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. 2014 Aug;88(15):8597-614.
doi: 10.1128/JVI.00983-14. Epub 2014 May 21.

Effects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccine

Naoko Iwata-Yoshikawa  1 , Akihiko Uda  2 , Tadaki Suzuki  1 , Yasuko Tsunetsugu-Yokota  3 , Yuko Sato  1 , Shigeru Morikawa  2 , Masato Tashiro  4 , Tetsutaro Sata  1 , Hideki Hasegawa  1 , Noriyo Nagata  5
Affiliations

Effects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccine

Naoko Iwata-Yoshikawa et al. J Virol. 2014 Aug.

Abstract

Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b(+) cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants.

Importance: Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.

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Figures

FIG 1
FIG 1
Immunization with UV-V induces eosinophilic immune pathology in adult mice after SARS-CoV challenge. Adult female BALB/c mice were vaccinated with UV-V, UV-V with alum (UV-V+Alum), or vehicle (PBS with Alum, PBS+Alum) and subsequently challenged with 1,000 TCID50 of F-musX. (A) Body weight changes following the challenge inoculation (n = 5). Dead mice are marked with crosses. Error bars indicate standard deviations. Significant differences (P < 0.05, one-way ANOVA) between groups are marked with an asterisk. (B) Virus titers in the lungs and lung wash fluids on day 3 postchallenge (n = 3). The dashed line indicates the limit of detection (101.5 TCID50/ml). Error bars indicate standard deviation. Significant differences (P < 0.05, one-way ANOVA) between groups are marked with an asterisk. LW, lung wash fluid. (C) Neutralizing serum antibody titers against SARS-CoV on days 50, 29, and 1 before challenge (n = 11) and on days 3 and 10 after challenge (n = 5 to 6). Serum samples were 2-fold serially diluted beginning at 1:2. Error bars indicate standard deviations. Significant differences (P < 0.05, one-way ANOVA) between groups are marked with an asterisk. (D) Numbers of lymphocytes, macrophages, neutrophils, and eosinophils in lung sections (n = 3) on day 3 after challenge. Five 240-μm2 regions in the extrabronchioles of lung per mouse were examined at magnification ×40. Asterisks indicate P < 0.05 by the Bonferroni test. Error bars indicate standard deviations. (E) Representative images of lung sections from UV-V- and UV+Alum-immunized mice on day 10 postchallenge. Hematoxylin-and-eosin (magnification, ×10) and C.E.M. kit (inset; magnification, ×100) staining were used. Br, bronchi; *, blood vessel.
FIG 2
FIG 2
Histopathological findings in the lungs of dead mice after SARS-CoV challenge. Lungs were obtained for pathological examination (A, C, and E) and immunohistochemical analysis of SARS-CoV virus antigens (B, D, and F) from mice that died 5 days after challenge. Br, bronchi; *, blood vessel. Severe inflammatory infiltrates containing eosinophils were observed in the lungs of the UV-V-immunized mouse (A, inset). A few virus antigens were present in the bronchi (B). The UV-V+Alum-immunized mouse also showed eosinophilic inflammatory reactions, but no viral antigen-positive cells were present in the lungs (C, inset, and D). Congestion, hemorrhage, and pulmonary edema with mononuclear cell infiltration were observed in the mock-vaccinated mouse (PBS+Alum) (E, inset). Cells positive for viral antigen were seen throughout the lung (F). Hematoxylin-and-eosin (magnification, ×10) and C.M.E kit (inset; magnification, ×100) staining, a reliable and specific stain for eosinophils (A, C, and E), or immunohistochemical staining with an anti-SARS-CoV antibody (magnification, ×20) (B, D, and F) were used.
FIG 3
FIG 3
