Overview of Nonhuman Primate Models of SARS-CoV-2

Abstract

COVID-19, the disease caused by the SARS-CoV-2 betacoronavirus, was declared a pandemic by the World Health Organization on March 11, 2020. Since then, SARS-CoV-2 has triggered a devastating global health and economic emergency. In response, a broad range of preclinical animal models have been used to identify effective therapies and vaccines. Current animal models do not express the full spectrum of human COVID-19 disease and pathology, with most exhibiting mild to moderate disease without mortality. NHPs are physiologically, genetically, and immunologically more closely related to humans than other animal species; thus, they provide a relevant model for SARS-CoV-2 investigations. This overview summarizes NHP models of SARS-CoV-2 and their role in vaccine and therapeutic development.

Coronaviruses are enveloped, single-stranded, positive-sense, RNA viruses in the subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales. There are 4 coronavirus genera, that is, Alphacoronavirus and Betacoronavirus, which infect mammals; and Gammacoronavirus and Deltacoronavirus, which primarily infect birds, with some able to infect mammals.¹³³ From these natural reservoirs, coronaviruses may infect other animals and humans. Human transmission typically requires an intermediate host.

Prior to the 2002 SARS-CoV epidemic, only 2 human coronaviruses (HCoVs) had been identified - an alphacoronavirus (HCoV-229E) transmitted from bats to humans by alpacas, and a betacoronavirus (HCoV-OC43) transmitted from rodents to humans by cattle.^16,18 In 2004, HCoV-NL63 (alphacoronavirus, bat reservoir) and in 2005, HCoV-HKU1 (betacoronavirus, rodent reservoir) were identified.^39,132 Together, these 4 HCoVs cause an estimated 15% to 30% of common cold cases in humans, but can cause severe infections in infants, juvenile children, and the elderly.^23,64 However, in 2002, a new betacoronavirus caused an epidemic that originated in China, resulting in 8,000 confirmed cases with a mortality rate of 9.6%. The virus was named SARS-CoV and was transmitted from bats to humans by a palm civet intermediate host.^59,63,83 Ten years later in June 2012, MERS-CoV, a novel betacoronavirus transmitted from bats to humans by dromedary camels, emerged in Saudi Arabia.^17,25 MERS-CoV was also responsible for a 2015 outbreak in South Korea. Although human-to-human transmission of MERS-CoV was limited, the virus resulted in more than 2,000 confirmed cases and a mortality rate of approximately 35%.⁹ Elderly people and those with comorbidities were more likely to develop severe disease.⁴³

Seven y later, in December 2019, another novel betacoronavirus named SARS-CoV-2, emerged in Wuhan City, Hubei Province China.^19,26 The animal reservoir responsible for transmission to humans has not been definitively identified but has been reported to be bats.^4,143 In February 2020, the World Health Organization named the disease associated with SARS-CoV-2, Corona virus disease 19 (COVID-19) and declared it a pandemic on March 11, 2020.^22,62,95 COVID-19 causes fever and pneumonia that can progress to acute respiratory distress syndrome (ARDS), multiple organ dysfunction and failure, coagulopathy, and death.³¹ Common gross findings in human autopsy specimens include lung consolidation, pulmonary edema, increased lung weight, pleurisy, white mucous and pink froth in airways, and hemorrhage. Histopathologic changes of human COVID-19 follow a timeline relative to the onset of symptoms.⁸⁶ During early infection, microvascular damage, thrombi, exudate formation, and intra-alveolar fibrin deposits occur. Epithelial changes can be present at all stages of disease, specifically diffuse alveolar damage (DAD), which includes hyaline membrane formation, epithelial denudation and pneumocyte hyperplasia. Finally, interstitial fibrosis develops about 3 wk after symptom onset.¹¹⁰ The clinical presentation of those infected with SARS-CoV-2 ranges from mild to severe to critical in 81%, 14%, and 5% of cases, respectively.^135,145 Similar to SARS-CoV and MERS-CoV, severe disease from SARS-CoV-2 is more likely in elderly individuals or in those with comorbidities.^12,72,127 In a New York City hospital study, deaths among hospital patients at the study endpoint were 3.3% or lower in patients in their 40s or younger, 4.8% among those in their 50s, 6.4% in their 60s, 12.6% in their 70s, and 25.9% in their 80s or above. Age related death rates reported by China, Italy and France are similar to the United States. Reported rates of asymptomatic infection range from 4% to 32%; however,¹²⁷ a systematic review concluded that true asymptomatic infection could be uncommon.^8,82,111,127

The contagiousness of an infectious disease is referred to as the R₀, or reproduction number, and indicates the average number of people who will contract a contagious disease from someone infected with that disease. SARS-CoV (R₀ of 1.5 to 1.9)^12,72,127 and MERS-CoV (R₀ of less than 1) have R₀ values lower than SARS-CoV-2 (initial R₀ was calculated to be 2.0 to 2.5, now revised upward to 5.7) and a lower fatality rate (2.3%).^84,97 As of December 26, 2020, 78,604,532 confirmed SARS-CoV-2 cases and 1,744,235 COVID-19 related deaths have been reported worldwide.¹²⁹ The global impact of COVID-19 has been catastrophic, with adverse effects on physical and mental health, an overwhelming need for health care resources, and increased poverty and economic insecurity.⁴⁷ Effective vaccines and therapeutics are key to controlling the SARS-CoV-2 pandemic. The success of these efforts depends in part on animal models that replicate human COVID-19 disease.^52,81,105

The ideal animal model for SARS-CoV-2 should be permissive to infection, have the same receptors for viral entry as in humans, and replicate the full spectrum of human COVID-19 disease and pathology.¹⁰⁹ Several publications review and compare common animal models for SARS-CoV-2 and conclude that current models simulate mild infection with full recovery, but not severe COVID-19 disease.^{13,31,52,78,81,100,105} Disease features not expressed in current animal models include ARDS, coagulopathy, systemic sequelae, and mortality.³¹

This review will focus on why NHPs provide a valuable model for SARS-CoV-2 research. Using NHPs has several drawbacks as compared with small animal models, including higher purchase cost, limited availability, higher housing cost, larger space requirement, and need for specialized staff. In addition, NHPs are outbred, leading to greater variation in results among individual animals, sometimes making data analysis and interpretation difficult.^40,68 The preexisting shortage of NHPs available for biomedical research, combined with the high demand for COVID-19 research and China’s ban on the sale, transport, and export of NHPs to curtail the spread of COVID-19 (instituted January 26, 2020) has affected NHP research globally.^{3,116,138,141} Nevertheless, the scientific benefits of using NHPs for SARS-CoV-2 research outweigh these drawbacks. NHPs are physiologically, genetically, and immunologically more closely related to humans than are small animals.⁶⁸ Furthermore, the main receptor for SAR-CoV-2 binding, angiotensin l converting enzyme 2 (ACE2), in catarrhines (apes, Asian monkeys, and African monkeys) is identical to the human ACE2 receptor.^24,74 Moreover, like most humans, macaques infected with SARS-CoV-2 develop mild to moderate respiratory disease. Thus, macaques offer a relevant model to study SARS-CoV-2 pathogenesis, therapeutics, vaccines, and the impact of age and other comorbidities on disease outcome.

NHP Models of SARS-CoV-2 Pathogenesis

African Green Monkeys (Chlorocebus aethiops, [AGM]).

Currently, 6 publications have used the AGM model for SARS-CoV-2.^{6,20,45,53,108,134} Table 1 summarizes the age, sex, and number of AGM used per study, the virus strain and dose, clinical disease outcome, pathology, and detection of viral RNA (vRNA) days post infection (dpi). Of 33 AGM infected with SARS-CoV-2, 31 reached study endpoints which range between 3 dpi to 57 dpi, but 2 16-y old AGM experienced acute respiratory distress syndrome (ARDS) requiring euthanasia.⁷ The majority (29 AGM) experienced mild disease, 2 experienced moderate, and 2 experienced severe disease. Eighteen AGM were infected via multiple mucosal routes (intranasal [IN], intratracheal [IT], conjunctival [CJ], oral), 9 via small particle aerosols, and 6 IN using a mucosal atomization device that is US FDA approved for intranasal delivery of drugs. Except for 2 studies that did not present seroconversion data, all NHP seroconverted by 14 dpi. Viral RNA (vRNA) was detected in mucosal swabs and bronchoalveolar lavage fluid (BALF) by 7 dpi. vRNA was detected until study endpoint (28 dpi) in pharyngeal and nasal swabs in 2 16-y old AGM with mild disease.⁶ In another report, infectious virus was shed from the respiratory and gastrointestinal tracts (GI) of 6 juvenile adult AGM from 2 to 7 dpi, with a recrudescence at 14 to 21 dpi and vRNA was detected until study endpoint (28 to 35 dpi).⁴⁵ Single-cell sequencing of lung tissue from 8 AGM identified pneumocytes as the likely dominant cell type supporting viral replication, although a high percentage of macrophages contained vRNA, likely due to phagocytosis.¹⁰⁸ Gross and histopathologic findings were dependent on the time of euthanasia after infection (Table 1). Gross lung lesions in AGM with mild to moderate disease ranged from no lesions to lung consolidation, hemorrhage, edema, and hilar and mediastinal lymphadenopathy. Histologically, interstitial pneumonia was the primary finding. In more severely affected AGM, hyaline membrane formation, type II pneumocyte hyperplasia, and alveolar damage was observed. One adult AGM infected by aerosolized virus developed adhesions between several lung lobes.⁵³ Six adult AGM infected IN with a mucosal atomization device developed fibrin in medium to small vessels without thrombus formation.²⁰

Table 1.

