A Double-Inactivated Severe Acute Respiratory Syndrome Coronavirus Vaccine Provides Incomplete Protection in Mice and Induces Increased Eosinophilic Proinflammatory Pulmonary Response upon Challenge

The icGD03-S (AY525636) (47), icMA15 (FJ882957), and icHC/SZ/61/03-S (45) strains of SARS-CoV were propagated on Vero E6 cells in Eagle's MEM supplemented with 10% fetal calf serum, kanamycin (0.25 μg/ml), and gentamicin (0.05 μg/ml) at 37°C in a humidified CO₂ incubator. For virus growth, cultures of Vero E6 cells were infected at an approximate multiplicity of infection (MOI) of 1 for 1 h, and the monolayer was washed twice with 2 ml of phosphate-buffered saline (PBS) and then overlaid with complete medium. At 30 h postinfection, the supernatant was clarified by centrifugation at 1,600 rpm for 10 min, aliquoted, and frozen at −70°C. Virus stocks were titrated by plaque assay.

Due to the poor availability of aged mice, two slightly divergent mouse strains were utilized during the course of this research. BALB/c mice (Harlan Laboratoriess, Indianapolis, IN) were challenged with lethal viruses (icMA15 and icHC/SZ/61/03-S), while the National Institute of Aging (NIA) provided BALB/cBy mice for nonlethal/epidemic strain challenges. Prior studies in our laboratory have shown conserved susceptibility phenotypes in these mouse strains following SARS-CoV challenge, with slightly increased morbidity and mortality in the NIA (BALB/cBy) strain. Therefore, in the nonlethal icGD03-S challenge, we expected slightly more morbidity than normally would have been predicted in Harlan mice.

Female BALB/cAnNHsd mice (young [6 to 8 weeks old] and aged [12 to 14 months old]; Harlan Laboratories, Indianapolis, IN) were separated into 4 groups of 12 young and 12 aged mice. The mice within each group were vaccinated by footpad injection with 20-μl volumes consisting of 0.2 μg of double-inactivated SARS-CoV vaccine, 0.2 μg of double-inactivated SARS-CoV vaccine with alum, 0.2 μg of inactivated influenza virus (iFlu), or 0.2 μg of inactivated influenza virus with alum. The mice were boosted with the same regimen 22 to 28 days later. Aged female BALB/cBy mice (12 to 14 months old; NIA) were vaccinated with PBS-, alum-, or VAP (VEE [Venezuelan equine encephalitis virus] replicon [VRP] adjuvanting particle)-adjuvanted iFlu (n = 8, 10, and 9, respectively) or DIV (n = 10, 9, and 10, respectively) immunogen. The vaccine formulations consisted of 0.2 μg of DIV or iFlu plus either PBS, 0.69 mg/ml alum, or 10⁵ infectious units (IU) of VAP, a dose which had previously been demonstrated to provide protective immunity in young mice (25, 54). These mice were then boosted with the same regimen 22 to 28 days later.

We collected blood from tail veins prior to challenge with icMA15, icHC/SZ/61/03, or icGD03-S on day 36 postvaccination. Mice were anesthetized with a ketamine (1.3 mg/mouse)-xylazine (0.38 mg/mouse) mixture administered intraperitoneally in a 50-μl volume. The mice were intranasally inoculated with 10⁵ PFU of icMA15, icHC/SZ/61/03, or icGD03-S in 50-μl volumes and weighed daily. At 2 or 4 days postinfection, the mice were euthanized with isoflurane, and lung and serum samples were collected for analysis. For studies involving VRP vaccinations, 5-week-old female BALB/c mice were immunized with 10⁵ IU of VRPs expressing SARS-CoV N, a bat coronavirus (BtCoV.279) N, or hemagglutinin (HA) in a 10-μl volume by footpad injections. Three weeks later, blood was collected by the tail nick method for enzyme-linked immunosorbent assay (ELISA), and the mice were boosted with 10⁵ IU of the respective VRPs. Three weeks postboost, blood was collected by tail nick for assessing antibody responses. The mice were moved to a satellite facility under BSL3 conditions, acclimatized, and challenged with 10⁵ PFU of recombinant MA15 (rMA15) icGDO3 virus (48) by intranasal inoculation as described above.