Reinfection with SARS-CoV in aged mice. Aged mice were infected with the HKU39849 isolate or mock vaccinated (no vaccination) and subsequently infected with 1,000 TCID50 of F-musX. (A) Mice were weighed daily after challenge. All mock-vaccinated mice died by day 5, but all reinfected mice survived. Dead mice are marked with crosses. Error bars indicate standard deviations. (B) Virus titers in the lungs and lung wash fluids 3 days after challenge (n = 3). The dashed line indicates the limit of detection (101.5 TCID50/ml). Error bars indicate standard deviations. Significant between-group differences (P < 0.05 by one-way ANOVA) are marked with an asterisk. LW, lung wash fluid. (C) Neutralizing serum antibody titers against SARS-CoV on days 0, 3, and 10 after challenge (n = 6 to 12). Serum samples were 2-fold serially diluted beginning at 1:2. Error bars indicate standard deviations. Significant between-group differences (P < 0.05 by one-way ANOVA) are marked with an asterisk. (D) Numbers of lymphocytes, macrophages, neutrophils, and eosinophils in lung sections (n = 3) 3 days after challenge. Five 240-μm2 regions in the extrabronchioles of each mouse lung were examined at magnification ×40. Asterisks indicate P < 0.05 by the Bonferroni test. Error bars indicate standard deviations. (E) Representative images of the lungs of SARS-CoV-reinfected mice. Br, bronchi; *, blood vessel. Lung samples taken 3 and 10 days after infection were sectioned and stained with hematoxylin and eosin (magnification, ×10) and the C.E.M. kit (inset; magnification, ×100).
FIG 4
FIG 4
Immunization with UV-V and TLR agonists inhibits excessive eosinophilic infiltration after SARS-CoV challenge. Adult female BALB/c mice were vaccinated with UV-V, UV-V with TLR agonists (UV-V+TLR), or vehicle (PBS) and subsequently challenged with 1,000 TCID50 of F-musX. Dead mice are marked with crosses. (A and B) Mice were weighed daily and monitored for morbidity (n = 6 to 7). (C) SARS-CoV titers in the lungs and lung wash fluids 3 days after intranasal challenge with SARS-CoV (n = 4). Significant differences (P < 0.05, one-way ANOVA) between groups are marked with an asterisk. The dashed line indicates the limit of detection (101.5 TCID50/ml). Error bars indicate standard deviations. LW, lung wash fluid. (D) SARS-CoV-specific neutralizing serum antibody titers 52, 10, and 0 days before challenge (n = 13 to 14) and 3 and 10 days after challenge (n = 6 to 7, respectively) with SARS-CoV. Serum samples were 2-fold serially diluted beginning at 1:2. Error bars indicate standard deviations. (E) Representative images of lung sections from mice immunized with UV-V, UV-V+LPS, or UV-V+TLR on days 3 and 10 after challenge with F-musX. Hematoxylin-and-eosin (magnification, ×10) and C.E.M. kit (inset magnification, ×100) staining was used. Br, bronchi; *, blood vessel. (F) Numbers of lymphocytes, macrophages, neutrophils, and eosinophils in the lung sections (n = 3). Five 240-μm2 regions in the extrabronchioles of lung per mouse were examined at magnification ×40. Asterisks indicate P < 0.05 by the Bonferroni test. Error bars indicate standard deviations.
FIG 5
FIG 5
Cytokine and chemokine protein concentrations in lung homogenates of mice immunized with UV-V and challenged with SARS-CoV. The concentrations of cytokines and chemokines in lung homogenates were determined on days 3 and 10 after challenge (n = 4). Asterisks indicate significant differences (P < 0.05, one-way ANOVA). Error bars indicate standard deviations.
FIG 6
FIG 6
Type I IFN gene expression in lung homogenates of mice immunized with UV-V and challenged with SARS-CoV. Type I IFN mRNA expression profiles in UV-V- and UV-V+TLR-immunized mice (A) or amounts of viral RNA present during infection (B) are shown. RNA was taken from the lungs of UV-V- and UV-V+TLR-immunized mice 1 day after challenge. Type I IFN mRNAs and SARS-CoV genome (nsp11 region) were measured by quantitative real-time RT-PCR. Results are expressed as log10 fold change from results for mock-vaccinated, challenged mice. *, P < 0.05. Error bars indicate standard deviations.
FIG 7
FIG 7
Immunization with UV-V or UV-V+TLR induces eosinophilic immune pathology in adult mice after long-term SARS-CoV challenge. Adult female BALB/c mice were vaccinated with UV-V or UV-V+TLR or mock vaccinated (PBS) and subsequently challenged with 1,000 TCID50 of F-musX. (A) Body weight changes following the challenge inoculation (n = 7). Dead mice are marked with crosses. Error bars indicate the standard deviations. (B) Titers of virus in the lungs and lung wash fluids on day 3 postchallenge (n = 4). The dashed line indicates the limit of detection (101.5 TCID50/ml). Error bars indicate standard deviations. Significant between-group differences (P < 0.05 by one-way ANOVA) are marked with an asterisk. LW, lung wash fluid. (C) Neutralizing serum antibody titers against SARS-CoV 