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. 2011 Dec;85(23):12201-15.
doi: 10.1128/JVI.06048-11. Epub 2011 Sep 21.

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Meagan Bolles  1 , Damon Deming, Kristin Long, Sudhakar Agnihothram, Alan Whitmore, Martin Ferris, William Funkhouser, Lisa Gralinski, Allison Totura, Mark Heise, Ralph S Baric
Affiliations

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Meagan Bolles et al. J Virol. 2011 Dec.

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.

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Figures

Fig. 1.
Fig. 1.
DIV vaccination and nonlethal heterologous challenge in aged animals. (A) Log10 half-maximum ELISA titers for anti-N and anti-S total IgG antibodies following DIV immunization. One-year-old aged NIA mice were immunized with DIV (n = 10), DIV plus alum (n = 9), or DIV plus VAP (n = 9). The values were statistically compared by a Mann-Whitney test. The error bars indicate standard deviations. (B) Log10 half-maximum ELISA titers of IgG1 versus IgG2a subtypes. Each point represents log10 IgG1 and IgG2a half-max titers for a single mouse. (C) Representative images (×400 magnification) of eosinophil infiltration in icGD03-S-challenged mice following DIV or iFlu vaccination regimens. Lungs taken 4 days postinfection were sectioned and stained with Congo red, a reliable and specific stain for eosinophils (arrows). (D) Box and whisker counts of eosinophils proximal to airways in icGD03-S-challenged aged mice, with the range shown by the whiskers. Eosinophils were counted in 4 160-μm2 regions proximal to airways (5 airways per mouse). The counts were statistically compared to those of adjuvanted controls by a t test with Welch's correction. (E) Mice challenged with the icGD03-S virus were weighed daily and visually assessed for morbidity. DIV and DIV-plus-alum immunogens significantly reduced the morbidity associated with icGD03-S challenge by day 4 postchallenge. (F) Mice were harvested on day 4, and one-quarter of the lung was homogenized, with the titer determined by plaque assay. Lung titers were sporadically reduced for both the DIV and DIV-plus-alum groups, with only DIV plus alum reaching statistical significance by the Mann-Whitney test. *, P < 0.05; **, P <0.01; †, P <0.001; ‡, P <0.0001.
Fig. 2.
Fig. 2.
Neutralizing antibody titers of vaccinated mice. Shown are PRNT50 values of sera collected from young and aged mice vaccinated with DIV or DIV plus alum. Neutralizing titers were significantly reduced in both aged vaccination groups compared to the young groups (DIV, P < 0.01; DIV plus alum, P < 0.0001; Fisher exact test). Alum adjuvant significantly increased neutralization titers for both young and aged animals (**, P < 0.01; ‡, P < 0.0001; 2-tailed Mann-Whitney test). No neutralizing antibody was detectable for young or aged mice vaccinated with iFlu (n = 15 and n = 16, respectively) or iFlu plus alum (n = 14 and n = 15, respectively) (data not shown). PRNT50 values below the limit of detection were assigned a value of 50 and those above the upper limit of quantification (ULOQ) a value of 3,200. (lower limit of quantification [LLOQ] = 100; ULOQ = 1,600).
Fig. 3.
Fig. 3.
Morbidity and mortality of lethal mouse-adapted and zoonotic challenges following DIV immunization. (A and B) Young and aged mice were vaccinated with iFlu, iFlu plus alum, DIV, or DIV plus alum (n = 6 per group) and subsequently mock infected (data not shown) or challenged with 105 PFU of icMA15 or icHC/SZ/61/03-S. The mice were weighed daily and monitored for morbidity (A) and mortality (B). (C) Four days postinfection, lungs were harvested, and the viral load was assessed by plaque assay. Values were statistically compared by Mann-Whitney test. The error bars indicate standard deviations.
Fig. 4.
Fig. 4.
Pathology in young mice challenged with icMA15 or icHC/SZ/61/03-S. Shown are representative images of H&E-stained lung panorama (×100), airway (×400), and parenchyma (×400) sections from young mice vaccinated with the indicated immunogen and challenged with icMA15 or icHC/SZ/61/03-S. TB, terminal bronchiole; AD, alveolar duct; black arrows, denuded airway; blue arrows, acute alveolitis and septal congestion; green arrows, peribronchovascular cuffing; red arrows, hyaline membrane.