All mice were housed under sterile conditions in individually ventilated Sealsafe cages using the SlimLine system (Tecniplast, Exton, PA). Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of North Carolina, Chapel Hill, NC.

One-quarter of each lung was taken for determination of the viral titer. Samples were weighed and homogenized for 60 s at 6,000 rpm in four equivalent volumes of PBS to generate a 20% solution. The solution was centrifuged at 13,000 rpm under aerosol containment in a table top centrifuge for 5 min, the clarified supernatant was serially diluted in PBS, and 200-μl volumes of the dilutions were placed onto monolayers of Vero E6 cells in six-well plates. Following 1 h of incubation at 37°C, the cells were overlaid with 0.8% agarose-containing medium. Two days later, the plates were stained with neutral red, and the plaques were counted.

We heat inactivated mouse serum at 55°C for 30 min and then serially diluted it to 1:50, 1:100, 1:200, 1:400, and 1:800 in PBS to a volume of 125 μl. Next, we added 125 μl of PBS containing 125 PFU of icSARS-CoV to each serum dilution, incubated the virus-serum mixtures at 37°C for 30 min, added 200 μl of each mixture to confluent cultures of Vero E6 monolayers, and allowed them to incubate at 37°C for 1 hour. Following the 1-h infection, we covered each monolayer with 4 ml of 0.8% agarose melted in standard Vero E6 cell medium and resolved the plaques with neutral red staining 2 days later. Finally, we calculated the 50% plaque reduction neutralization titer (PRNT₅₀) values, the serum dilutions at which plaque formation was reduced by 50% relative to that of virus not treated with serum.

One-half of each lung was fixed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) for at least 7 days, embedded in paraffin, sectioned to 5 μm, and stained with hemotoxylin and eosin (H&E). Sections were blindly evaluated by W. Funkhouser for the extent of tissue damage and characterization of inflammation.

Lung tissues were prepared as described above and stained with H&E or Congo red (plus hematoxylin) (30). For each slide, an initial assessment of gross lung pathology was followed by selection of a lung section and enumeration of eosinophils within the viewing field at ×400 magnification. Representative images were minimally and identically processed to enhance contrast in Adobe Photoshop CS4. For both H&E- and Congo red-stained slides, multiple 160-μm² sections proximate to airways were assessed, and the eosinophils counted were averaged per lung.

One-quarter of a lung from each mouse was placed into RNAlater (Ambion) for 4 days at 4°C and then frozen at −70°C. Lung samples were transferred from RNAlater to TRIzol and homogenized for 60 s at 6,000 rpm, and RNA was extracted by chloroform/isopropanol precipitation. cDNA was prepared by standard protocols using random hexamers and SuperScript II Reverse Transcriptase (Invitrogen). Quantitative PCR was conducted on a Lightcycler 480II (Roche) using ABI TaqMan Gene Expression Assays specific for mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or mouse interleukin 4 (IL-4), IL-5, gamma interferon (IFN-γ), IL-13, CCL11 (eotaxin), or Cxcl1 Keratinocyte-derived chemokine (KC). Relative quantification was calculated as the log₁₀ fold change (2^ΔΔCT) relative to mock-vaccinated, mock-challenged controls.

