Primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge - PubMed (original) (raw)

Primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge

Candice Clay et al. J Virol. 2012 Apr.

Abstract

Our knowledge regarding immune-protective and immunopathogenic events in severe acute respiratory syndrome coronavirus (SARS-CoV) infection is limited, and little is known about the dynamics of the immune response at the primary site of disease. Here, an African green monkey (AGM) model was used to elucidate immune mechanisms that facilitate viral clearance but may also contribute to persistent lung inflammation following SARS-CoV infection. During primary infection, SARS-CoV replicated in the AGM lung for up to 10 days. Interestingly, lung inflammation was more prevalent following viral clearance, as leukocyte numbers peaked at 14 days postinfection (dpi) and remained elevated at 28 dpi compared to those of mock-infected controls. Lung macrophages but not dendritic cells were rapidly activated, and both cell types had high activation marker expression at late infection time points. Lung proinflammatory cytokines were induced at 1 to 14 dpi, but most returned to baseline by 28 dpi except interleukin 12 (IL-12) and gamma interferon. In SARS-CoV homologous rechallenge studies, 11 of the 12 animals were free of replicating virus at day 5 after rechallenge. However, incidence and severity of lung inflammation was not reduced despite the limited viral replication upon rechallenge. Evaluating the role of antibodies in immune protection or potentiation revealed a progressive increase in anti-SARS-CoV antibodies in lung and serum that did not correlate temporally or spatially with enhanced viral replication. This study represents one of the first comprehensive analyses of lung immunity, including changes in leukocyte populations, lung-specific cytokines, and antibody responses following SARS-CoV rechallenge in AGMs.

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Figures

Fig 1

Fig 1

Experimental design for SARS-CoV challenge and rechallenge studies. African green monkeys were instilled with 1 × 107 PFU of SARS-CoV strain HKU-39849 intranasally (first inverted triangle) followed by sacrifice at 1, 3, 5, 10, and 14 days postinoculation. In rechallenge studies, animals were reinoculated intranasally with the same dose and strain of SARS-CoV 28 days after primary infection (second inverted triangle), and animals were sacrificed at days 5 or 28 after primary or secondary challenge. (For each study, n = 6 animals at the indicated time points, except at 1 dpi [n = 5 animals]).

Fig 2

Fig 2

Spatiotemporal analysis of SARS-CoV replication and antibody responses following primary and secondary challenge. (A to F) SARS-CoV replication was assessed in various respiratory tract tissues by plaque-forming assays. Shown in each graph are the virus levels of individual animals at the time of euthanasia for nasal swabs (A), pharyngeal swabs (B), as well as homogenized nasal turbinates (C), trachea (D), and portions of the right caudal lung lobe (proximal [E], distal [F]). Note that the data shown in panel A for B9292 (open gray triangle) at day 5 after rechallenge is not the necropsy time point for this animal but is included, as this was the only SARS-CoV plaque-positive sample recovered from any animal following rechallenge. (G) Anti-SARS-CoV S protein-specific IgG was measured in lung tissue by ELISA. The asterisk indicates that the mean titers are significantly different (P < 0.05). (H) SARS-CoV neutralizing antibodies were measured in the sera throughout infection, with the arrow marking the reinfection time point. Data are shown for animals that were followed until day 28 after rechallenge only (in gray), with AGM numbers indicated in the legend. The unique symbols representing each animal at the different time points are kept consistent in all of the graphs so as to enable tracking of virus and antibody in the distinct samples of each individual animal. Values are plotted on a log scale.

Fig 3

Fig 3

Representative histologic changes in the lung of AGM at specific times after SARS-CoV infection. H&E-stained sections from lung lesions at 1 day (A), 3 days (B), 5 days (C), 10 days (D), 14 days (E), and 28 days (F). Lung lesions from AGMs with SARS-CoV infection, 5 days after rechallenge (G) and 28 days after rechallenge (H). All photos were taken at 400× the original magnification.

Fig 4

Fig 4

Flow cytometric characterization and leukocyte quantitation of SARS-CoV-induced inflammatory lung infiltrates. (A) The number of total lung leukocytes was determined per gram of tissue from standardized collected lung samples in mock-infected animals and at specific time points after primary and secondary SARS-CoV infection. The unique symbols at each time point represent the same animals in which virus and antibody levels were reported in Fig. 2. (B and C) Average percent frequencies of CD3+ T cells (B) and CD14+CD11c+ aMϕs (C) of total lung leukocytes were determined for mock (open bars) and SARS-CoV (solid bars)-infected animals by flow cytometry. Asterisks indicate that values are significantly different from those of mock-infected controls (P < 0.05).

Fig 5

Fig 5

Cytokine and chemokine profile in the lung during SARS-CoV primary and secondary infection. Protein levels of cytokines and chemokines were measured in lung tissue homogenates across the primary infection time course (1, 3, 5, 10, 14, 28 dpi) and at days 5 and 28 after rechallenge with bead-based arrays. The fold-induction of average protein levels over those of mock-infected controls is represented in a heat map, with black representing no change, red indicating a 4-fold induction, and green indicating a 4-fold reduction in the average cytokine levels (n = 6 animals for each time point, except at 1 dpi [n = 5 animals]). Average cytokine and chemokine levels (pg/ml) ± standard errors of the means (SEM) at each time point are given in Table 5.

Fig 6

Fig 6

Expression of activation markers on lung Mϕs and DCs in primary and secondary SARS-CoV challenge. Mϕs (CD14+) and DCs (CD14−CD11c+) isolated from the lungs of SARS-CoV-infected and mock-infected animals were evaluated by flow cytometry for activation marker CD86, antigen-presenting molecule HLA-DR, and SARS-CoV receptor CD209/DC-SIGN. (A) Representative FACS plots are shown for Mϕs and DCs from mock- and SARS-CoV-infected animals (14 dpi), depicting the gates used for evaluation of these cell surface antigens. The percent frequencies of receptor-positive cells of total Mϕs or DCs are indicated in each plot. (B to D) Flow cytometric results are summarized for activated Mϕs (solid bars) and DCs (open bars) during primary and secondary SARS-CoV infection. Percent frequencies of CD86 (B), HLA-DR high (C), and CD209/DC-SIGN (D) of total Mϕs or DCs are plotted. Asterisks indicate that values are significantly different from those of mock-infected controls for that particular cell type (P < 0.05).

Fig 7

Fig 7

Impact of SARS-CoV on lymphocyte numbers and activation in the lung-draining lymph node. (A) The number of lymph node cells was determined per gram of tracheobronchial lymph node tissue. Lymph node cell numbers at necropsy time points after primary and secondary infection are graphed with the same symbols for individual animals as those used in Fig. 2 and 4. (B and C) The relative frequency of lymph node T cells (CD3+) and mDCs (CD14−CD11c+) for mock (open bars) and SARS-CoV (closed bars)-infected animals was determined by flow cytometry. (D to F) mDCs and lymph node T cells were further characterized for expression of activation markers, including HLA-DR (D), proliferation marker Ki67 (E), and Granzyme B (F). Asterisks indicate that values are significantly different from those of mock-infected controls (P < 0.05).

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