Summary of studies using African green monkey models of COVID-19.

Number, species,§, gender, age	Virus strain, inoculation route†, (number exposed)	Clinical signs, (dpi)*	Gross and histologic lung pathology#, (dpi* euthanized)	Viral detection‡, (dpi)*	(Ref)
4 AGM, 2 M/ 2F, 16 y	2019-nCoV/USA-WA1/2020, Aerosol (2), Combined IT, IN, PO, CJ (2)	4/4 ↑Temperature ↓ Appetite, 2/4 Acute respiratory distress syndrome	AGMs with ARDS: Edema, severe consolidation, bronchointerstitial pneumonia, DAD, 2/4 (28 or 25 dpi) No gross lesions, minimal interstitial inflammation	Mouth, throat, bronchus, rectum (3-7) Nose, pharynx (3-28)	6
6 AGM, Female, Adult	SARS-CoV-2/INMI1-58 Isolate/2020/Italy, IN with mucosal atomization device	6/6 ↓Appetite, 2/6 ↑Temperature (3)	3/6 (5 dpi) bronchointerstitial pneumonia, ulcerative tracheobronchitis, 3/6 (34 dpi) Moderate multifocal chronic interstitial pneumonia	6/6 Mouth (2)	20
				1/6 Nose (2-15)
				2/6 Rectum (28)
				6/6 BALF (3-7)
6 AGM	SARS-CoV-2/München-1.1/2020/929	6/6 ↓Tidal volume (7)	28 or 35 dpi: Multifocal interstitial infiltration	Nose, mouth, rectum:	45
Male	Aerosol (4)	3/6 ↑Temperature		first peak (2-7)
3.5 y	Combined IT, IN, PO, CJ (2)	3/6 ↓Temperature 5-7 dpi onwards		second peak (14-21)
				Conjunctiva (2-5)
3 AGM	2019-nCoV/USA-WA1/2020	3/3 ↑Respiratory sounds and effort	3/3 (8 dpi) Focal to diffuse red discoloration of lung lobes, alveolar fibrosis, type II pneumocyte hyperplasia, 1/3 Adhesions between lung lobes	Nose, throat (3)	53
M/F	Aerosol (3)	1/3 Cough		Rectal swab (≥ 7)
Adult (Sex ratio not listed)		1/3 Tachycardia
10 AGM	nCoV-WA1-2020	10/10 ↓Appetite (↓appetite in controls likely from repeated sedations)	Controls (3 dpi) no gross or histologic lesions	Nose, throat, rectum, BALF (1)	108
4M/ 4F	2/10 were controls and received killed virus	5/8 tachypnea	4/8 (3 dpi) ↑ mediastinal LN, subtle alveolar thickening
Adult	Combined IT, IN, PO, CJ	2/8 hunched posture, one coughed	4/8 (10 dpi) ↑ mediastinal LN, alveolar thickening, 2/4 interstitial pneumonia
6 AGM 2M/ 4F Adult	SARS-CoV-2/INMI1-58 Isolate/2020/Italy, Combined IT, IN	5/6 ↓ Appetite 5/6, 2/6 ↑Temperature	3/6 (5 dpi) and 3/6 not euthanized 3/3 Pulmonary consolidation, hyperemia 1/3 pneumonia, DAD 1/3 interstitial pneumonia	6/6 Nose, BALF (3) 3/6 Mouth (3) 3/6 Rectum (3-21)	134

§ AGM African green monkey, F female, M male, † IT intratracheal, IN intranasal, PO per oral, CJ conjunctival, * dpi days postinfection, # ARDS acute respiratory distress syndrome, DAD diffuse alveolar damage, LN lymph node, ‡ BALF bronchoalveolar lavage fluid.

Cytokine, chemokine, and CBC analyses in some SARS-CoV-2 infected AGM mirrored findings predictive of respiratory failure and mortality in humans. In humans, elevated IL6 signals impending respiratory failure.^11,46 IL6 upregulates acute phase fibrinogen synthesis, resulting in coagulation abnormalities that correlate with disease severity in humans.^14,89,144 The serum and BALF of all AGM (6/6) infected by combined IT and IN routes with an Italian SARS-CoV-2 isolate had elevated IL6, IL8, IL10, IL12, IP10, MCP1, IFN↓, and 4/6 had elevated fibrinogen. Necropsied AGM exhibited lung pathology, even though the clinical disease was mild.¹³⁴ In another study, 6/6 AGM had elevated APTT and fibrinogen.²⁰ The 2 aged AGM that developed ARDS had significant elevations of INF↖, IL4, IL6, IL8, IL13 and IL1↓ in plasma. Two additional AGM in the same study had mild elevations in these cytokines in association with mild disease.⁶ In contrast, most cytokines were undetectable at all timepoints in 6 juvenile AGM infected with a SARS-CoV-German isolate.⁴⁵ The CBC results of most SARS-CoV-2 infected AGM showed lymphopenia, thrombocytopenia, and neutrophilia, similar to that of human COVID-19 patients.^35,60

Both thoracic radiography and PET/CT imaging were used longitudinally to detect pneumonia. PET/CT was the most sensitive imaging modality, detecting lung inflammation in 3/6 AGM by 4 dpi and lymph node inflammation in 6/6 by 11 dpi, whereas thoracic radiography of the same animals detected only 1 AGM with lung abnormalities.⁴⁵ Radiographs detected lung involvement in the 2 aged AGM that developed ARDS, but not in 2 aged AGM with a mild clinical presentation.⁶ Further, thoracic radiography did not detect lung involvement in 6 adult AGM, thus failing to show the degree of gross lung pathology, including hemorrhage, seen at necropsy.¹³⁴ These findings are consistent with current practices for detecting COVID-19 lung disease in asymptomatic humans, as nearly 60% of asymptomatic COVID-19 patients have no radiographic abnormalities.^87,126

To validate newly developed SARS-CoV-2 diagnostic assays, 1 study prescreened 4 AGM, 4 cynomolgus macaques, and 4 rhesus macaques for prior SARS-CoV-2 exposure.⁵³ Unexpectedly, 1 AGM was RT-PCR positive for SARS-CoV-2 in a nasal swab, indicating prior exposure to SARS-CoV-2. Sequence analysis revealed that the virus was in a separate clade than the nCoV-WA1-2020 virus stock used in the laboratory, indicating community-acquired exposure. The viral isolate sequenced in this AGM was phylogenetically similar to viruses sequenced in the US (Arizona, Georgia, Virginia) and Greece and was likely the result of a transmission event during importation.⁵³ Preexposure to SARS-CoV-2 significantly impacted the virus load and immune response in this AGM as compared with the other NHP in the study and implies that NHP should be prescreened for SARS-CoV-2 prior to use on SARS-CoV-2 studies.⁵³

Cynomolgus Macaques (Maca fascicularis), [CM]).

To date, 6 publications have used a CM model of SARS-CoV-2 infection (see Table 2).^{51,53,58,67,92,96} These studies infected a combined total of 35 CM with SARS-CoV-2. Experimental endpoints varied, with 15 CM euthanized between 2 to 13 dpi, 4 CM between 14 to 19 dpi, and 3 CM at 28 dpi. The remaining 13 CM were not euthanized. Most were infected by combined mucosal routes (IN, IT, CJ, oral) and 6 CM were exposed by aerosol. An analysis of the clinical data reported for the 35 CM used in these 7 publications indicates that fever and weight loss were the most common clinical signs observed; however, 54% of CM exhibited no clinical signs. Seroconversion occurred by 14 dpi, and vRNA was detected by 2 dpi in most CM in nasal, throat, and rectal swabs. vRNA detection was delayed to 4 dpi in 2 aged CM.⁹² One adult CM shed virus in feces for 21 dpi.⁶⁷ At necropsy, the most frequent gross finding was focal to diffuse hemorrhage in the lungs, with hilar and mediastinal lymphadenopathy mentioned in one report.⁶⁷ Histologic lung findings included interstitial pneumonia, bronchopneumonia, endotheliitis, hemorrhage, consolidation, and necrosis. Adhesions were present between the pleura and thoracic wall in one adult CM.⁵³ A pulmonary vessel thrombus was found in the lungs of one 15 y-old CM.⁵¹

Table 2.

Summary of publications using cynomolgus macaque models of COVID-19.