Mice vaccinated with PBS, DIV, or DIV plus alum were challenged with 10⁵ PFU of icHC/SZ/61/03, weighed and monitored daily for morbidity, and euthanized 4 days postinfection by isoflurane inhalation. The lungs were perfused with 10 ml PBS by cardiac puncture, dissected, manually minced, and vigorously agitated for 2 h in digestion medium (RPMI, 10% fetal bovine serum [FBS], 15 mM HEPES, 1.7 mg/ml DNase 1 [Sigma], 2.5 mg/ml collagenase A [Roche], 1× streptomycin, 1× gentamicin). The lungs were then passed through a 75-μm cell strainer, resuspended in RPMI medium (RPMI, 10% FBS, 15 mM HEPES), and overlaid on a density gradient of iodixanol diluted to a density of 1.079 gm/cm³ with RPMI 1640 containing 10% FBS (Optiprep, Sigma-Aldrich Co., St. Louis, MO). Following centrifugation, cells were collected from the interface and washed, and viable cells were counted with a Countess automated cell counter (Invitrogen). The cells were then incubated with the following panel of antibodies: allophycocyanin (APC) anti-leukocyte common antigen (LCA), PE-Cy7 anti-CD11b, and phycoerythrin (PE)-Cy5 anti-major histocompatibility complex (MHC) class II antigens, all from eBioscience (San Diego, CA); fluorescein isothiocyanate (FITC) anti-Gr-1 and PE anti-SiglecF, from BD-Pharmingen (San Diego, CA); and PE-Texas Red anti-CD11c (Molecular Probes Invitrogen, Carlsbad, CA). Following staining, the cells were washed with fluorescence-activated cell sorter (FACS) wash buffer (1× Hanks balanced salt solution [HBSS], 1% FBS) and fixed with 2% formalin, and flow cytometry was conducted on a CyAn ADP (Beckman-Coulter) with 300,000 live-cell events gathered per lung sample. Analysis was performed with Summit software (version 5.2; Beckman-Coulter). First, we gated on LCA⁺ and CD11c⁺ cells by plotting those parameters against forward scatter and gating on positive cells. Then, Gr-1 was plotted against SigLecF (see Fig. 8A). SigLecF-high, Gr-1-intermediate cells were selected, and CD11b signal versus CD11c signal was plotted for cells in that region. CD11b⁺, CD11c⁻ cells are classified as eosinophils, while alveolar macrophages are CD11c⁺ (see Fig. 8b). After gating on LCA⁺ and CD11c⁺ cells, Gr-1-positive cells are classified as neutrophils (see Fig. 8a). Of the cells that remain after gating out SiglecF⁺ cells and neutrophils, we classify MHC class II-negative, CD11b⁺, and B220-negative cells as monocyte-derived dendritic cells (mDCs). The cell counts per lung were calculated as the product of the total viable lung cell population by the percentage of gated cells in live-cell events. We used a two-factor analysis of variance (ANOVA) to assess the statistical significance of age and vaccine on the overall number of cells. If the ANOVA determined a factor was significant, post hoc analyses using Tukey's honestly significant differences (HSD) were used to further determine the effects of treatment on cell counts.

Antigen-specific IgG and IgG subisotype titers were determined by ELISA. Briefly, purified recombinant SARS N protein or S protein was coupled to high-binding 96-well ELISA plates (Greiner) in basic carbonate buffer (pH = 9.6). After washing with ELISA wash buffer (EWB) (PBS with 0.016% Tween 20), diluted serum was added to the wells in EWB with 10% blocking buffer (Sigma-Aldrich). After 2 h at 4°C, the plates were washed again, and horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2a was added to the appropriate wells diluted in EWB plus blocking buffer. After another 2 h, chromogenic substrate (o-phenylenediamine in citrate buffer with added hydrogen peroxide) was added to each well. After 30 min, the reaction was stopped with the addition of 0.1 M sodium fluoride and read at 450 nm. A sigmoidal curve was fit to each set of optical density (OD) versus log₁₀ serum dilution values using the curve-fitting software of the SigmaPlot graphics package (Systat Software, Inc.), and the inflection point (where the OD is one-half of the maximum value recorded for that isotype-antigen combination) is calculated and reported as the “half-max titer.”

The efficacy of double-inactivated SARS-CoV vaccines has not been evaluated in aged animals, which show immunosenescence, increased susceptibility to clinical disease, and increased pathology, reflecting conditions in the more vulnerable SARS-CoV-infected aged populations (48, 50). One-year-old National Institute of Aging mice were vaccinated with double-inactivated SARS vaccine (DIV) or nonspecific immunogen (iFlu) in unadjuvanted form, adjuvanted with alum, or adjuvanted with VAP. SARS-specific total IgG responses, as measured by ELISA, show a significant increase in the alum-adjuvanted group compared to the PBS-adjuvanted group for both N-specific (3.580 versus 2.625 log₁₀ half-max titer) and S-specific (3.743 versus 2.781 log₁₀ half-max titer) antibodies (Fig. 1A). Unexpectedly, the VAP adjuvant nearly ablated total IgG antibody compared to PBS, reducing total S- and N-specific antibodies to titers of 0.7432 and 1.182 log₁₀ half-max, respectively (Fig. 1A). The alum-adjuvanted DIV induced a strong skew in the N- and S-specific antibodies toward IgG1, a subtype associated with Th2 immune responses, while the nonadjuvanted DIV resulted in a more balanced or IgG2a-skewed antibody population (Fig. 1B).

