Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant

Abstract

Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract.IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of nonlive RSV vaccine candidates. The findings presented here underscore the need to evaluate a nonlive RSV vaccine candidate during preclinical development over a wide dose range in the cotton rat RSV enhanced-disease model, as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself and to safe development of nonlive RSV vaccines for seronegative infants and children.

Keywords: GLA-SE; RSV; RSV F; cotton rat; enhanced RSV disease; respiratory syncytial virus; vaccine.

FIG 1

ELISA confirmation of pre-F antigen conformation. Post-F and pre-F conformations were assessed by sandwich ELISA. Plates were coated with motavizumab, recognizing both conformations, or with D25, which is specific for the pre-F conformation. Bound antigens were then detected using HRP-coupled 1331H antibodies, which recognize post- and pre-F conformations. The error bars indicate standard deviations (SD).

FIG 2

GLA-SE-adjuvanted post-F and pre-F induce neutralizing antibodies (NAb) and confer protection from RSV A and B infection in cotton rats. Animals (n = 4 to 7 per group) were immunized i.m. with different doses of antigens with or without GLA-SE adjuvant at day 0 and day 21 and challenged i.n. at day 35 with 10⁶ PFU of RSV A2 or RSV B. (A and B) Serum neutralization titers against RSV A2 (A) or RSV B (B) were measured prior to challenge at day 35. The data are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with the LLOD of 4 indicated by dotted lines. (C to F) RSV A2 (C and E) and RSV B (D and F) viral loads were measured by plaque assay in lung and nasal turbinates, respectively, 4 days postchallenge. The means of individual results ± SD are shown. For statistical analyses, groups immunized with post-F and pre-F were compared; *, P < 0.05. The data are representative of the results of 2 independent experiments.

FIG 3

GLA-SE-adjuvanted post-F and pre-F at a prophylactic dose do not induce alveolitis in the cotton rat RSV vaccine-enhanced disease model. Animals (n = 4 to 8 per group) were immunized i.m. at day 0 and day 21 and challenged i.n. at day 49 with 10⁵ PFU of RSV A Long. Naive animals were left untreated. (A) Serum neutralization titers against RSV A2 were measured prior to challenge at day 49. The data are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with the LLOD of 4 indicated by the dotted line. (B) Lung viral loads were measured by plaque assay 4 days postchallenge. (C) Lung alveolitis (macrophages, eosinophils, neutrophils, and/or lymphocytes in the alveoli) was analyzed blindly by a trained pathologist on sections of paraffin-embedded tissue stained with hematoxylin and eosin (H&E). Scoring: 0.5, minimal; 1, mild; 2, moderate; 3, marked. The means of individual results ± SD are shown. For statistical analyses, in panels A and B, groups with values above the LLOD were compared to FI-RSV; in panel C, all groups were compared to FI-RSV. *, P < 0.05. The data are representative of the results of 2 independent experiments.

FIG 4

Alum-adjuvanted post-F induces lung pathology similarly to FI-RSV in an extended cotton rat RSV vaccine-enhanced disease model. Animals (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 and challenged i.n. at day 91 with 10⁵ PFU of RSV A Long. Naive animals were left untreated. (A) Serum neutralization titers against RSV A2 were measured prior to challenge at day 91. The data are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with the LLOD of 4 indicated by the dotted line. (B) Lung viral loads were measured by plaque assay 4 days postchallenge. (C) Lung alveolitis (macrophages, eosinophils, neutrophils, and/or lymphocytes in the alveoli) was analyzed blindly by a trained pathologist on sections of paraffin-embedded tissue stained with H&E. Scoring: 0.5, minimal; 1, mild; 2, moderate; 3, marked. The means of individual results ± SD are shown. For statistical analyses, in panels A and B, groups with values above the LLOD were compared to FI-RSV; in panel C, all groups were compared to FI-RSV; *, P < 0.05.

FIG 5

GLA-SE-adjuvanted post-F and pre-F induce a lifetime persistent immune response in cotton rats. Animals (n = 8 per group) were immunized i.m. at day 0 and day 21 and bled once a month for over a year. Neutralization titers are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with an LLOD of 4. The dotted line indicates the neutralization titer level required for complete protection from RSV challenge based on historical data generated in cotton rats (28). The means of individual results ± SD are shown.

FIG 6

GLA-SE and alum-adjuvanted post-F at nanogram doses induce alveolitis in the cotton rat RSV vaccine-enhanced disease model. Animals (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 and challenged i.n. at day 50 with 10⁵ PFU of RSV A Long. Naive animals were not treated. (A) Serum neutralization titers against RSV A2 were measured prior to challenge at day 50. The data are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with the LLOD of 4 indicated by the dotted line. (B and C) Lung (B) and nasal turbinate (C) viral loads were measured by plaque assay 4 days postchallenge. (D) Lung alveolitis (macrophages, eosinophils, neutrophils, and/or lymphocytes in the alveoli) was analyzed blindly by a trained pathologist on sections of paraffin-embedded tissue stained with H&E. Scoring: 0.5, minimal; 1, mild; 2, moderate; 3, marked. The means of individual results ± SD are shown. For statistical analyses, in panels A, B, and C, groups with values above the LLOD were compared to FI-RSV; in panel D, all groups were compared to FI-RSV; *, P < 0.05. The data are representative of the results of 2 independent experiments.

FIG 7

Alveolitis induced by post-F at nanogram doses adjuvanted with GLA-SE and alum in the cotton rat RSV vaccine-enhanced disease model. Lung tissue was harvested 4 days post-RSV challenge. Representative images of lung sections with pathological findings are presented. (A) Naive cotton rats (no challenge) or buffer-, RSV A Long-, FI-RSV-, and FI-mock-immunized cotton rats challenged with 1 × 10⁵ PFU of RSV A Long at day 49 post-prime immunization (magnification, ×10). (B) Cotton rats that were immunized with 0.3, 0.03, 0.003, or 0.0003 μg of RSV post-F in the presence of GLA-SE or alum adjuvant and also challenged with 1 × 10⁵ PFU of RSV A Long at day 49 post-prime immunization (magnification, ×10). (C) Higher-magnification (×20) of a section of lung tissue of 0.3, 0.03, 0.003, or 0.0003 μg of RSV post-F plus GLA-SE-immunized cotton rats, focusing on the alveolar histopathology.

FIG 8

GLA-SE- and alum-adjuvanted pre-F at nanogram doses induce alveolitis in the cotton rat RSV vaccine-enhanced disease model. Animals (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 and challenged i.n. at day 49 with 10⁵ PFU of RSV A Long. Naive animals were not treated. (A) Serum neutralization titers against RSV A2 were measured prior to challenge at day 49. The data are presented as the log₂ dilution of serum that provides 50% reduction in viral entry, with the LLOD of 4 indicated by the dotted line. (B) Lung viral loads were measured by plaque assay 4 days postchallenge. (C) Lung alveolitis (macrophages, eosinophils, neutrophils, and/or lymphocytes in the alveoli) was analyzed blindly by a trained pathologist on sections of paraffin-embedded tissue stained with H&E. Scoring: 0.5, minimal; 1, mild; 2, moderate; 3, marked; 4, severe. The means of individual results ± SD are shown. For statistical analyses, in panels A and B, groups with values above the LLOD were compared to FI-RSV; in panel C, all groups were compared to FI-RSV; *, P < 0.05. The data are representative of the results of 2 independent experiments.