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. 2020 May 20;eabc4776.
doi: 10.1126/science.abc4776. Online ahead of print.

SARS-CoV-2 Infection Protects Against Rechallenge in Rhesus Macaques

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Free PMC article

SARS-CoV-2 Infection Protects Against Rechallenge in Rhesus Macaques

Abishek Chandrashekar et al. Science. .
Free PMC article

Abstract

An understanding of protective immunity to SARS-CoV-2 is critical for vaccine and public health strategies aimed at ending the global COVID-19 pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against re-exposure. We developed a rhesus macaque model of SARS-CoV-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. Following initial viral clearance, animals were rechallenged with SARS-CoV-2 and showed 5 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with primary infection. Anamnestic immune responses following rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against re-exposure in nonhuman primates.

Figures

Fig. 1
Fig. 1. Viral loads in SARS-CoV-2 challenged rhesus macaques.
Rhesus macaques were inoculated by the intranasal and intratracheal route with 1.1 × 106 PFU (Group 1; N = 3), 1.1 × 105 PFU (Group 2; N = 3), or 1.1 × 104 PFU (Group 3; N = 3) SARS-CoV-2. (A) Log10 viral RNA copies/ml (limit 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following challenge. (B and C) Log10 viral RNA copies/swab (B) and log10 sgmRNA copies/swab (limit 50 copies/swab) (C) were assessed in nasal swabs (NS) at multiple timepoints following challenge. Red horizontal bars reflect median viral loads.
Fig. 2
Fig. 2. Immune responses in SARS-CoV-2 challenged rhesus macaques.
(A to D) Humoral immune responses were assessed following challenge by binding antibody ELISA (A), pseudovirus neutralization assays (B), live virus neutralization assays (C), and systems serology profiles (D) including antibody subclasses and effector functions to receptor binding domain (RBD), soluble spike (S) ectodomain, and nucleocapsid (N) proteins on day 35. Antibody-dependent complement deposition (ADCD), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent NK cell degranulation (NK CD107a) and cytokine secretion (NK MIP1β, NK IFNγ) are shown. (E and F) Cellular immune responses were also assessed following challenge by IFN-γ ELISPOT assays (E) and multiparameter intracellular cytokine staining assays (F) in response to pooled S peptides. Red and horizontal bars reflect mean responses.
Fig. 3
Fig. 3. SARS-CoV-2 induces acute viral interstitial pneumonia.
(A to F) H&E sections of fixed lung tissue from SARS-CoV-2 infected rhesus macaques 2 days following challenge showing interstitial edema and regional lung consolidation (A), intra-alveolar edema and infiltrates of neutrophils (B), bronchiolar epithelial sloughing and necrosis [(C) and (D)], bronchiolar epithelial syncytial cell formation (E), and hyaline membranes within alveolar septa (F). (G and H) IHC for SARS nucleocapsid showing virus infected cells within interstitial spaces including a viral syncytial cell within the lumen (G) and virus infected alveolar lining cells (H). (I) Inflammatory infiltrate showing multiple cells containing SARS-CoV-2 RNA by RNAscope in situ hybridization. (J to L) bronchial respiratory epithelium showing inflammation within the submucosa and transmigration of inflammatory cells into the ciliated columnar respiratory epithelium of a bronchus (J), SARS-CoV-2 RNA (K), and SARS nucleocapsid (L). Scale bars (A) = 200 microns; (C, I, K-L) = 100 micron; (G) = 50 micron; (B, D-F, J) = 20 microns, and (H) = 10 microns. H&E = hematoxylin and eosin; IHC = immunohistochemistry; RNAscope = SARS-CoV-2 RNA staining.
Fig. 4
Fig. 4. SARS-CoV-2 infects alveolar epithelial cells in rhesus macaques.
Cyclic immunofluorescence (CyCIF) staining of fixed lung tissue from SARS-CoV-2 infected rhesus macaques 2 days following challenge. (A) Whole slide image of a lung stained with Hoechst 33342 to visualize cell nuclei (greyscale); regions of nuclear consolidation (arrows), and foci of viral replication (box) are highlighted. (B) Higher magnification image of inset box in (A) showing staining for SARS nucleocapsid protein (SARS-N; green) and cell nuclei (grey scale). (C) Higher magnification image of inset box in (B) showing SARS-N (green) and cell nuclei (blue). (D) Bright-field IHC for SARS-N from corresponding lung region depicted in (C). (E to K) CyCIF staining for DNA (blue; all panels) and SARS-N [(E), (F), and (H-K); green], CD206 [(E) and (K); magenta], pan-cytokeratin (pan-CK) [(G) and (H); red], CD68 (I; yellow), or Iba-1 (J; greyscale) showing virus infected epithelial cells and macrophages near an infected epithelial cell. Scale bars (F-K) = 50 microns.
Fig. 5
Fig. 5. Viral loads following SARS-CoV-2 rechallenge in rhesus macaques.
On day 35 following initial infection (Fig. 1), rhesus macaques were rechallenged with SARS-CoV-2by the intranasal and intratracheal route with 1.1 × 106 PFU (Group 1; N = 3), 1.1 × 105 PFU (Group 2; N = 3), or 1.1 × 104 PFU (Group 3; N = 3). Three naïve animals were included as a positive control in the rechallenge experiment. (A) Log10 viral RNA copies/ml (limit 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following rechallenge. One of the naïve animals could not be lavaged. (B) Comparison of viral RNA in BAL following primary challenge and rechallenge. (C and E) Log10 viral RNA copies/ml (C) and log10 sgmRNA copies/swab (limit 50 copies/ml) (E) were assessed in nasal swabs (NS) at multiple timepoints following rechallenge. (D and F) Comparison of viral RNA (D) and sgmRNA (F) in NS following primary challenge and rechallenge. Red horizontal bars reflect median viral loads. P-values reflect two-sided Mann-Whitney tests.
Fig. 6
Fig. 6. Anamnestic immune responses following SARS-CoV-2 rechallenge in rhesus macaques.
Binding antibody ELISAs, pseudovirus neutralization assays, live virus neutralization assays, and IFN-γ ELISPOT assays are depicted prior to and 7 days following SARS-CoV-2 rechallenge. Red lines reflect mean responses. P-values reflect two-sided Mann-Whitney tests.

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