To assess protection from heterologous SARS infection, the aged DIV-vaccinated mice were challenged with 10⁵ PFU of icGD03-S, a recombinant heterologous human strain that closely resembles zoonotic strains circulating in 2004. Both DIV- and DIV-plus-alum-vaccinated groups showed significant, though incomplete, reductions in morbidity (as measured by weight loss) by day 4 postinfection, while none of the nonspecific-vaccination groups showed any reduction in morbidity or lung viral titer by 4 days postinfection (Fig. 1E and F). The VAP-adjuvanted DIV group predictably showed no reduction in morbidity or titer, consistent with the failure of DIV plus VAP to induce SARS-specific antibody responses in the aged animals (data not shown). When the viral titers in the lungs were assessed, only the DIV-plus-alum group showed significant reductions in day 4 lung titers, while all other groups, including non-adjuvanted DIV, showed high levels of viral replication (Fig. 1F). These results demonstrate that while DIV does provide some heterologous protection in highly susceptible aged populations, the vaccine is unable to provide complete protection from either viral replication or virus-induced morbidity.

Previous work by our group and others has demonstrated that vaccination with SARS N protein fails to protect from SARS replication while driving a vaccine-induced eosinophilic pathology. Therefore, given that none of the DIV vaccine strategies resulted in complete protection from viral replication in the aged animals, we assessed whether any of the vaccine groups exhibited signs of eosinophilic immune pathology. Importantly, both the DIV and DIV-plus-alum groups showed increased numbers of eosinophils in the lungs following challenge (Fig. 1C). Mice vaccinated with iFlu and iFlu-plus-alum showed a low number of eosinophils in regions proximate to airways by Congo red staining: both iFlu and iFlu-plus-alum groups had a median count of 1.0 eosinophil per region (Fig. 1D). Both DIV- and DIV-plus-alum-vaccinated groups showed significant increases in eosinophil counts over the comparable nonspecific immune groups at 5.0 and 35.0 eosinophils per region, respectively. Further, adjuvanting with alum significantly increased the eosinophil influx compared to DIV alone.

Few candidate vaccines have been tested in aged animals following homologous and heterologous lethal challenge, and no whole-virus vaccines have been so tested. Therefore, we assessed whether the DIV formulations would protect against heterologous and homologous lethal challenges in young- and aged-animal models. As a control, vaccination with inactivated influenza virus with or without alum did not induce detectable levels of anti-SARS (Urbani) antibody in either 12-week-old young or 59-week-old aged mice (data not shown). When young mice were vaccinated with 0.2 μg of the SARS-CoV DIV, 8/16 generated detectable levels of SARS neutralizing antibody titers. Vaccination with DIV plus alum induced detectable levels of neutralizing antibody in 15/15 mice and at significantly increased PRNT levels compared to DIV alone (Fig. 2). Specifically, the mean (±standard deviation [SD]) PRNT₅₀ value for DIV alone was 221 ± 220, which significantly differed from the DIV-plus-alum group's neutralizing titer of 2,710 ± 992 (Fig. 2). Importantly, DIV alone did not induce detectable levels of anti-SARS antibodies in aged mice, while the addition of alum increased the response in aged animals, with 8/14 aged animals generating detectable levels of neutralizing antibodies. The mean PRNT₅₀ value was 425 ± 806, significantly reduced by about 6-fold compared with DIV-plus-alum-vaccinated young animals. Thus, in young and aged animals, the presence of alum in the DIV vaccine formula significantly improved the induction of SARS-CoV neutralizing antibody: from moderate to high levels in young animals and from unmeasurable to moderate levels in aged animals.