Number, species,§, gender, age	Virus strain, inoculation route†, (number exposed)	Clinical signs, (dpi)*	Gross and histologic lung pathology#, (dpi* euthanized)	Viral detection‡, (dpi)*	(Ref)
3 CM, 2 F/ 1 M, 10-15 y	SARS-CoV-2 JP/TY/WK-521/2020, Combined IT, IN, PO	3/3 Fever 1-2 dpi, 1/3 10% weight loss, ↓ appetite, lymphopenia, neutrophilia	All euthanized 28 dpi 3/3 Focal hemorrhage, interstitial pneumonia, 1/3 Congestion, thrombus, bronchopneumonia	Nose, mouth, trachea conjunctiva, (1)	51
4 CM M/ F Adult (Sex ratio not listed)	2019-nCoV/USA-WA1/2020, Aerosol	4/4 Fever,↑respiratory sounds, 2/4 Tachypnea, ↑ respiratory effort, 3/ 4 Tachycardia	Euthanized 18 dpi, 4/4 multifocal red to gray discoloration of one or more lung lobes, 1/4 Adhesions between pleura and thoracic wall, 3/4 DAD	Nose, throat (3), Rectal swab (≥ 7)	53
8 CM 4M/ 4F, 3-6 y	SARS-CoV-2 Korean isolate Combined IT, IN, PO, CJ, IV	4/8 Decreased activity and fever 8/8 Lymphopenia	4/8 (3 dpi): all had acute interstitial pneumonia, endotheliitis	Nose, throat, rectum conjunctiva, (1)	58
6 CM 3 M/ 3 F 2-4 y	SARS-CoV-2/ Victoria/01/2020 IT, IN	None	Euthanized: 2 NHP at 4/5 dpi, 14/15 dpi and 18/19 dpi Mild to moderate pulmonary consolidation Interstitial pneumonia, DAD	Nose, throat (3), BALF (4-5), Rectum (1-9), 2/6 Whole blood (6)	96
6 CM 3 M/ 3 F, Adult	SARS-CoV-2 from CDC, Guangdong Province China, Combined, IT, IN, CJ	4/6 Fever 5/6 Weight loss	Pulmonary hemorrhage, ↑hilar and mediastinal lymphadenopathy, Interstitial pneumonia, diffuse hemorrhage and lung necrosis	Nose, throat (2), Rectum, feces (2), 1/6 Rectum, feces (2- 21)	67
CM 8F, Half, 4-5 y, Half, 15-20 y	SARS-CoV-2 German isolate Combined IT, IN	No clinical signs	2 of each age group euthanized at 4 dpi, All had interstitial pneumonia, DAD	Nose, throat: juvenilejuvenile (1-2), aged (4), 1/4 Rectum (14)	92

§ CM cynomolgus macaque, F female, M male, † intratracheal, IN intranasal, PO per oral, CJ conjunctival, IV intravenous, * dpi days postinfection, # DAD diffuse alveolar damage, ‡ BALF bronchoalveolar lavage fluid.

To date cytokine, chemokine, and CBC analyses have been limited in CM. However, 6 CM infected via the respiratory and conjunctival mucosa exhibited serum elevations in several cytokines, but not in IL6.⁶⁷ In another study, 8/8 CM developed lymphopenia.⁵⁸ One of 3 10–15 y-old CM had marked plasma elevations in IL6, MCP1 and IL10 at 1 dpi; IL4, IL13, IL17, and MIP1⟨ were elevated at 10 dpi in plasma.⁵¹

CT and thoracic radiography were used in 4 studies to determine if CM develop lung abnormalities typical of COVID-19 infected human patients, including an interstitial pattern, ground glass opacities (GGOs), and nodules.^51,53,67,96 In one study, thoracic radiographs of all CM and rhesus macaques (RM) at 12 dpi displayed an interstitial pattern, which was more severe in RM than CM.⁶⁷ In another study, thoracic radiographs detected pneumonia in one of 3 10-15 y-old CM.⁵⁶ A third study reported that 4 CM exhibited radiographic signs of lung opacity between 3-11 dpi.⁵³ CT imaging detected GGOs and peripheral lung consolidation in 2 CM at 18 dpi.⁹⁶

Rhesus macaques (Macaca mulatta, [RM]).

At present, 17 publications have used RM as a NHP model for SARS-CoV-2 pathology and reinfection (see Table 3, Table 4 and Table 5).^{2,6,10,27,28,34,53,58,67,79,93,96,101,106,107,140,142} Most of these studies have focused on model characterization. One publication performed transcriptomic and proteomic analysis on tissues from 5 RM used in another study to characterize the immune response to SARS-CoV-2.^93,106 Another paper examined the cellular events in SARS-CoV-2 infected AGM and RM.³⁴ Three reports investigated immune protection after viral reinfection.^10,28,101

Table 3.

Summary of publications using rhesus macaque models of COVID-19.

Number, species,§, gender, age	Virus strain, inoculation route†, (number exposed)	Clinical signs, (dpi)*	Gross and histologic lung pathology#, dpi* euthanized	Viral detection‡, (dpi)*	(Ref)
13 RM M/F 6-12 y (Sex ratio not listed)	2019-nCoV/USA-WA1-A12/2020 Combined IN, IT	Refer to Table 4 Reference 11	2 euthanized 2 and 4 dpi Lung consolidation, inflammation, endothelialitis, Microthrombi and fibrin in alveolar septae, endothelial hyperplasia narrowing vessel lumina, collagen I deposition in lung parenchyma, -vWF throughout lung	Refer to Table 4 Reference 11	2
4 RM 3 M/ 1 F 13-15 y	2019-nCoV/USA-WA1-A12/2020 Aerosol (2) Combined IT, IN, PO, CJ (2)	Mild transient change in temperature and SpO2- Appetite	Euthanized 27-28 dpi 3/ 4 grossly normal 1/ 4 focal pulmonary scar in right caudal lung lobe surrounded by acute hemorrhage 4/4 minimal to mild interstitial inflammation	Nose, throat, rectum (2-7) Shorter viral shedding from mucosal sites when compared with AGMs	6,34
5 RM 5M 3-5 y	SARS-CoV-2/WH-09/ human/ 2020/CHN CJ (2) IT (1) IG (2)	No clinical signs reported	1 of each route euthanized at 7 dpi CJ: mild, focal, interstitial pneumonia, minimal alveolar exudate IT: moderate, diffuse interstitial pneumonia, - alveolar exudate and inflammation GI: None	CJ /IT: nose/ throat (1-7) CJ: conjunctiva (1) IG none CJ - virus load in nasolacrimal and ocular tissues IT- virus in lung	27
4 RM M/ F Adult (Sex ratio not listed)	2019-nCoV/USA-WA1/2020 Aerosol	4/4 - respiratory sounds 3/ 4 Mild hypoxia, periocular erythema 1/4 increased respiratory effort	Euthanized 8 dpi Gross: Not described Histopathology: 3/3 DAD	Nose, throat (3) Rectal swab (3 7)	53
8 Chinese RM 4 M/ 4 F 3-6 y	SARS-CoV-2 Korean isolate Combined IT, IN, CJ, IV	3/ 4 -activity (data acquired on 4/8 RM with activity collars) 6/8 -temperature (1-3)	4 euthanized at 4 dpi Multifocal interstitial pneumonia, endotheliitis	Highest in respiratory swabs and lung tissue early in infection	58
14 RM	SARS-CoV-2 from CDC, Guangdong Province China Combined IT, IN, CJ	11/14 -temperature 11/14 weight loss at 10 dpi (5.88% to 28.57%)	Euthanized	Nose (2-14)	67
4 Juvenile			1 adult at 4, 7 and 12 dpi
6 Adult			1 Juvenile and 1 old at 14 dpi	Blood (2-10)
4 Old (Sex ratio not listed)			Interstitial pneumonia, diffuse hemorrhage and necrosis in lung, hilar and mesenteric lymphadenopathy	Prolonged viral shedding in feces
			More interstitial pattern on radiographs in old than juvenile RM
8 RM 4 M/ 4 F Adult	SARS-CoV-2 isolate nCoV-WA1–2020 Combined IT, IN, PO, CJ	8/8 piloerection, - appetite, hunched posture, tachypnea/ dyspnea, weigh loss 8/8-Temperature 1 dpi, then returned to normal	4 euthanized 3 dpi and 4 at 21 dpi Pulmonary edema, mild to moderate interstitial pneumonia, DAD	Serum (10)	79
6 RM 3 M/ 3 F 2-4 y	SARS-CoV-2/ Victoria/01/2020 IT, IN	None	Euthanized:	Nose and Throat (2)	96
			2 NHP at 4/5 dpi	BALF (4-5)
			2 NHP at 14/15 dpi	Rectum (1-9)
			2 NHP at 18/19 dpi	1/6 Whole blood (3)
			Gross: Mild to moderate consolidation
			Histopathology: Multifocal to coalescing interstitial pneumonia, DAD
16 RM 8 Juvenile	SARS-CoV-2 isolate USA-WA1/2020	4 used acutely (2 juvenile/ 2 old) euthanized 3 dpi: No fever or weight loss, -serum C-reactive protein and serum CO2 12 euthanized 14–17 dpi (6 juvenile, 6 old juvenile): Same as acute study, but majority lost weight	Acute 3 dpi:	Acute 3 dpi: Nose, mouth, BALF	106
4 M/ F	Combined		3/ 4 no gross abnormalities	(1-3)
8 Old	IT, IN, CJ		1/ 4 multifocal to coalescing red discoloration left lung lobes	14-17 dpi: 10/12 BALF (3)
2 M/ 4 F	** IN, IT via mucosal atomization device		Histopathology: multifocal mild to moderate interstitial pneumonia, fibrin deposition, edema, vasculitis, necrosis, consolidation	6/12 Nose (3-17)
			14-17 dpi group: grossly unremarkable, histopathology similar	2/12 Rectum (3-9) None in serum
5 RM 3 juvenile 3-5 y 2 Aged 15 y (Sex ratio not listed)	BetaCoV/Wuhan/IVDC-HB-01/2020 IT	4/5 weight loss, lethargy	1 juvenile and 1 aged euthanized 7 dpi Juvenile: interstitial pneumonia	Rectum (3-11)	140
			Aged: Diffuse, severe, interstitial pneumonia, more viral antigen in lung than juvenile RM	Respiratory tract (3)
8 Chinese RM 4 M 5 y 4 M >16 y	SARS-CoV-2/ strain 107/ Guangdong ChinaIT	Juvenile Adults: no clinical signs, - respiratory rate, -pCO2, fever 11 dpi Aged Adults: Weight loss 5-11 dpi, fever 7 dpi	2 euthanized at 7 dpi	Trachea (3)	107
			Both groups – abnormal blood exudation, congestion and inflammation around trachea. More severe pathology in juvenile than aged.
			6 euthanized at 15 dpi
			Juvenile - interstitial pneumonia, pulmonary fibrosis
			Aged – No interstitial pneumonia detected, but inflammation around trachea persisted
14 RM 14 M 8-12 mo	SARS-CoV-2-KMS1/2020IN	14/14 Fever, -appetite and activity Clinically normal by 10 dpi	Euthanized:	Throat (2 – 14)	142
			2 NHP at 3 and 5 dpi	Nose – peaked at 2 and 5 dpi, shed until 27 dpi
			3 NHP at 7 and 9 dpi	Rectum (9-27)
			2 NHP at 21 dpi	Blood (5)
			2 NHP not euthanized
			Gross:
			12/12 Red lesions in lungs
			1/2 Severe scattered pleural adhesions 5 dpi
			Histopathology:
			Patchy edema, mild to marked interstitial pneumonia, DAD

