Cynomolgus Macaque Model for COVID-19 Delta Variant

Animals and study design

A total of 6 female Cambodian-origin cynomolgus macaques (Macaca fascicularis), aged 6–7 years, were selected by institutional veterinary experts based on their general health. Animals were housed in cages in the animal biosecurity level 3 laboratory in Korea National Primate Research Center (KNPRC) at Korea Research Institute of Bioscience and Biotechnology (KRIBB). Animals were anaesthetized with a combination of ketamine hydrochloride (10 mg/kg) and tiletamine/zolazepam (5 mg/kg) for viral challenge, swabs, and blood collection. All animals were inoculated with 10.5 ml virus (2.1×10⁶ 50% tissue culture infection doses/ml [TCID₅₀/ml]) through the mucous membranes, including the mouth, trachea, nose, and conjunctiva. Nasopharyngeal and oropharyngeal swabs were collected longitudinally at the indicated time points as shown in Fig. 1A. Swab samples in universal viral transport medium were centrifuged (1,600 g for 10 min) and filtered with 0.2-µm pore size syringe filters for virus quantification. A total of 3 animals were euthanized at days 3 and 21 after infection. Respiratory tissue samples were harvested for viral detection and microscopic analysis. Lung and bronchus tissue samples were homogenized using a Precellys homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) and centrifuged. All experimental protocols were approved by the KRIBB Institutional Animal Care and Use Committee (permit number: KRIBB-AEC-21289).

Figure 1
Scheme of experimental design and viral RNA and infectious virus in mucosal swabs and lung tissue samples. A total of 6 macaques were selected to investigate the characteristics of the SARS-CoV-2 Delta variant. (A) Baseline samples were collected on day 0 and all macaques were inoculated with SARS-CoV-2. Clinical signs, viral kinetics, and humoral and cellular immune responses to SARS-CoV-2 infection were observed at the indicated time points. (B) Viral RNA (copies/ml) and infectious virus (TCID₅₀/ml) were determined in nasal and throat swab samples and lung tissues, and recorded at the indicated dpi. Circles indicate individual data and bar values represent the mean ± SEM.

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SARS-CoV-2 virus

The SARS-CoV-2 virus (NCCP. 43390 for the GK clade [B.1.617.2 lineage]) were obtained from the National Culture Collection for Pathogens (Cheongju, Korea). The virus was propagated by inoculating VERO cells to generate viral stocks. TCID₅₀/ml was measured in VERO cells and calculated using the Reed and Muench method.

Virus identification and quantification

VERO cells inoculated with swab samples and supernatants of tissue homogenates were incubated for 3 days at 37°C for infectious virus quantification to calculate the TCID₅₀/ml values. To quantify viral RNA copies, RNA was extracted from the samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed with a primer/probe set to detect the ORF1b gene, as described in a previous study (9, 10, 13).

Histopathology and immunohistochemistry

For histological analysis, lung samples were fixed in 4% paraformaldehyde and embedded in paraffin, as described previously (8). Briefly, 4 to 5 µm sections were stained with hematoxylin and eosin and examined under a microscope. The lung sections were blindly examined and scored. The severity of lesions ranged from 0 to 4: no specific lesion, 0; alveolar wall thickening infiltrated with mononuclear cells, 1; infiltration of macrophages, mononuclear cells, and neutrophils in alveolar septa and alveolar lumina without vascular changes, 2; infiltration of inflammatory cells with vascular walls and pulmonary edema, 3; and hyaline membrane, 4 (8). For the distribution of lesions from 0 to 3: no specific lesion, 0; <30%, 1; 30%–50%, 2; and >50%, 3.

For detection of SARS-CoV-2 antigens, lung tissue sections were deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval. Tissue sections were pretreated with 0.3% hydrogen peroxide, washed twice in distilled water, and blocked with 4% BSA in TBS and 1 μg dextran for 30 min at room temperature. The slides were incubated overnight at 4°C with rabbit nucleocapsid monoclonal antibodies (Sino Biological, Beijing, China) diluted 1:10,000 in TBS containing goat serum, washed twice then incubated in blocking buffer with enzyme-conjugated secondary antibody at room temperature for 1 h. Finally, the sections were developed with a chromogen and counterstained with hematoxylin.