Fig. 5.
Fig. 5.
Pathology following immunization and subsequent lethal challenge in aged Harlan mice. Shown are representative H&E-stained sections of panorama (×100), airway (×400), vasculature (×400), and parenchyma (×400) lung regions from aged mice: (A) mock infected; (B) iFlu plus alum vaccinated, icHC/SZ/61/03-S challenge; (C) DIV plus alum vaccinated, icHC/SZ/61/03-S challenge; (D) DIV plus alum vaccinated, icMA15 challenge. See the legend to Fig. 4 for arrow definitions.
Fig. 6.
Fig. 6.
Cytokine and chemokine mRNA expression profiles in aged mice. RNA was taken from the lungs of aged mice vaccinated with DIV plus alum and challenged with icHC/SZ/61/03 at 2 and 4 days postinfection. Cytokine and chemokine mRNAs were measured by quantitative real-time RT-PCR. The values are shown as log10 fold change over an unvaccinated, unchallenged control. *, P < 0.05; **, P < 0.01. The error bars indicate standard deviations.
Fig. 7.
Fig. 7.
Visual identification of eosinophils following lethal challenge. Eosinophils proximal to airways were counted in H&E-stained sections of lung from aged icHC/SZ/61/03-S-infected mice. (A) Representative images of regions counted, with eosinophils highlighted by arrows. (B) Box and whisker counts of eosinophils proximal to airways in icGD03-S-challenged aged mice, with ranges shown by the whiskers. Eosinophils were counted in 4 160-μm2 regions proximal to airways; n = 31 fields per group. Both DIV- and DIV-plus-alum-immunized mice had visibly more eosinophils present following challenge than the nonspecific-immunogen groups. ‡, P < 0.0001; t test with Welch's correction.
Fig. 8.
Fig. 8.
Flow cytometry gating strategy for cell populations. (A) Representative (PBS-vaccinated) plot of LCA+ lung cell populations. Neutrophils were defined as live cells, LCA+, Siglec−, and GR-1+ (green gate). SiglecF+ populations (black gate) from young and old mice were further analyzed, as shown in panel B. (B) Representative images of eosinophil (CD11b+ and CD11c−; blue gate) and alveolar macrophage (CD11c+; gray gate) gates for young and aged mice vaccinated with PBS, DIV, or DIV plus alum.
Fig. 9.
Fig. 9.
Flow cytometric analysis of additional lung immune cell populations in young and aged mice following immunization and subsequent lethal challenge. Mice challenged with icHC/SZ/61/03-S were sacrificed 4 days postinfection, and the lungs were stained with an eight-color panel. Each point represents the cell population for an individual mouse. (A) Eosinophil counts for each of the vaccination groups (n = 6 per group). Regardless of the age of the mice, the eosinophil counts were significantly increased in both DIV- and DIV-plus-alum-immunized groups compared to mock-vaccinated groups (F2,30 = 15.81; P < 1 × 10−4). (B) For neutrophil counts, only age was a significant factor (F1,30 = 24.7150; P = 2.525e−5). (C) Alveolar macrophage counts were significantly affected by immunogens in young, but not aged, animals, with post hoc analysis indicating that both DIV and DIV-plus-alum groups significantly differed from mock-vaccinated groups (F2,30 = 3.5534; P = 0.04121). (D) mDCs were also significantly affected by age (F1,30 = 20.3622; P = 9.193e−05), as well as immunogen (F2,30 = 9.5681; P = 0.0006107), with post hoc analysis indicating significant reductions in mDC counts in DIV-plus-alum groups relative to both DIV and mock-vaccinated groups.
Fig. 10.
Fig. 10.
Eosinophilia influx is conserved across group 2b N proteins. Young mice immunized with VRPs expressing the SARS N protein (VRP N); the N protein from another group 2b bat coronavirus, BtCoV.279 (VRP 279 N); or an irrelevant antigen (VRP-HA) were challenged with icGD03, and the lungs were taken 4 days postinfection. (A) Representative images of lung sections (×400) stained with Congo red. The inset shows areas of dense inflammatory infiltrate at higher magnification. (B) Both the SARS N and BtCoV.279 N proteins induce a significant eosinophilic inflammatory influx compared to the irrelevant antigen. ‡, P < 0.0001.
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