To directly assess vaccine-mediated protection from lethal disease in young and old animals, mice were challenged either with the homologous mouse-adapted icMA15 virus or with the heterologous zoonotic virus icHC/SZ/61/03-S. In young animals, DIV provided partial protection, increasing survival following challenge with icMA15 from 0% to 83.3% and significantly reducing morbidity by day 3 (P < 0.01; 2-tailed Mann-Whitney test). In contrast, DIV plus alum provided complete protection from morbidity and mortality by 4 days postinfection in icMA15-challenged mice (Fig. 3A and B). When young mice were challenged with icHC/SZ/61/03-S, both the DIV alone and DIV-plus-alum groups were provided complete protection from mortality. The DIV-plus-alum group showed complete protection from morbidity, while DIV-immunized groups showed mild weight loss. The nonspecifically vaccinated groups (iFlu and iFlu-plus-alum) lost ∼20% of their starting weight by day 3 (icMA15) or day 4 (icHC/SZ/61/03-S) postinfection, respectively. Furthermore, the iFlu- and iFlu-plus-alum-vaccinated icMA15-infected animals showed 0% survival by day 4, while the same groups challenged with icHC/SZ/61/03-S showed 67% and 50% survival, respectively. In short, although DIV-mediated protection was not complete, there was partial protection from homologous and heterologous lethal challenges in young animals, and this protection from weight loss and death was enhanced by the inclusion of alum adjuvant.

When viral titers were assessed in the lungs at day 4 postinfection, young mice vaccinated with DIV plus alum showed no detectable viral titer following either viral challenge (Fig. 3C). In contrast, for DIV alone, lung titers following icMA15 challenge were reduced to undetectable levels in only 1 of 5 surviving mice, and the remaining 4 had titers ranging from 5.65 to 6.41 log₁₀ PFU/g. Following icHC/SZ/61/03-S challenge, viral titers in DIV-vaccinated mice were reduced to undetectable levels for all but 1 mouse (3.74 log₁₀ PFU/g). In comparison, the iFlu-immunized groups had titers ranging from 3.65 to 4.83 log₁₀ PFU/g (iFlu) and 4.72 to 5.14 log₁₀ PFU/g (iFlu plus alum). Therefore, in agreement with morbidity data, DIV plus alum was able to provide complete protection in young animals, while DIV alone reduced but did not eliminate viral replication following homologous or heterologous challenge.

In stark contrast to the encouraging results seen in young animals, 1-year-old aged animals vaccinated with either DIV alone or DIV-plus-alum did not show complete protection from mortality, and both groups demonstrated significant morbidity, with weight loss and high levels of viral replication. As in young animals, iFlu-vaccinated groups showed an ∼20% weight loss by day 3 (icMA15) or 4 (icHC/SZ/61/03-S) postinfection. Survival following icMA15 challenge was 16.7% and 0% on day 4 for iFlu and iFlu-plus-alum, respectively; following icHC/SZ/61/03-S challenge, the rate was 16.7% for each treatment. Unlike young animals, aged groups vaccinated with DIV alone did not show promising reductions in morbidity, mortality, or viral titer following these lethal challenges (Fig. 3). icMA15- or icHC/SZ/61/03-S-challenged groups vaccinated with DIV alone had greater than 20% weight loss by day 4, and the DIV immunization provided no significant increases in survival for either virus challenge (16.7% for icMA15 and 66.7% for icHC/SZ/61/03-S). The one surviving mouse following icMA15 challenge had a lung viral titer of 6.70 log₁₀ PFU/g, and following icHC/SZ/61/03-S challenge, the surviving mice had lung titers ranging from 3.89 to 4.44 log₁₀ PFU/g, which was slightly reduced compared to the peak titers seen in surviving iFlu-vaccinated control mice (4.83 and 4.98 log₁₀ PFU/g). In contrast to DIV alone, DIV plus alum did significantly increase survival rates for aged mice after both icMA15 and icHC/SZ/61/03-S challenges compared to the iFlu-plus-alum-immunized groups. Following icMA15 challenge, aged mice vaccinated with DIV plus alum showed 83.3% survival by day 4 (versus 0% for iFlu plus alum; P < 0.01; 2-tailed Fisher exact test), and the comparable group challenged with icHC/SZ/61/03-S showed 100% survival (versus 16.7% for iFlu plus alum; P < 0.01; 2-tailed Fisher exact test). However, mice in both the icMA15- and icHC/SZ/61/03-S-challenged groups had significant weight loss compared to mock-infected controls (P < 0.05 for both) (Fig. 3A). Additionally, DIV plus alum reduced lung titers to undetectable levels by day 4 in only 3 of the 5 surviving icMA15-challenged animals and did not eliminate virus from any of the surviving icHC/SZ/61/03-S-challenged animals despite 100% survival in those animals (Fig. 3C).