Blood and fluorescence activated cell sorting analysis

Blood was collected from the inguinal veins at the indicated time points (Fig. 1A) and hematological examination was conducted using an autohematology analyzer (Mindray BC-5000; Mindray, Shenzhen, China). Flow cytometric analysis was performed using a BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo v10.7.1 (BD Biosciences), as previously described (8). To exclude dead cells, blood was first stained with Fixable Viability Stain 575V (BD Biosciences) for 20 min at room temperature. For surface staining, cells were stained with the following antibodies for 30 min at 4°C: CD3 (Alexa Fluor 700; Invitrogen, Waltham, MA, USA), CD20 (APC/Cyanine7; Invitrogen), CD27 (PE/Cyanine7; Invitrogen), IgD (PE; BioLegend Inc., San Diego, CA, USA), IgM (FITC; SouthernBiotech, Birmingham, AL, USA), IgG (V450; BD Bioscience), CD4 (V500; Invitrogen), CD8 (V450; Invitrogen), CD95 (PE/Cyanine5; Invitrogen), and CD28 (ECD; Beckman Coulter, Brea, CA, USA). The cells were washed with permeabilization wash buffer and fixed with 1% paraformaldehyde. Data were acquired using an LSR Fortessa system (BD Biosciences) and analyzed using FlowJo v10.7.1 (BD Biosciences).

ELISA

The Delta variant-specific antibody in plasma was determined as previously described (8). Briefly, 96-well plates (Thermo Fisher Scientific, Waltham. MA, USA) were coated with 100 ng/well of delta variant spike (S1+S2) or receptor-binding domain (RBD) protein (all from Sino Biological) and incubated overnight at 4°C. The ELISA plates were blocked with 200 µl blocking buffer (0.05% Tween 20 and 10% FBS in PBS) for 1 h at room temperature. Diluted plasma was then added to the plate and incubated for 2 h at room temperature. After incubation, the plates were washed 6 times with 200 µl PBS-T and incubated with anti-monkey IgG and IgM-HRP conjugated antibodies (1:10,000; Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h at room temperature. After washing, the substrate tetramethylbenzidine was prepared and added to each well. After incubation, the development was stopped with 2N sulphuric acid and the absorbance at 450 nm was read using an ELISA plate reader (BioTek, Winooski, VT, USA).

Enzyme-linked immunospot (ELISPOT) assay

The membranes of high protein-binding PVDF 96-well plates (Millipore, Burlington, MA, USA) were coated with NHP-specific IFN-γ antibody (MabTech, Stockholm, Sweden) overnight at 4°C. Splenocytes (5×10⁵/well) were stimulated for 18 h with 2 µg/ml of S peptide pool consisting of PepTivator SARS-CoV-2 Prot_S (Miltenyi Biotec, Bergisch Gladbach, Germany). After incubation, the coated plate was washed once with PBS. The spleen cells were then seeded into the coated plate with 200 μl of CTL-Test™ B Medium (Cellular Technology Ltd. [CTL], Shaker Heights, OH, USA) and incubated for 4 h in a humidified incubator providing 5% carbon dioxide at 37°C. The wells were subsequently washed twice with 200 µl PBS and twice with 200 µl PBS-T. Plates were incubated with anti-human biotin-conjugated IFN- γ antibodies (1:1,000; MabTech) in PBS-T-F (0.05% Tween 20 and 1% FBS in PBS) at room temperature for 2 h. The plates were washed twice with 200 µl PBS-T, and 80 µl of streptavidin-AP (1:1,000; CTL) in PBS-T-F was added to the wells and was incubated at room temperature for 1 h. The wells were washed twice with 200 µl PBS-T and distilled water prior to addition of developer solution (CTL), followed by incubation for 15 min at room temperature. To stop the reaction, the wells were washed with tap water and left to dry completely. Spots were scanned and counted using the immunospot CTL reader (S6 Universal Analyzer; CTL). The results were presented as spot-forming cells per 10⁶ Spleen cells.

Statistical analysis

Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test using Prism version 9.3.1 (GraphPad Software, Inc., San Diego, CA, USA). All results are expressed as mean ± SEM. Statistical significance was defined as p<0.05.

