reduces viral load in vivo

Abstract

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.

FIG. 1

Reduction of RSV infection in the lungs of mice following immunization with S1S-MAP. The animals were challenged i.n. with RSV (10⁶ PFU/50 μl) 4 to 5 weeks after the last boost. The lungs were removed 4 days later (at the peak of the viral load in the lungs) for quantification of viral titers. The results are expressed as log₁₀ RSV PFU/g of lung tissue ± 1 standard deviation. The lowest level of virus detectable in this assay was 2.10 log₁₀ PFU/g of lung tissue (12 PFU/animal). The animals in the RSV group (the positive control for protection) were infected with RSV (10⁶ PFU/50 μl) 9 days prior to rechallenge with RSV. The open bars represent BALB/c mice (5 animals/group) immunized i.p. and boosted 3 weeks later with S1S-MAP plus Th, S1S-MAP–Th, or Th (5 nmol/animal). Anti-RSV IgG log₁₀ titers for each group prior to challenge were as follows: S1S-MAP plus Th, 3.29 ± 0.13; S1S-MAP–Th, 3.01 ± 0.11; Th, <1.90. The shaded bars represent BALB/c mice (4 animals/group) immunized following the schedule described above but which received a second boost 6 weeks later. Anti-RSV IgG log₁₀ titers for each group prior to challenge were as follows: S1S-MAP plus Th, 3.61 ± 0.20; S1S-MAP–Th, 3.38 ± 0.15; Th, <1.90. A significant reduction in RSV titers was observed in the lungs of all animals immunized with S1S-MAP plus Th and S1S-MAP–Th in comparison to that in the Th control group (∗, P < 0.01; ∗∗, P < 0.001 [Student t test]). Over five experiments, no significant differences were observed in RSV titers recovered from the lungs of Th-immunized and mock-immunized animals (P > 0.2 by one-way analysis of variance).

FIG. 2

Sections of lung tissue stained with hemotoxylin and eosin showing reduced cellular infiltration in mice immunized with S1S-MAP plus Th constructs (A and B) compared to that in control mice immunized with Th alone (C and D) following challenge with RSV. As controls, sections from RSV-immunized and challenged mice showing no infiltration are also shown (E and F). The histological analyses were performed 7 days after challenge with RSV (10⁶ PFU/50 μl). Magnification, ×100 (A, C, and E) and ×250 (B, D, and F).