§ RM rhesus macaque, F female, M male, † IT intratracheal, IN intranasal, PO per oral, CJ conjunctival, IV intravenous, IG intragastric, * dpi days postinfection, # CM cynomolgus macaque, DAD diffuse alveolar damage, vWF von Willebrand factor, ‡ AGM African green monkey, BALF bronchoalveolar lavage fluid.

Table 5.

Summary of SARS-CoV-2 nonhuman primate rechallenge studies.

Number, species, § gender, age	Virus strain, inoculation route†, (number exposed)	Clinical signs, (dpi)*	Gross and histologic lung pathology#, dpi* euthanized	Viral detection‡, (dpi)*	(Ref)
13 RM 4 infection controls 9 rechallenged 6-12 y (Sex ratio not listed)	SARS-CoV-2 USA-WA1/2020 Combined IT, IN Rechallenged 35 dpi*	Primary infection: Mild ↓ appetite and responsiveness Neutralizing antibody titers significantly higher 14 d post challenge than 14 d after initial infection	2 euthanized 2 dpi post primary infection: Multifocal interstitial pneumonia, consolidation, edema, DAD 2 euthanized 4 d post primary challenge: Viral pneumonia and inflammation diminished	Initial challenge: Nose, BALF (1) Not detected in plasma At necropsy: vRNA in respiratory, lower GI tract, liver, kidney, hilar LN Rechallenge ↓ vRNA than primary challenge: 3/9 BALF (1) 4/9 sgRNA Nose (1)	10
7 RM 3 infection controls 4 challenged 28 d after primary infection 3-5 y (Sex ratio not listed)	SARS-CoV-2/ WH-09/ human/ 2020/CHN IT for primary and rechallenge	Primary challenge: 4/7 lost 200-400 g weight 6/7 reduced appetite No fever Rechallenge: Transient increase in temperature, not seen during primary infection	Primary challenge: 1 NHP euthanized 5 dpi and 7 dpi, Mild to moderate interstitial pneumonia, epithelial degeneration, fibrin deposition Rechallenge: 2 NHP euthanized 5 d after rechallenge had no pathology	Primary infection: Nose, Throat, Rectum (3) Rechallenge: No vRNA detected in nose, throat, or rectal swabs	28
8 RM 3 M/ 3 F 6-11 y (2 negative controls)	SARS-CoV-2/ WH-09/ human/ 2020/CHN IT	Primary challenge: 1/6 decreased appetite 2/6 experienced 7% to 8% weight loss by 14 dpi Rechallenge: 2/2 No clinical signs	Primary challenge: 2 NHP euthanized 3 dpi and 6 dpi Interstitial pneumonia, DAD #, thrombus in one NHP, began resolving by 5 dpi	Primary challenge:	101
				3/6 Nose (2-5)
				3/6 Throat (1-6)
				3/6 Rectum (2-4)
				Rechallenge:
				2/2 No vRNA detected

§ RM rhesus macaque, F female, M male, † IT intratracheal, IN intranasal, * dpi days post infection, # DAD diffuse alveolar damage, ‡ BALF bronchoalveolar lavage fluid, GI gastrointestinal, LN lymph node, vRNA viral ribonucleic acid, sgRNA subgenomic ribonucleic acid.

Table 4.

Summary of studies using other NHP models of COVID-19.

Number, species, gender§, age	Virus strain, inoculation route†, (number exposed)	Clinical signs, (dpi)*	Gross and histologic lung pathology#, (dpi* euthanized)	Viral detection‡, (dpi)*	(Ref)
6 Marmosets Callithrix jacchus 3M/ 3F Adult	SARS-CoV-2 from CDC, Guangdong Province China IN (6)	3/6 ↑ Temperature	2/6 (13 dpi) gross lesions in lung, liver, and spleen. ↑ type II pneumocytes, RBC and macrophages alveolar space, mild hemorrhage in spleen, swollen hepatocytes, active proliferation of germinal centers in spleen 4/6 not euthanized	Nose (2) – lower compared with RM and CM No vRNA in tissues at necropsy No nAb produced	67
6 Marmosets Callithrix jacchus Adult (Sex ratio not listed)	USA-WA1/2020IN, PO, CJ	No clinical signs	2/6 (3 dpi) and 4/6 (14 dpi) Mild pathology Interstitial pneumonitis, but not as prevalent as in baboons and RM	6/6 Nose (2-10)	106
				6/6 Mouth (2-8)
				6/6 Rectum (2-8)
				6/6 Blood (ranged between 2-8)
				approximately 2 logs lower vRNA in lungs at necropsy than baboons or RM
6 Baboons Papio hamadryas M/F Juvenile (Sex ratio not listed)	USA-WA1/2020 IN, PO, CJ	No clinical signs described	6/6 (14-16 dpi) Extensive pathology including, red discoloration of lungs, minimal to moderate interstitial pneumonia, expanded alveolar septa (3/6), fibrosis (2/6), type II pneumocyte hyperplasia (4/6), syncytial cells in alveolar lumen (6/6), bronchitis (6/6)	Similar to RM:	106
				Nose (3-12)
				BALF ‡ (3)
				Mouth (9)
				Unlike RM:
				RS (3-12) – long term persistent viral shedding compared with RM

§ F female, M male, † IT intratracheal, IN intranasal, PO per oral, CJ conjunctival, * dpi days postinfection, ‡ BALF bronchoalveolar lavage fluid, RM rhesus macaque, CM cynomolgus macaque

A total of 129 RM (16 juvenile [1.5 to 2 y], 91 adults [3 to 14 y], and 22 aged adults [>15 y]) were infected with varying strains of SARS-CoV-2; 82 of these NHP (13 juvenile, 51 adults, and 18 aged adults) were euthanized. Of the 129 RM in the 17 publications summarized, 43% of the RM were euthanized between 2 to 7 dpi, 51% between 8 to 19 dpi, and 4 aged RM (6%) at 27 to 28 dpi.^{2,6,10,27,28,34,53,58,67,79,93,96,101,106,107,140,142} The primary route of viral inoculation was a combined mucosal exposure (oral, IN, IT, CJ). Although some studies used only the IN or IT routes, 3 studies used aerosolized virus administered via the IN and IT routes, and one study inoculated via the intragastric (IG) route. Except for 2 RM inoculated by the IG route, all RM became infected. Neutralizing antibodies (nAb) to the viral spike (S) protein were detected as early as 3 dpi, but detection was dependent on sampling schedule. In one report, aged and adult RM developed higher antibody titers than did juvenile RM.⁶⁷ Another study detected antibodies in aged RM earlier (5 dpi) than juvenile RM (7 dpi).¹⁰⁷ vRNA in mucosal swabs (nasal, oropharyngeal, tracheal, rectal) and BALF was present within 1 to 3 dpi.¹⁰⁷ In one study, inoculation route impacted viral load.²⁷ RM infected by the CJ route had more virus in the nasolacrimal system, while RM infected by the IT route had higher virus loads in the lungs.²⁷ Another publication concluded that RM were superior to CM and marmosets for SARS-CoV-2 research based on increased inflammatory cytokine expression in serum and increased lung pathology.⁶⁷

The most frequent gross findings reported in RM necropsied between 2 to 12 dpi were lung consolidation, edema, hemorrhage, necrosis, and hilar and mediastinal lymphadenopathy.^{2,7,11,67,79,96,101,106,107} Histopathologic findings were consistent with multifocal, mild to moderate interstitial pneumonia.^{2,7,27,53,58,67,79,96,106,107,140,142} A range of vascular changes such as, endothelialitis, endothelial disruption, intimal proliferation, microthrombi in alveolar septae, and thrombus formation were observed.^2,58,101 Typical epithelial pathology included alveolar and terminal bronchiolar epithelial necrosis, hyaline membrane formation, and DAD.^{10,28,79,96,101,106,142} In summary, RM infected with SARS-CoV-2 display lung pathology comparable to infected humans, but none progress to ARDS.

