SARS-CoV-2 infection protects against rechallenge in rhesus macaques

Abstract

An understanding of protective immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for vaccine and public health strategies aimed at ending the global coronavirus disease 2019 (COVID-19) pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against reexposure. 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. After the 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 after the primary infection. Anamnestic immune responses after rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against reexposure in nonhuman primates.

Fig. 1. Viral loads in SARS-CoV-2–challenged rhesus macaques.

Rhesus macaques were inoculated by the IN and IT routes 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) of SARS-CoV-2. (A) Log₁₀ viral RNA copies/ml (limit 50 copies/ml) were assessed in BAL at multiple time points after challenge. (B and C) Log₁₀ viral RNA copies/swab (B) and log₁₀ sgmRNA copies/swab (limit 50 copies/swab) (C) were assessed in NS at multiple time points after challenge. Red horizontal bars reflect median viral loads.

Fig. 2. Immune responses in SARS-CoV-2–challenged rhesus macaques.

(A to D) Humoral immune responses were assessed after 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 RBD, soluble S ectodomain, and N proteins on day 35. Antibody-dependent complement deposition, antibody-dependent cellular phagocytosis, antibody-dependent neutrophil phagocytosis, and NK CD107a and cytokine secretion (NK MIP1β, NK IFNγ) are shown. (E and F) Cellular immune responses were also assessed after challenge by IFNγ ELISPOT assays (E) and multiparameter intracellular cytokine-staining assays (F) in response to pooled S peptides. Red horizontal bars reflect mean responses.

Fig. 3. SARS-CoV-2 induces acute viral interstitial pneumonia.

(A to F) Hematoxylin and eosin–stained sections of fixed lung tissue from SARS-CoV-2–infected rhesus macaques 2 days after 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) Immunohistochemistry for SARS-N 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-N (L). Scale bars: (A), 200 μm; (C), (I), (K), and (L), 100 μm; (G), 50 μm; (B), (D), (E), (F), and (J), 20 μm; (H), 10 μm.

Fig. 4. SARS-CoV-2 infects alveolar epithelial cells in rhesus macaques.

Shown is CyCIF staining of fixed lung tissue from SARS-CoV-2–infected rhesus macaques 2 days after challenge. (A) Whole-slide image of a lung stained with Hoechst 33342 to visualize cell nuclei (grayscale); 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-N (green) and cell nuclei (grayscale). (C) Higher-magnification image of inset box in (B) showing SARS-N (green) and cell nuclei (blue). (D) Bright-field immunohistochemistry for SARS-N from corresponding lung region depicted in (C). (E to K) CyCIF staining for DNA (all panels, blue) and SARS-N [(E), (F), and (H) to (K), green], CD206 [(E) and (K), magenta], pan-CK [(G) and (H), red], CD68 [(I), yellow], or Iba-1 [(J), grayscale] showing virus-infected epithelial cells and macrophages near an infected epithelial cell. Scale bar for (F) to (K), 50 μm.

Fig. 5. Viral loads after SARS-CoV-2 rechallenge in rhesus macaques.

On day 35 after the initial infection (Fig. 1), rhesus macaques were rechallenged by the IN and IT routes 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) of SARS-CoV-2. 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 BAL at multiple time points after rechallenge. One of the naïve animals could not be lavaged. (B) Comparison of viral RNA in BAL after 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 NS at multiple time points after rechallenge. (D and F) Comparison of viral RNA (D) and sgmRNA (F) in NS after primary challenge and rechallenge. Red horizontal bars reflect median viral loads. P values reflect two-sided Mann-Whitney tests.

Fig. 6. Anamnestic immune responses after SARS-CoV-2 rechallenge in rhesus macaques.

Results of binding antibody ELISAs, pseudovirus neutralization assays, live virus neutralization assays, and IFNγ ELISPOT assays are depicted before and 7 days after SARS-CoV-2 rechallenge. Red lines reflect mean responses. P values reflect two-sided Mann-Whitney tests.