Decrease in Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice

<div><p>Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study. </p> </div

Vaccine enhanced disease in FI-A2 vaccinated mice after RSV Challenge.

<p>Mice were i.m. vaccinated with formalin inactivated RSV A2, rested for at four weeks and i.n challenged with either live RSV A2 (FI-A2+A2), UV-inactivated RSV A2 (FI-A2+UV-A2) or unifected vero cell lysate (FI-A2+vero). Unimmunized mice challenged with live RSV A2 (A2) provide a comparison to results after acute infection. Unvaccinated RSV challenged mice are compared to FI-RSV vaccinated mice challenged with live RSV mice and to FI-RSV vaccinated mice challenged with UV-inactivated RSV A2. Data from a separate experiment compares the inflammatory responses to FI-RSV vaccinated and vero cell lysate challenged mice. On day 5 post-infection, total BAL cell counts (A), total eosinophil counts (B) and viral RSV M gene mRNA (C) in the lungs of mice were determined. Total eosinophil counts were calculated by multiplying the total BAL cell counts by the percent eosinophils and presented as total eosinophils in BAL. Significance was calculated using a Student's <i>t</i> test, comparing mice in each group to mice vaccinated with FI-A2 alone and then challenged with RSV . * <i>p</i><0.05 A2 compared to FI-A2+UV-A2 or FI-A2+vero; ** <i>p</i><0.05 significance FI-A2 + A2 compared to either A2 or FI-A2+UV-A2 or FI-A2+vero. ND= not detected.</p

Decreased pulmonary cell inflammatory response in FI-A2 and G-A2 peptide vaccinated mice after RSV challenge.

<p>Mice were i.m. vaccinated with formalin inactivated RSV A2 (FI-A2) and then s.c. vaccinated with RSV A2 G peptide (FI-A2+G-A2) and control peptide (FI-A2 + L393). Two weeks following the final peptide boost, groups were challenged with live RSV A2 and the mean numbers of BAL cells/lung (A), total eosinophil counts in the BAL (B), IL-4 levels (pg/ml) (C) and IFN gamma (D) in the cell-free BAL supernatant, and viral message levels (relative genome equivalent PFU/ml) (E) at day 5 post-challenge (peak of cell infiltration) were determined. Representative data from five independent experiments are shown. * <i>p<0</i>.<i>05</i>.</p

Decreased weight loss in FI-A2 and G-A2 peptide vaccinated mice after RSV challenge.

<p>Mice were i.m. vaccinated with formalin inactivated RSV A2 (FI-A2) and then s.c. vaccinated with G-A2 or control peptides. Two weeks following the final peptide boost, FI-A2 and RSV A2 G peptide (FI-A2+G-A2) and control L393 peptide (FI-A2+L393) mice were challenged with 10⁶ PFU live RSV A2 and percent body weight loss were determined. Five independent experiments were performed and combined (representative) data from two experiments are shown. * <i>p<0</i>.<i>05</i>.</p

Immunization and challenge timeline.

<p>Timeline of vaccination and challenge schedule as described in the Materials and Methods.</p

Antibody response against immunizing G peptides in FI-A2 vaccinated mice.

<p>Two weeks following the final boost and prior to RSV challenge, whole RSV A2 IgG specific titers (A) and G-A2 peptide specific IgG titers (B) in sera from mice vaccinated with formalin-inactivated RSV A2 alone (FI-A2), FI-A2 and RSV A2 G peptide (FI-A2+G-A2), FI-A2 and control L393 peptide (FI-A2+L393), FI-Vero alone (FI-Vero), or FI-vero and RSV A2 G peptide (FI-Vero+G-A2) were detected by indirect ELISA. Shown are the absorbance values (OD₄₀₅) after subtraction of background OD₄₀₅ values against control antigen. Results are means of 5 to 10 mice per group and are representative of three independent experiments. (C) Neutralizing antibodies against live RSV A2 in vaccinated animals (1:200 dilution) were detected by a microneutralization assay. Serum from a naïve mouse and anti-RSV F monoclonal antibody (143-6C) were used as negative and positive controls, respectively. * <i>p<0</i>.<i>05</i>.</p

Pulmonary cell infiltration following FI-RSV and FI-Vero vaccination.

<p>(A) Total BAL cell counts in the lungs of mice i.m. vaccinated with FI-RSV A2 and live RSV A2 challenged (FI-A2+A2), mice vaccinated with FI-Vero cell lysate supernatant and then challenged with Vero cell lysate supernatant (FI-Vero+Vero), or mice vaccinated with FI-Vero lysate and G-A2 peptide and challenged with either RSV A2 (FI-Vero+G-A2+A2) or uninfected, Vero lysate (FI-Vero+G-A2+Vero) (day 5 post-infection). (B) Total neutrophils (PMN), lymphocytes, (LYM), macrophages (MAC) and eosinophils (EOS) counts in BAL. (C) Total BAL cell counts in the lungs of mice vaccinated with or without FI-Vero supernatant at day 5 post-RSV A2 challenge. (D) Total PMN, LYM, MAC and EOS counts in the BAL of mice infected with A2 and mice vaccinated with FI-Vero cell lysate and then challenged with RSV A2 (FI-Vero+A2) at day 5 post-challenge are shown. Significance was calculated using a Student's <i>t</i> test, comparing mice in each group to mice vaccinated with FI-A2 alone and challenged with RSV. * <i>p<0</i>.<i>05</i>.</p

Lung histopathology in vaccinated mice.

<p>Lung histopathology was examined by hematoxylin and eosin staining in (A) mice i.m. vaccinated with FI-A2 and then challenged with RSV A2 (FI-A2), (B) mice vaccinated with FI-A2 and control L393 peptide and challenged with RSV A2 (FI-A2+L393), or (C) mice vaccinated with FI-A2 and G-A2 peptide and challenged with RSV A2 (FI-A2+G-A2) at day 3 post-challenge. The arrows mark areas in inflammatory infiltrates. Control groups were immunized with FI-Vero cell lysate and challenged with either (D) RSV A2 (FI-Vero) or (E) uninfected, Vero lysate (FI-Vero+Vero). (F) Lung histopathology of an uninfected, unimmunized BALB/c mouse. The perivascular areas (asterisks) are devoid of inflammatory infiltrates. 40X original magnification, Scale bar= 200mm. </p