Clinical signs and viral load kinetics of SARS-CoV-2 Delta variant infection

A total of 6 female cynomolgus macaques were challenged with SARS-CoV-2 Delta variant via multiple routes. Clinical signs of SARS-CoV-2 infection, such as changes in body temperature, body weight, and respiratory rate, were recorded. qRT-PCR was performed to assess viral kinetics in the nasal cavity, throat, and lungs throughout the experiment (Fig. 1B). None of the macaques showed notable changes in body weight or respiratory rate during infection, but the body temperature of 4 of the 6 macaques increased at 1 day post-infection (dpi) (Supplementary Fig. 1). As shown in Fig. 1B, viral loads in nasal swab samples were high at early time points (7.33±0.37 log₁₀ copies/ml) and viral RNA was detected up to 7 dpi. Levels of viral RNA in the throat peaked on 1 dpi (6.63±0.11 log₁₀copies/ml) and then gradually decreased over time; 7 dpi was detected in 2 subjects. Infectious virus in nasal swabs from all macaques was highest at 2 dpi (3.58±0.4 log₁₀TCID₅₀/ml) and sustained up to 5 dpi, whereas levels of viable virus in throat samples were peaked at 1 dpi (2.82±0.11 log₁₀TCID₅₀/ml) and remained up to 3 dpi. We found high viral loads in all lung lobes and bronchi of all macaques at 3 dpi, and viral particles were observed at 21 dpi as well. Infectious virus was detected in one to 2 of the 6 lobes from all subjects at 3 dpi, but could not be isolated from whole lobes and bronchi at 21 dpi. These results suggest that the SARS-CoV-2 Delta variant caused productive infection in the upper and lower respiratory tracts in the early phase of infection, and the infectious virus remained longer in the nasal cavity than in the throat.

Pathological changes

A total of 2 out of the 6 macaque groups (short- and long-term groups) were euthanized at the indicated time points, and necropsies were performed (Fig. 1A). Grossly, consolidations were observed in 2–3 lobes of the lung at 3 dpi, and the size of the lesions varied from lobe to lobe and individual to individual (Fig. 2A). At 21 dpi, consolidations were noted at the edges of the lung, and the size and distribution of lesions tended to decrease compared to those at 3 dpi. As shown in Fig. 2B, acute diffuse alveolar damage (DAD) was the major histologic lesion in the pulmonary tissue of infected monkeys. Neutrophils, alveolar macrophages, lymphocytes, fibrin, and eosinophilic edema were observed in the lumen of alveoli. The alveolar wall was dissociated due to edema and pulmonary necrosis, and monocytes and neutrophils were also observed infiltrating the wall. In one of 3 cases, a large number of neutrophils migrated around the alveolar lumen and wall. A hyaline membrane was seen in some damaged alveoli. Degeneration of vascular endothelial cells was observed, and neutrophils and monocytes aggregated around them. In the lesions except severe pulmonary edema, there was no alveolar lumen edema, but the alveolar wall was moderately thickened with infiltration of neutrophils and mononuclear leukocytes. One of 3 macaques showed pulmonary edema at 3 and 21 dpi. There was no significant difference in the severity and distribution of lesions between days 3 and 21 post-infection (Fig. 2D). The SARS-CoV-2 viral nucleocapsid protein antigen was detected in type 1 and 2 pneumocytes and alveolar macrophages in the lungs of macaques at 3 and 21 dpi (Fig. 2C). These results revealed that infection with the SARS-CoV-2 Delta variant could infect the lung, resulting in DAD, and could last for 21 dpi without alleviation of lung damage.

Figure 2
Histopathological changes in macaques during SARS-CoV-2 Delta variant infection. Macaques of the short-term (n=3) and long-term (n=3) groups were euthanized at 3 and 21 dpi, respectively. (A) Lungs from macaques presented local pulmonary consolidation (black arrow) at 3 and 21 dpi. (B) Histological changes were examined in the lung tissues at 3 and 21 dpi. The thickened alveolar walls and lumina were filled with immune cells. Scale bar, 400 µm; inlet, 50 µm. (C) Immunohistochemistry showing detection of SARS-CoV-2 antigens in the lungs. Scale bar, 40 µm. (D) Semi-quantitative analyses of the severity and distribution of lung lesions in macaques at 3 and 21 dpi. Data are presented as the scores of the 6 lung lobes of each animal per group.

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Cellular immune responses to SARS-CoV-2 Delta variant

Cellular immune responses and hematological changes induced by the SARS-CoV-2 Delta variant were assessed using flow cytometry and a hematological analyzer. As shown in Fig. 3A, neutrophils, an important component of innate immunity against early viral infection, significantly increased at 1 dpi and returned to normal levels at 2 dpi. All macaques exhibited transient lymphopenia at 1 or 2 dpi. We observed a significant increase in basophils and eosinophils at 5 dpi, which gradually decreased over time. No remarkable changes in white blood cells and monocytes were observed during the infection.