1 day before challenge (n = 14) and 3 and 10 days after challenge (n = 7 each). Serum samples were 2-fold serially diluted beginning at 1:2. Error bars indicate standard deviations. Significant between-group differences (P < 0.05 by one-way ANOVA) are marked with an asterisk. (D) Numbers of lymphocytes, macrophages, neutrophils, and eosinophils in lung sections (n = 3). Five 240-μm2 regions in the extrabronchioles in the lungs of each mouse were examined at magnification ×40. Asterisks indicate P < 0.05 by the Bonferroni test. Error bars indicate standard deviations. (E) Representative images of lung sections from UV-V-immunized (left panel) and UV-V+TLR-immunized (right panel) mice 3 days after challenge. Hematoxylin-and-eosin (magnification, ×10) and C.E.M. kit (inset; magnification, ×100) staining was used. Br, bronchi; *, blood vessel.
FIG 8
FIG 8
Global gene expression profiles of mice immunized with UV-V after SARS-CoV challenge. An ANOVA was performed to assess differences among all groups. All genes with a greater than 2.0-fold change (P < 0.05) in expression, relative to the median of the unchallenged groups, are depicted. Each row represents the lungs of a group of mice (n = 3, mock immunization with PBS (PBS); n = 6, inoculation with HKU39849 isolate (HKU), UV-V (UVV) or UV-V+TLR (UVVTLR)). The heat map shows the relative levels of expression of 305 probes (242 genes), confirmed statistically by direct comparisons between the UV-V and UV-V+TLR groups. The heat map was generated using the software program GeneSpring GX 12.1. (A) Uncentered Pearson correlation was used as the distance metric with average linkage for unsupervised hierarchical clustering. In the heat map, red represents high expression, black represents median expression, and green represents low expression. The color scale bar at the bottom indicates the relative level of expression. The sidebar on the right indicates genes that are closely related to each other. (B) A gene interaction network including 39 genes was constructed from 242 genes connected by IPA software. The solid and dotted lines indicate direct and indirect interactions, respectively. Genes shown in red were upregulated, and those shown in green were downregulated, compared with the PBS group. The central node is IL-4, a key cytokine in inflammation associated with eosinophils. Network 1 was composed of genes associated with eosinophilia. Network 2 was composed of genes associated with “inflammation of the lungs.” The same network is shown for UV-V-immunized (upper panel) and UV-V+TLR-immunized (lower panel) mice.
FIG 9
FIG 9
A network of genes in mice immunized with UV-V after SARS-CoV challenge. A direct comparison of gene expression profiles in the lungs of UV-V- and UV-V+TLR-immunized mice is shown. The diagrams show the TLR3 and TLR4 signaling pathways. Genes shown in red were upregulated, and those in green were downregulated, compared with expression for the PBS group. Several genes downstream of TLR3 and TLR4 signaling were upregulated in UV-V+TLR-immunized mice (B) compared with expression in UV-V-immunized mice (A). We overlaid gene expression data on the formed network using Ingenuity Pathway Analysis software.
FIG 10
FIG 10
Pathway analysis of the gene-to-gene networks of TLR3, TLR4, and poly(I·C) in mice immunized with UV-V after SARS-CoV challenge. Direct comparison of gene expression profiles in CD11b+ cells isolated from the lungs of UV-V- and UV-V+TLR-immunized mice. (A and D) FACS analysis of enriched populations of CD11b+ lung cells in UV-V-immunized (A) or UV-V+TLR-immunized (D) mice. Cells were prepared as described in Materials and Methods. (B and E) Conventional Giemsa staining of cytospins from populations of CD11b+ lung cells in UV-V-immunized (B) or UV-V+TLR-immunized (E) mice (magnification, ×100). (C and F) Diagram showing the pathways of TLR3 and TLR4 signaling. Genes shown in red were upregulated, and those in green were downregulated. Several genes downstream of TLR3 and TLR4 signaling were upregulated in UV-V-immunized mice (C) compared with expression in UV-V+TLR-immunized mice (F). We overlaid gene expression data on the formed network by using Ingenuity Pathway Analysis software.
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