DIV alone failed to provide complete protection from SARS replication and disease in both young and old animals, while DIV plus alum failed to prevent weight loss in aged animals, suggesting that neither vaccine formulation would protect from SARS-induced respiratory pathology. We therefore evaluated the lungs of animals from each of the vaccination groups for vaccine-induced pathology. Lethal challenges to naïve mice result in a denuding bronchiolitis and apoptotic debris in the airways of both young and old animals. Young mice challenged with icHC/SZ/61/03-S displayed diffuse parenchymal alveolitis, while challenge with icMA15, and both challenges to aged mice, demonstrated additional pathology, including diffuse alveolar damage (DAD) and hyaline membrane formation, both hallmarks of acute respiratory distress syndrome (ARDS) in SARS cases (young, Fig. 4; aged, Fig. 5).

Young animals vaccinated with DIV suffered significant pathological changes that were generally worse in icMA15- than icHC/SZ/61/03-S-challenged animals. Compared to mock-vaccinated groups, the DIV-vaccinated groups showed increased perivascular and peribronchial cuffing, as well as scattered infiltrates in the parenchyma, including neutrophils, alveolar macrophages, and eosinophils (Fig. 4). DIV-vaccinated aged animals showed greater pathology than young animals, particularly perivascular cuffing and interstitial thickening (Fig. 5). These results indicate that not only did the DIV vaccine fail to protect from viral replication and morbidity/mortality, but the vaccine actually promoted the development of enhanced inflammatory damage within the lungs.

The adjuvanted vaccine, DIV plus alum, well protected young mice from icMA15 challenge, showing minimal pathological changes. However, despite good protection from weight loss or lethality, DIV-plus-alum icHC/SZ/61/03-S-challenged mice showed histopathology more extensive than that in unvaccinated mice, with increased peribronchiovascular cuffing consisting of mature lymphocytes, macrophages, and eosinophils (Fig. 4). In aged animals, DIV plus alum provided little protection against either challenge, demonstrating pathology beyond that in the unvaccinated mice, with additional perivascular and peribronchial cuffing, as well as fibrinous exudates in the alveolar parenchyma (Fig. 5C and D).

Given the Th2-associated skew in antibody profiles (Fig. 2) and the broad influx of inflammatory cells in vaccinated mice (Fig. 4 and 5), we used quantitative RT-PCR to analyze in vivo cytokine mRNA expression following vaccination and challenge (Fig. 6). Aged mice vaccinated with DIV plus alum and challenged with icHC/SZ/61/03 had elevated mRNA levels of Th2 effector cytokines, including IL-13 and IL-5, at 2 and 4 days postinfection (P < 0.01 for each). The eosinophil chemoattractant CCL11 (eotaxin) was significantly increased in the lungs of vaccinated mice at both 2 and 4 days postinfection (P < 0.01). In contrast, IL-4 and IFN-γ showed no significant changes at these time points (the earliest of which is day 2), and the neutrophil-associated chemokine, Cxcl1, was elevated in mock-vaccinated rather than DIV-vaccinated groups (P < 0.05).

The presence of high numbers of eosinophils among the inflammatory infiltrates following vaccination and subsequent challenge has been a consistent observation associated with respiratory syncytial virus (RSV)- and measles vaccine-induced immunopathology and was observed in vaccination experiments with the SARS-nucleocapsid immunogen, peaking at 4 days postinfection (11, 61). To assess the eosinophilic influx following whole-virus immunization and lethal challenge, we blindly assessed H&E-stained lung sections of aged mice challenged with HC/SZ/61/03-S and enumerated eosinophils proximate to airways. Representative images (×400 magnification) of regions proximate to airways are shown in Fig. 7A, with counts of eosinophils per 160 μm² in Fig. 7B. A median 9.0 eosinophils per 160 μm² were present around the airways of DIV-vaccinated groups, comparable to the median 11.0 present in DIV-plus-alum groups. These counts are significantly higher than in the iFlu (median, 1.0) and iFlu-plus-alum (median, 0.0) groups, respectively (Fig. 7B). These data suggested that increased eosinophilia occurs following DIV and DIV-plus-alum vaccination in young and aged animals, especially following heterologous challenge.