Figure 3
Cellular immune response in SARS-CoV-2 Delta variant infected cynomolgus macaques. (A) Hematologic features of SARS-CoV-2 infection in whole blood of all macaques following SARS-CoV-2 infection up to 21 days (n=6 at 0, 1, 2, 3 dpi; n=3 at 5, 7, 14, 21 dpi). (B) Immune cell dynamics in whole blood of macaques after SARS-CoV-2 infection up to 21 days (n=6 at 0, 1, 3 dpi; n=3 at 7, 14, 21 dpi). Circles indicate individual data measured from macaques and data are presented as mean±SEM. One-way ANOVA with Tukey’s multiple comparison test.
WBC, white blood cell.

^*p<0.05, ^**p<0.01.

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As shown in Supplementary Fig. 2, the population of immune cells (CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, CD20⁺ B cells, and NK cells) decreased in the acute phase of infection (1 and 2 dpi) and then gradually recovered thereafter. Notably, NK cells were significantly reduced at 1 dpi, maintained up to 3 dpi, and then increased to normal levels (Fig. 3B). More specifically, the number of CD4⁺T subsets (CD4⁺ naïve, central memory, effective memory) rapidly returned to baseline after 1 day of SARS-CoV-2 infection, and the CD8⁺ T subset (CD8⁺ naïve, central memory, effective memory) showed the same pattern. No statistically significant changes were observed in the CD20⁺ B subset (naïve, double-negative, and switched memory) cells. Among monocyte subtypes, changes in classic monocytes were not observed before and after SARS-CoV-2 inoculation, but the number of intermediate monocytes (3 of 6 macaques) increased and decreased at 3 and 21 dpi, respectively. The number of non-classic monocytes significantly increased at 7 dpi and then decreased to normal levels. Natural killer T cell and plasma blast frequencies decreased at early infection time points and returned to baseline thereafter. We did not observe significant changes in hematological parameters, including red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelet, mean platelet volume, platelet distribution width, and plateletcrit after the SARS-CoV-2 Delta variant challenge (Supplementary Fig. 3). SARS-CoV-2-specific T cell responses increased at 21 dpi compared to those at 3 dpi (3dpi: 2±1.1, 21dpi: 80.67±36) (Supplementary Fig. 4).

Antibody responses to SARS-CoV-2 Delta variant infection

Spike protein (comprising the S1 and S2 domains) of SARS-CoV-2 plays a major role in direct viral attachment to membrane receptors and its fusion, and entry into host cells (14). RBD of the S1 domain initiates viral infection by binding to angiotensin-converting enzyme 2 in host cells (14). To evaluate humoral immune responses to SARS-CoV-2 Delta variant infection, we measured the plasma levels of SARS-CoV-2 spike antigen (S1+S2) and RBD-specific IgM, IgG, and IgA by ELISA, following infection. As shown in Fig. 4, the levels of IgM specific for the spike and RBD were significantly increased at 14 dpi (1.57-fold and 2.53-fold, respectively). The switched immunoglobulins, including IgG and IgA, against SARS-CoV-2 Delta variant were gradually elevated compared to that before the virus challenge, and peaked at 14 dpi (S1+S2 specific IgG: 3.38-fold, RBD specific IgG: 9.4-fold, S1+S2 specific IgA: 3.47-fold, RBD specific IgA: 2.84-fold). These results suggest that all macaques seroconverted to spike and RBD proteins of the SARS-CoV-2 Delta variant.

Figure 4
Seroconversion in cynomolgus macaques infected with SARS-CoV-2 Delta variant. (A) SARS-CoV-2 spike (S1+S2) protein specific IgM, IgG, IgA antibodies were measured by ELISA at 0, 3, 7, 14, 21 dpi and fold change in IgM, IgG and IgA antibodies was determined by dividing with baseline (0 dpi). (B) SARS-CoV-2 RBD specific IgM, IgG, IgA antibodies were measured by ELISA at same time points and fold change in IgM, IgG, and IgA antibody was determined by dividing with baseline (0 dpi). n=6 at 0, 3 dpi; n=3 at 7, 14, 21 dpi. Circles indicate individual data measured from macaques and bars show the group mean±SEM. One-way ANOVA with Tukey’s multiple comparison test.
^*p<0.05, ^**p<0.001, ^***p<0.0001.

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