Cytokine, chemokine, and CBC analyses were presented in most papers that used RM as the NHP model. Eleven of the 17 papers reported cytokine and chemokine data in varying detail.^{2,6,34,58,67,79,93,96,106,107,142} Modest changes in cytokines were noted in 2 aged RM, in contrast to the significant elevations observed in aged AGM.⁶ Transcriptomic and proteomic analyses detected a significant induction of genes associated with IFN signaling, neutrophil degranulation, innate immune pathways, and downregulation of collagen formation pathways.²⁹³ Marked enrichment of signatures associated with coagulation, thrombosis and vascular disease were detected in BALF, peripheral blood, and serum. Further, vWF (von Willebrand factor) was upregulated in blood and serum.² Proinflammatory cytokines were also elevated (for example, IL6, IL8, IL10, IL15, IL1Ra, MCP1, MCP↓, IFN⟨, IFN↖).^{2,34,67,79,106,142} One study did not detect elevations in IL6.⁶⁷ No statistical differences in cytokine concentrations were found between juvenile and aged Chinese RM at 7 dpi; however, at 14 dpi, cytokine concentrations were significantly higher in aged Chinese RM.¹⁰⁷ Nine of the 17 papers^{28,34,53,58,79,93,101,107,142} measured the neutrophil to lymphocyte ratio (NLR) and found that the NLR was elevated in the CBC of SARS-CoV-2 infected RM, which is consistent with 82% of COVID-19 infected humans.^{28,34,53,58,67,101,107,142} In contrast, the CBC results in a subset of studies showed leukopenia, lymphopenia, and neutropenia.^{10,79,101,106,140,142} One investigation³⁴ described a multiphasic immune response during the first 28 dpi, concluding that acute infection is characterized by inflammation and monocyte recruitment in the lung, followed by a gradual switch from a type 1 to a type 2 T-cell response, and a final phase that ends in either disease resolution or disease progression. Disease resolution is correlated with an increase in the antiinflammatory cytokine IL10 and a reduction in IL6. Disease progression is linked to upregulation of proinflammatory cytokines.³⁴

Thoracic radiography was the primary imaging modality employed, with CT used in 2 studies.^96,106 Typical findings on thoracic radiographs included GGOs, mild to moderate interstitial pneumonia, pulmonary infiltrates, and interstitial-to-alveolar patterns with soft tissue opacities.^{27,28,53,67,79,101,106,107,140} GGOs presented in one lung lobe immediately after infection and then progressed to other lobes. GGOs and interstitial pneumonia resolved by 12 dpi in one report and by 28 dpi in another.^28,140 Aged RM demonstrated a more extensive interstitial pattern on radiograph than did juvenile RM.⁶⁷ In addition, RM had more severe lung changes on radiographs than did CM.⁶⁷ Pneumonia was diagnosed in all RM by CT and was more severe in aged RM than in juvenile RM.¹⁴⁰ CT imaging of 2 juvenile RM at 18 dpi detected GGOs involving less than 25% of the lung.⁹⁶

Viral rechallenge experiments have been performed in RM to elucidate immune correlates of protection.^10,28,101 Results varied from partial to complete protection. Nine RM that were rechallenged by IT/IN 35 d after primary infection with the same viral strain, dose, and route had reduced vRNA in BALF and nasal swabs and exhibited minimal to no clinical signs.¹⁰ nAb titers after viral rechallenge were significantly higher than titers generated after primary infection. The authors concluded that protection was likely mediated by rapid immune control and not sterilizing immunity (situation where the immune system prevents the virus from replicating in the body).¹⁰ Conversely, in 2 other reports, viral rechallenge with the same viral strain, dose and route was completely protective.^28,101 In the first of these 2 publications, 4 RM that were rechallenged by the IT route at 28 dpi developed a transient fever that was not present during primary infection; however, thoracic radiographs were normal. During the 14 d follow up period, no vRNA was detected in nasopharyngeal or anal swabs, and RM developed higher antispike IgG and nAb titers than they developed after primary challenge.²⁸ In the second publication, after IT rechallenge, 2 RM remained clinically normal with no vRNA detected in nose, throat, or anal swabs.¹⁰¹ These results suggest primary infection protected from rechallenge.

Other NHP Models.

A limited number of pigtail macaques (Macaca nemestrina), hamadryas baboons (Papio hamadryas), olive baboons (Papio cynocephalus anubis), and common marmosets (Callithrix jacchus), have been used to investigate SARS-CoV-2 pathogenesis and/or the safety and immunogenicity of COVD-19 vaccine candidates (See Table 4 and Table 6).^{32,67,106,112,120} Common marmosets infected with SARS-CoV-2 by a combined IT/IN route developed fever (3 of 6) but not respiratory signs or weight loss.⁶⁷ Viral gRNA was detected in nasal swabs and blood, but not in lung tissues at necropsy.⁶⁷ In another study, SARS-CoV-2 infection was compared in baboons, marmosets, and RM.¹⁰⁶ Although RM exhibited moderate disease and baboons had extensive pathology with prolonged viral shedding; marmosets had mild disease. The apparent resistance of marmosets to SARS-CoV-2 infection may result from the 4 amino acid difference in their ACE2 receptor as compared with the human ACE2 receptor.⁶⁷ Both olive baboons and pigtail macaques (PTM) offer a useful alternative to RM and CM for SARS-CoV-2 vaccine studies.^32,112,120

Table 6.

Overview of nonhuman primate vaccine studies.

Vaccine/NHP species§	Summary NHP study of findings‡	Vaccine Name/ Human trial status/ Developer or manufacturer	(Ref)
Viral Vectors
LION/repRNA-CoV2S (nonreplicating alphavirus vector) PTM	Immunization of NHP as a prime only or prime/boost, produced limited T cell responses and antibody titers were comparable to titers in human convalescent serum. The vaccine induced TH1-biased antibody and T cell responses, which reduces risk of vaccine related antibody dependent disease enhancement.	HDT-301/ Phase I and II Approved HDT Bio / Gennova Biopharmaceuticals	32
Ad5-S-nb2 (nonreplicating adenovirus vector) RM (Chinese)	A single intramuscular dose elicited spike protein specific antibody and cell-mediated immune responses. Intranasal vaccination elicited systemic and pulmonary antibody responses, but weaker cell mediated responses. Macaques were protected against intratracheal SARS-CoV-2 challenge 30 d after a single vaccination by either route.	Ad5-nCoV/ Phase III CanSino Biologic/ Beijing Institute of Biotechnology/Petrovax	38
Sad23L-nCoV-S and Ad49L-nCoV-S (nonreplicating adenovirus vector)RM	RM were vaccinated with Sad23L-nCoV-S and boosted 4 wk later with Ad49L-nCoV-S. The vaccines were well tolerated with no clinical or pathologic changes. High antibody titers to the spike protein and receptor binding domain were elicited and were greater than convalescent COVID-19 patient sera.	Preclinical	69
Ad26.COV2.S (nonreplicating adenovirus vector)RM	A single immunization induced high neutralizing antibody titers and completely protected 5/6 RM from SARS-CoV-2 intratracheal and intranasal challenge. 1/6 RM had low levels of virus in nasal swabs. Neutralizing antibody and T cell responses did not expand after challenge, suggesting minimal to no virus replication after challenge.	Ad26.COV2-S/ Phase III Janssen Pharmaceutical Companies/ Beth Israel Deaconess Medical Center/ Emergent BioSolutions/ Catalent/ Biologic E/ Grand River Aseptic Manufacturing (GRAM)	75
YF-S0 (YF17D live-attenuated yellow fever vaccine vector)CM	CM were vaccinated at days 0 and 7 and challenged 3 wk later by combined intratracheal and intranasal routes. No adverse effects were observed. All CM developed high neutralizing antibody titers that reduced vRNA levels compared with infection controls, indicating YF-S0 vaccine was protective.	Preclinical Clinical trials planned for second quarter 2021 KU Leuven	98
ChAdOx1nCoV-19 (nonreplicating adenovirus vector) RM	RM vaccinated with one dose or a prime-boost at days 0 and 28 and challenged 28 d later by combined intratracheal, intranasal, conjunctival, and oral routes, exhibited no clinical signs or pathology. vRNA was significantly reduced in bronchoalveolar lavage fluid after challenge but not in nasal swabs. The vaccine will not prevent SARS-CoV-2 infection or transmission but could significantly reduce illness.	AZD1222/ Phase III University of Oxford/AstraZeneca	114,115
Inactivated Vaccines
PiCoVacc RM	RM were immunized intramuscularly at day 0, 7 and 14 with high or low dose vaccine. The vaccine was safe with no clinical signs or pathology. RM were challenged intratracheally 1 wk after the last immunization. The high dose vaccine provided complete protection; the low dose provided partial protection.	CoronaVac + alum/ Phase III Sinovac/ Instituto Butantan/ Bio Farma	41
BBIBP-CorV CM and RM	Prime-boost immunization induced high neutralizing antibody titers in CM and RM and protected against intratracheal challenge. The vaccine was safe.	BBIBP-CorV/ Phase III Beijing Institute of Biologic Products/ Sinopharm	123
DNA Vaccines
INO-4800 (plasmid vaccine with electroporation) RM (Chinese)	RM were vaccinated intradermally at wk 0 and 4 and challenged intratracheally and intranasally 4 wk after the last vaccination. vRNA was reduced in the lungs and nasal swabs of immunized macaques compared with controls.	INO-4800 / Phase I/II Inovio Pharmaceuticals/ Beijing Advaccine Biotechnology/ VGXI/ Richter-Helm BioLogics/ Ology Bioservices/ International Vaccine Institute/ Seoul National University Hospital/ Thermo Fisher Scientific	42
SARS-CoV-2 S protein RM	RM were vaccinated intramuscularly without adjuvant at wk 0 and 3 and challenged intratracheally and intranasally 3 wk after last vaccination. The vaccine elicited both cellular and humoral immune responses. Neutralizing antibody titers were comparable to convalescent COVID-19 patients. Following viral challenged, vaccinated RM had >3 log reductions in viral load in bronchoalveolar lavage fluid and nasal swabs.	Preclinical	139
RNA Vaccine
mRNA-1273 RM	RM were vaccinated at wk 0 and 4 with high or low dose mRNA-1273, then challenged 8 wk postvaccination. Viral load was significantly reduced in the bronchoalveolar lavage fluid and nasal swabs of vaccinated RM. At necropsy, 1-wk postchallenge, there was minimal inflammation or vRNA in the lungs of the RM receiving the high dose vaccine.	LNP-encapsulated mRNA (mRNA 1273)/Authorized Moderna/ NIAID/ Lonza/ Catalent/ Rovi/ Medidata/ BIOQUAL	15
BNT162b2	RM were vaccinated with high or low dose BNT162b2 at days 0 and 21. The neutralizing antibody titers produced were higher than convalescent COVID-19 patients. A strong TH1 response was also elicited. RM challenged 55 d after last vaccination had no vRNA in bronchoalveolar lavage fluid. vRNA was detected only at day 1 in nasal and throat swabs.	BNT162/ Authorized	118
RM		BioNTech/ Pfizer/ Fosun Pharma/ Rentschler Biopharma
Protein Subunit
NVX-CoV2373 CM Olive Baboons	CM were vaccinated with high or low dose NVX-CoV2373 with 50 µg Matrix-M at days 0 and 21. Vaccination produced higher antispike protein antibody titers than convalescent COVID-19 patient serum. After intratracheal and intranasal challenge, vaccinated CM had no sgRNA in bronchoalveolar lavage fluid or nasal swabs, except for one CM in the low dose vaccine group Vaccinated CM necropsied 7 d after challenge had little to no lung inflammation.	Full-length recombinant SARS COV-2 glycoprotein nanoparticle vaccine adjuvanted with Matrix M (NVX-CoV2373)/Phase III	44,112
	Baboons vaccinated with NXV-CoV2373 developed higher neutralizing antibody titers levels than convalescent COVID-19 patient serum.	Novavax/Emergent Biosolutions/ Praha Vaccines/ Biofabri/ Fujifilm Diosynth Biotechnologies/ FDB/ Serum Institute of India/ SK bioscience/ Takeda Pharmaceutical Company Limited/ AGC Biologics/ PolyPeptide Group/ Endo
SARS-CoV-2 S1-Fc fusion protein Macaques (species not specified)	CFA was used to prime 2 RM at the first immunization and AD11.10 for all booster injections. RM were vaccinated intramuscularly with 250 ug SARS-CoV-2 S1-FC on day 4, 9, 22, and 26. Both high titers of anti-S1 spike protein antibodies and neutralizing antibodies were induced.	Preclinical, Recombinant S1-Fc fusion protein, AnyGo Technology	91
RBD protein in aluminum hydroxideRM	RM were vaccinated intramuscularly at days 0 and 7 and challenged 3 wk after last vaccination. RM were necropsied 7 d post challenge. No detectable gRNA or sgRNA was present in the lungs of vaccinated RM. The vaccine reduced viral gRNA in throat swabs and anal swabs, but no sgRNA was detected. Lungs were normal grossly and histopathologically.	RBD (baculovirus production expressed in Sf9 cells), Phase I. West China Hospital, Sichuan University	136
RBD-12GS-153-50 PTM	Two PTM were primed with 250 µg of RBD-12GS-153-50 and boosted 4 wk later. This vaccine displays 60 SARS-CoV-2 spike RBDs and is expected to limit the emergence of viral escape mutants as it elicits antibodies to multiple distinct RBD epitopes. The vaccine induced nAb 10-fold higher than prefusion stabilized S protein vaccines, with a 5-fold lower vaccine dose.	Preclinical	120