To precisely characterize the inflammatory infiltrate following DIV vaccination, we challenged PBS-, DIV-, or DIV-plus-alum-immunized young and aged mice with icHC/SZ/61/03-S and examined whole lungs by flow cytometric analysis at 4 days postinfection. Eosinophils, defined as LCA⁺ SiglecF⁺ CD11b⁺ CD11c⁻ populations (Fig. 8), were significantly increased in all vaccinated compared to unvaccinated populations following lethal challenge (Fig. 8A [F_2,32 = 14.739; P < 1 × 10⁻⁴] and 9A). Post hoc analysis using Tukey's HSD showed that both DIV (P = 0.0003433) and DIV-plus-alum (P < 1 × 10⁻⁴) groups had significantly increased numbers of eosinophils compared to PBS-immunized animals. Adjuvant had no measurable effect on the eosinophilic influx, as DIV- and DIV-plus-alum-immunized mice did not have statistically significant differences in their overall numbers of eosinophils in either age group. Importantly, the elevated eosinophil counts were comparable between young and aged populations despite divergent clinical presentations. Further analysis of the inflammatory cell infiltrates of vaccinated mice showed a strong influence of age on the neutrophil (F_1,32 = 22.0339; P < 1 × 10⁻⁴) and monocytic dendritic cell (F_1,32 = 26.681; P < 1 × 10⁻⁴) influx, with both cell populations significantly increased in aged relative to young animals (Fig. 9B and D). Alveolar macrophage counts showed no significant differences compared to mock-vaccinated mice in either young or aged animals (Fig. 9C). Finally, the vaccinated animals showed significantly lower monocytic DC counts by day 4 postchallenge, independent of age (F_2,32 = 11.18; P = 0.0002079), and post hoc analysis showed the differences were enhanced by the alum adjuvant (P = 0.04402) (Fig. 9D). Vaccination with DIV clearly induces a strong eosinophilic infiltrate, independent of age and regardless of whether it protects from morbidity, and raises the possibility that DIV elicits immune pathology in the face of heterologous SARS challenge.

The data from our studies collectively demonstrated that eosinophilic infiltration occurs during DIV vaccination in both young and aged mouse populations, irrespective of the adjuvant and the challenge virus used. Previous data from our laboratory have shown that vaccination with VRPs expressing SARS N protein (VRP N) induces a similar eosinophilic phenotype while completely failing to protect against SARS CoV replication (11), suggesting that the eosinophilia may be driven by SARS N protein. To determine whether sequences intrinsic to SARS N protein or an N protein from a group 2b coronavirus closely related to SARS contribute to the induction of eosinophilia, we immunized mice with VRPs expressing SARS N, the N gene from BtCoV.279 (VRP 279 N), or an irrelevant antigen, VRP HA (containing the hemagglutinin gene from the PR8 strain of influenza virus). The BtCoV.279 N gene shares 95% sequence homology with the SARS N gene, with conservation across all the major domains of the N protein. Groups of mice immunized with 10⁵ IU of VRP N and VRP 279 N showed high antibody titers post boost (data not shown) and were challenged with 10⁵ PFU of rMA15 GDO3-S. This virus causes 15% weight loss in infected young BALB/c mice by day 4 postinfection (48). Mice from all the groups showed 15% weight loss by day 4 postinfection, with virus titers ranging up to 10⁵ PFU (data not shown), indicating failure of N gene-based vaccines to protect from virus replication. Histological analysis of Congo red-stained lung sections showed increased numbers of eosinophils proximal to airways in VRP N-immunized mice compared to VRP HA-immunized mice (Fig. 10A). Interestingly, enhanced influx of eosinophils was also found in regions proximal to the airways in lungs from mice immunized with VRP 279 N (Fig. 10A). Blind scoring of lung sections indicated a median count of 25 eosinophils per region in VRP N- and 279 N-immunized groups, whereas the VRP HA group showed a median count of 3 per region (Fig. 10B). These data clearly indicated that eosinophilia is driven by immune responses to sequences intrinsic to the SARS nucleocapsid protein and is conserved in N proteins across group 2b coronaviruses.