§ PTM pigtail macaque, RM rhesus macaque, CM cynomolgus macaque. ‡ TH1 type 1 T helper cell, vRNA viral ribonucleic acid, sgRNA subgenomic RNA, dpc days post challenge, CFA complete Freund’s adjuvant, AD11.10 saponin based adjuvant, gRNA genomic ribonucleic acid.

SARS-CoV-2 Therapeutic Studies in NHP

Worldwide, numerous human clinical trials are testing the safety and efficacy of various SARS-CoV-2 therapies; however, only a few of these treatments have been tested in relevant SARS-CoV-2 animal models to determine preclinical efficacy. At this time, 14 published NHP studies have reported outcomes of antiinflammatory and antiviral therapies (see Table 7).^{21,29,48,54,56,66,71,88,94,104,122,124,131,146} RM are the primary NHP used for these studies, with one study using AGM and one using CM.^21,71

Table 7.

Summary of therapeutics for COVID-19 disease tested in nonhuman primates.

Therapeutic‡(Reference)	Number and NHP species§, virus strain, challenge route†	Experimental groups, route†, dpi*	Clinical signs, virus load dpi*	Imaging gross and histologic lung pathology#	Conclusion
Baricitinib (48)	8 RM 2019-nCoV/USA-WA1/2020 Combined IT, IN	Group 1: 4 RM received 4 mg Baricitinib daily for 8-9 d starting 2 dpi	All developed:↓ Activity, appetite and weight, altered respiratory rate and SpO2, hunched posture, shivering, paleness, agitation vRNA levels similar in all	CXR: 2/4 Controls GGOs and pulmonary infiltration, 2/4 normal 8/8 Euthanized 10-11 dpi Treated group: ↓ alveolar damage and interstitial pneumonia	Baricitinib was safe and well tolerated, reduced pathology and inflammation, but did not impact SARS-CoV-2 replication kinetics.
		Group 2: 4 RM infection controls
Dalbavancin (122)	6 RM SARS-CoV-2 strain was a clinical isolate Combined IT, IN	Group 1: 3 RM treated with loading dose of Dalbavancin 60 mg/kg IV at time of infection, Dalbavancin 30 mg/kg IV repeated at 4 dpi Group 2 3 RM Infection controls	None noted	CXR: Less severe pneumonia in treated compared with controls 6/6 Euthanized 7 dpi: Treated group had minimal to moderate interstitial pneumonia Controls had severe interstitial pneumonia significantly ↓vRNA in lungs of treated compared with controls	Dalbavancin significantly inhibited viral replication and histopathologic injuries.
HCQ, AZH (71)	31 CM BetaCoV/France/IDF/0372/2020 Combined IT, IN	Group 1: 5 CM - ↑dose HCQ starting 1 dpi	Mild clinical signs in controls, that is, coughing, sneezing, early lymphopenia	CT scans: GGOs in treatment and controls vRNA load kinetics similar in all groups Neither HCQ nor HCQ/AZTH had a significant effect on viral load in tissues. All had increased inflammatory cytokines.	HCQ had no antiviral activity nor clinical efficacy regardless of timing of treatment initiation. Treatment with HCQ is unlikely to have antiviral activity in respiratory compartments.
		Group 2: 4 CM- ↓ dose HCQ starting 1 dpi
		Group 3: 5 CM – ↑dose HCQ + AZTH starting 1 dpi
		Group 4: 4 CM – ↓ dose HCQ starting 5 dpi
		Group 5: 5 CM – ↑ dose 7 d before infection
		Group 6: 8 CM – Infection controls
HCQ (94)	20 RM nCoV-WA1-2020 Combined IT, IN, PO, CJ	5 RM HCQ 6.5 mg/kg PO before infection 5 RM HCQ 6.5 mg/kg PO at 12 h postinfection 10 vehicle controls	No difference in clinical signs between control and treatment groups All RM had ruffled fur, pale appearance, irregular or ↑ abdominal respiration	No significant difference between treated and controls	HCQ used at the standard dosing regimen for malaria prophylaxis and treatment had no beneficial effect on SARS-CoV-2 replication, shedding, disease progression or outcome.
Remdesivir (131)	12 RMnCoV-WA1-2020 Combined IT, IN, PO, CJ	6 RM treated with loading dose of 10 mg/kg Remdesivir, then daily maintenance dose of 5 mg/kg 6 RM were vehicle controls	1/6 in Remdesivir group developed dyspnea, all controls had dyspnea	Treated RM: CXR had ↓pulmonary infiltrates, ↓ vRNA in BALF by 12 h after first treatment. By 3 dpi no infectious virus in BALF but detected in 4/6 controls Euthanized 7 dpi: ↓ vRNA and lung damage in treated compared with controls	Treatment of RM with Remdesivir during early infection has a clear clinical benefit. Data support use of Remdesivir in COVID-19 patients to prevent progression to severe pneumonia.
Remdesivir (29)	Analyzed data from Williamson paper using a mathematical model to predict Remdesivir effect.			Model predicted Remdesivir will lengthen SARS-CoV-2 infection.	Predicted Remdesivir will slow viral decay, slowing infected cell death and lengthening SARS-CoV-2 infection period, questioning potential clinical benefit.
Ly-CoV555 nAb (54)	7 RMSARS-CoV-2 (USA-WA1/2020) Combined IT, IN	4 RM treated with different doses of LY-CoV555 nAb (1, 2.5, 15, or 50 mg/kg) IV one day before infection 3 RM controls, received IgG1 before infection	Minimal to none Treated RM had dose-related↓ gRNA and sgRNA in respiratory tract, maximal protection at doses ≥ 2.5 mg/kg	Euthanized 6 dpi: Mild lobar congestion and hyperemia across control and treated groups	Ly-CoV555 nAb can prevent and treat SARS-CoV-2 infection,↓ viral replication in upper airway, may ↓transmission efficiency. Currently in human clinical trial.
Beta-galactosidase prodrug SSK1 (66)	9 RM (1 juvenile, 1 adult and 1 aged in each Group) SARS-CoV-2 from CDC, Guangdong Province China Combined IT, IN, CJ	Group 1: 3 RM vehicle treated infection controls Group 2: 3 RM low dose (0.5 mg/kg) SSK1 Group 3: 3 RM high dose (2 mg/kg) SSK1 Treatment started 22 dpi for 7 d	SSK1 prevented weight loss Adult and aged vehicle controls lost 17% body weight	Euthanized 5 d after treatment stopped 2/3 controls: red lung lesions, variable degree of thickened alveolar septum, edema and hemorrhage SSK1 treated: Slight lung lesions in 1/6	SSK1 effectively treated COVID-19 pneumonia by decreasing inflammation, clinical signs and pneumonia. SSK1 is a potential treatment for SARS-CoV-2 hyperinflammation.
Catalase [n(CAT)](88)	7 RMSARSCov-2/KM 1/2010 (Likely a misprint, should be 2020)IN	Group 1: 2 RM, infection controlsGroup 2: 3 RM, 5 mg n(CAT) nebulized 2, 4, 6 dpiGroup 3: 2 RM, 5 mg/kg n(CAT) IV2, 4, 6 dpi	Treated groups lost less weight than controls.	All euthanized 7 dpi, except for 1 RM in Group 2 euthanized at 21 dpi.n(CAT) repressed viral replication, protected alveolar cells from oxidative injury	Catalase was not toxic in RM and may provide an effective therapeutic for COVID-19 and treatment of hyperinflammation in general.
Convalescent Serum (21)	10 AGM SARS-CoV-2/ INMI1-Isolate/ 2020/Italy Combined IT, IN	Group 1: 2 AGM, untreated controls Group 2: 4 AGM ↑ dose (HD) pooled convalescent sera from infected AGM Group 3: 4 AGM, ↓ dose (LD) pooled sera from infected AGM	HD: 1/ 4 ↓appetite LD: 2/4 ↓ appetite, 1/ 4 tachypnea Infection Controls: None Convalescent sera ↓ viral replication and shedding	Euthanized 5 dpi: All had multifocal pulmonary consolidation, hyperemia, hemorrhage, less severe in HD and LD groups compared with controls HD Group: normalized coagulation times, fibrinogen, platelet count and cytokines	Data mirror results of human studies supporting convalescent plasma as an effective treatment strategy and indicate need to use donors with high potency nAb against SARS-CoV-2 for maximal benefit early in disease stage.
CT-P59 nAb (56)	8 RM NMC-nCoV02, clinical isolate from a Korean patient Combined IT, IN, PO, CJ	Group 1: 2 RM, 45 mg/kg CT-P59 IV one day after viral challenge Group 2: 3 RM, 90 mg/kg CT-P59, one day after viral challenge Group 3: 3 RM, vehicle controls	No clinical signs in any RM on study CT-P59 rapidly reduced virus loads and eliminated infectious virus in both treatment groups compared with controls.	Euthanized 6 dpi: No infectious virus detected in lungs of either vehicle controls or CT-P59 treated RM	CT-P59 significantly reduced viral titer in SARS-CoV-2 infected NHP suggesting that human mAb CT-P59 is a candidate for treatment of COVID-19.
CB6 (LALA) nAb (104)	9 RM Viral strain not provided IT	Treatment Group 1: 3 RM, treated with 50 mg/kg CB6(LALA) IV 1 and 3 dpi Prophylactic Group 2: 3 RM, treated with 50 mg/kg CB6(LALA) IV one dose 1 d prior to viral challenge Control Group 3: 3 RM	Did not report any clinical signs CB6 (LALA) given prior to infection prevented infection When give after infection, ↓ viral load immediately, by 4 dpi viral titer ↓ βψ 3 λoγσ χoμπαρεδ ωιτη χoντρoλ γρoυπ	Euthanized 1 RM from each group 5 dpi Control – interstitial pneumonia, thrombus in pulmonary capillary Treatment/ Prophylactic – limited lung damage	CB6, a nAb isolated from a patient recovering from COVID-19, could be a potential therapeutic for COVID-19.
MW05/LALA nAb (124)	9 RM SARS-CoV-2 (WIV04) IT	Group 1: 3 RM, 1 dose 40 mg/kg IV MW05/LALA 1 dpc Group 2: 3 RM, 1 dose 20 mg/kg IV MW05/LALA IV 1 d before challenge Group 3: 3 RM, treated with hIgG1	No clinical signs MW05/LALA prevented infection and as a therapeutic, immediately reduced virus load, clearing virus by 3 dpi	Euthanized 1 RM from each group at 5, 6, 7 dpi Controls – GGOs on thoracic radiographs, interstitial pneumonia, lungs had multifocal hemorrhagic lesions Milder lung pathology in treatment and prophylactic groups	Data show prophylactic and therapeutic efficacy of MW05/LALA in RM and demonstrates the effectiveness of nAb for prophylactic and therapeutic treatment of COVID-19.
COV2-2196 and COV2-2381 nAb (146)	12 RM	Group 1: 4 RM, IV COV2-2196, 3 d before challenge Group 2: 4 RM, IV COV2-2381 3 d before challenge	Clinical signs not described ↑ sgRNA in bronchoalveolar lavage fluid and nasal swabs of isotype mAb controls	Not euthanized	COV2-2196 and 2381 nAbs protected RM from infection and may provide COVID-19 immunotherapy in humans.
	2019-nCoV/USA-WA1/2020 Combined IT, IN	Group 3: 4 RM Infection control, treated IV with control mAb 3 d before challenge	No sgRNA detected in the nAb treatment groups		Therapeutic nAb cocktails more likely to prevent viral escape mutations than single nAb.
REGN-COV2 (REGN10987 + REGN10933) (5)	36 RM USA-WA1/2020 Combined IT, IN	Group 1 – Prophylaxis: 6 RM 50 mg/kg REGN-COV2, 6 RM placebo, treated 3 d before viral challenge Group 2 – 4 RM high dose and 4 RM low REGN-COV2, 4 RM placebo, treated 3 d before challenge with high dose virus Group 3 – 4 RM high dose and 4 RM low REGN-COV2, given 1 dpi with high dose viral challenge, 4 RM placebo	Clinical signs not described REGN-COV2 accelerated viral clearance in nasal and oropharyngeal swabs when used prophylactically or as a treatment	Euthanized 5 dpi As a treatment, REGN-COV2 reduced the incidence and severity of interstitial pneumonia compared with placebo	REGN-COV2 used prophylactically can completely block establishment of viral infection and reduces viral load in the upper and lower respiratory tract. REGN-COV2 is currently in clinical trials.

‡ HCQ Hydroxychloroquine, AZH azithromycin, siRNA small interfering ribonucleic acid, nAb neutralizing antibody. § RM rhesus macaque, CM cynomolgus macaque, AGM African green monkey. † IT intratracheal, IN intranasal, PO per oral, CJ conjunctival, IM intramuscular, IV intravascular. * dpi days post infection. # GGOs ground glass opacities, CXR chest radiograph, vRNA viral ribonucleic acid, BALF bronchoalveolar lavage fluid, sgRNA subgenomic ribonucleic acid.

Antiinflammatory Drugs.

Severe COVID-19 disease is characterized by an excessive inflammatory response resulting in acute lung injury and acute respiratory distress syndrome (ARDS).⁷⁷ Antiinflammatory therapies tested in NHP include Baricitinib, the ↓-galactosidase prodrug SSK1, and catalase. Baricitinib was tested in 4 RM, starting 2 d after SARS-CoV-2 infection. Although baricitinib did not reduce clinical signs of infection, the drug did reduce lung pathology and inflammation, but did not impact viral replication.⁴⁸ Macrophage infiltration contributes to the hyperinflammatory state in severe COVID-19 patients. To ameliorate macrophage-induced lung damage, 6 RM were treated with SSK1 targeting macrophages expressing ↓-gal in response to immune stimulation. SSK1 significantly reduced macrophage infiltration in the lungs of infected macaques as compared with uninfected controls. Also, SSK1 reduced inflammatory cytokines and upregulated IL18 in the blood of an aged SARS-CoV-2 infected RM, as also occurs in COVID-19 patients during recovery.⁶⁶ In another report, 5 RM were treated with nanocapsules containing the antioxidant catalase to reduce reactive oxygen species (ROS) and associated inflammation. After SARS-CoV-2 infection, RM were treated with catalase either by nebulization or IV injection. Catalase prevented oxidative lung injury and reduced viral replication, presumably by regulating cytokine production in leukocytes.⁸⁸

Antiviral Drugs.

Antivirals tested in NHP are limited to remdesivir, hydroxychloroquine (HCQ), dalbavancin, and siRNA. Remdesivir is a nucleotide analogue prodrug with broad antiviral activity that inhibits replication of MERS-CoV and SARS-CoV in human airway epithelial cell cultures and in SARS-CoV infected mice.¹⁰² When tested in SARS-CoV-2 infected RM shortly after infection, remdesivir reduced radiographic and pathologic signs of pneumonia and virus titers in BALF within 12 h of the first dose. By 3 dpi infectious virus was not detectable in BALF, yet was still detectable in control animals. The authors proposed that remdesivir may prevent progression to severe pneumonia in COVID-19 patients.¹³¹ Conversely, mathematical modeling of these data questions the potential clinical benefit of remdesivir as the analysis predicts remdesivir will lengthen the period of SARS-CoV-2 infection by slowing viral decay and cell death.²⁹ Consistent with the mathematical model, remdesivir did not reduce overall mortality, initiation of ventilation, or duration of hospital stay in human clinical trials.¹³⁰

The efficacy of HCQ as both a treatment and prophylaxis for SARS-CoV-2 was investigated in 2 separate studies.^71,94 In the first study, 18 CM were treated with either a high or low dose of HCQ, 7 d before combined IT/IN SARS-CoV-2 infection or starting at 1 to 5 dpi. One cohort of CM were treated with a combination of HCQ and azithromycin starting 1 dpi. In the first study, HCQ had no antiviral activity or clinical efficacy regardless of when treatment was initiated.⁷¹ In the second study, 10 RM were gavaged with HCQ, either before or 12 h after SARS-CoV-2 infection based on the standard human dosing regimen for malaria prophylaxis and treatment. Consistent with the first study, HCQ had no beneficial effect on SARS-CoV-2 replication, shedding, disease progression, or outcome.⁹⁴ The outcomes of several human clinical trials agree with these findings.^90,130 HCQ given before exposure did not prevent infection or reduce COVID-19 patient mortality rate.^90,130

Another antiviral drug tested in the NHP model is Dalbavancin. This lipoglycopeptide antibiotic blocks binding of the SARS-CoV-2 S protein to the ACE2 receptor.¹²² Dalbavancin has a high safety margin and long plasma half-life (8 to 10 d) in humans and macaques (5 to 7 d).¹²² Treatment of 3 RM with Dalbavancin yielded encouraging results.¹²² The drug significantly inhibited viral replication and histopathologic lung injury.¹²²

Convalescent Plasma.

Transfusion of convalescent plasma (CP) has been used to treat viral infections for over 100 y. CP has effectively treated Ebola, influenza, SARS-CoV, and MERS-CoV.^70,99 The use of CP to treat COVID-19 patients has shown therapeutic benefit, even in severe cases.^57,103 Large scale clinical trials are underway to evaluate the safety and efficacy of COVID-19 CP treatment. To date, one report has used CP in SARS-CoV-2 infected NHP. In this study, 2 groups of 4 AGM were treated with either high dose or low dose pooled AGM CP IV on the day of viral challenge by the combined IT/IN route. High dose CP decreased viral replication and shedding, reduced lung pathology, and normalized coagulation time, fibrinogen, platelet count, and cytokines,²¹ mirroring results of human studies.^57,103

Monoclonal Antibodies.

Although COVID-19 CP has clinical use, drawbacks include difficulty of collection, variability of binding and nAb titers, potential contamination with infectious agents, and risk of transfusion reactions.⁷³ On the other hand, monoclonal antibodies (mAb) can be mass produced with better quality control and safety profiles. Six studies tested the efficacy of antispike mAb from the blood of convalescent COVID-19 patients to treat or prevent SARS-CoV-2 infection in NHP.^{5,54,56,104,124,146} The mAbs targeted the receptor binding domain (RBD) of the SARS-CoV-2 S protein, blocking viral binding and fusion to the ACE2 receptor. To prevent antibody dependent enhancement (ADE) observed with SARS-CoV infection, 2 papers introduced 2 leucine to alanine substitutions at residues 234 and 235 in the Fc portion of the mAb, referred to as the LALA mutation.^{7,50,65,104,113,121,124,125,137} Two studies used mAb cocktails to avoid the production of viral escape mutants.^5,146 All studies employed RM and administered mAb either prophylactically before viral challenge or as a treatment after viral challenge. Treatment with mAb prior to SARS-CoV-2 infection reduced viral load and/or prevented infection.^{5,54,56,104,124,146} In all cases, treatment with mAb during early infection reduced viral titer.^5,56,104,124 A mAb cocktail given IV 1 dpi with SARS-CoV-2 reduced the incidence or severity of interstitial pneumonia as compared with placebo controls.⁵

SARS-CoV-2 Vaccine studies in NHP

According to the December 26, 2020 World Health Organization report on COVID-19 vaccine candidates, 61 vaccines are in clinical development, encompassing 5 primary vaccine platforms (that is, inactivated virus, DNA, RNA, protein subunit, and viral vectors (replicating or nonreplicating)).¹²⁸ Most COVID-19 vaccines are designed to block viral binding to host cells by stimulating nAb responses to the trimeric SARS-CoV-2 S protein.⁴⁹ Host proteases cleave the S protein into an S1 and S2 subunit. The ACE2 receptor binding domain (RBD), located in the S1 subunit, binds to the ACE2 receptor on host cells.^49,143 The S2 subunit contains the viral fusion peptide responsible for cell entry.³⁰ Antibodies targeting the S1 RBD may block viral binding, while antibodies targeting S2 may block viral fusion and entry into host cells.^30,55

NHP are ideal models for evaluating vaccine immunogenicity, protection, and safety; they serve a pivotal role in the clinical translation of vaccines. Characteristics making NHP ideal for this purpose include being outbred, having innate immune responses more like humans than rodents, and a larger size that allows use of clinically relevant vaccine doses. Currently, 16 publications have used NHP to test SARS-CoV-2 vaccine candidates. Vaccine strategies tested include viral vectors (adenovirus, alphavirus, flavivirus), DNA, RNA, protein subunit and inactivated virus (See Table 6).^{15,32,38,41,42,44,69,75,91,98,112,114,118,123,136,139}

Viral Vector Vaccines.

Nearly half of the 17 SARS-CoV-2 NHP vaccine studies use a viral vector platform; viral vectors comprise 21% of the COVID-19 vaccines in human clinical trials.¹²⁸ The predominant viral vector tested (5 of 7 studies) in NHP are nonreplicating adenoviral vectors. Strategies tested include homologous and heterologous prime-boost or prime only. Ad5-S-nb2, a recombinant serotype 5 adenovirus currently in Phase III clinical trials carrying the S protein, elicited S-specific Ab and cell-mediated immune responses after a single IM or IN vaccination in Chinese RM.^38,76,128 In addition, all RM were protected after an IT SARS-CoV-2 challenge 30 d after a single IM or IN vaccination. nAb titers did not increase in 2 of 3 RM after challenge. The lack of an anamnestic response in these 2 RM suggests that the vaccine eliminated the virus and controlled replication.³⁸ Ad26.COV2.S, a serotype 26 based adenoviral vector expressing the full-length S protein protected macaques after a single dose. Both nonreplicating genomic RNA (gRNA) and replicating subgenomic RNA (sgRNA) were measured via qPCR. After a combined IN/IT challenge, the vaccine protected both the upper and lower respiratory tract (no sgRNA in BALF or nasal swabs) in all but one NHP, which had detectable sgRNA in BALF and nasal swabs.⁷⁵ This vaccine is in Phase III clinical trials.^76,128 Preexisting immunity to Ad26 in human populations is not expected to impair the efficacy of the Ad26 viral vector, based on prior experience with an Ad26-HIV vaccine in the US, Africa, and Asia.^1,3 ChAdOx1nCoV-19, a chimpanzee adenoviral vector using a prime-only or a prime-boost regimen significantly reduced viral loads in the BALF and lungs of RM after SARS-CoV-2 challenge. Although pneumonia was not observed in vaccinated NHP, viral shedding in nasal swabs was not different between vaccinated and control SARS-CoV-2-infected RM. Thus, ChAdOx1nCoV-19, currently in Phase III clinical trials (as AZD1222), may not prevent infection or transmission of SARS-CoV-2, but could significantly reduce illness.^76,115,128 In another report, RM were vaccinated IM using a heterologous prime-boost with 2 novel adenovirus vectors (Sad23L and Ad49L) carrying the full-length S protein.⁶⁹ Immunization induced high titers of S, RBD, and nAb; no clinical or pathologic effects were noted.⁶⁹ The RM were not challenged.⁶⁹

Alphavirus Vector Vaccines.

Two alphavirus-based viral vaccine vectors have been tested in NHP. The first vaccine is LION/repRNA-CoV2S, which is a nonreplicating alphavirus derived replicon RNA vaccine that encodes the SARS-CoV-2 S protein with Lipid InOrganic Nanoparticles (LION).^32,119 LION particles enhance vaccine stability, delivery, and immunogenicity. repRNA acts as its own adjuvant, generating a superior immune response after a single dose, compared with conventional mRNA, which typically requires multiple, 1,000-fold higher doses.^32,119 After an IM prime or prime-boost with LION/repRNA-CoV2S, PTM developed nAb titers comparable to convalescent COVID-19 patients. Both T_H1-biased antibody and T-cell responses were induced, without evidence of ADE. PTM was not challenged.³³ In addition, PTM developed nAb within 10 d of a prime only immunization with 250 µg LION/repRNA-CoV2S that effectively neutralized pseudovirus (SARS-CoV-2 Wuhan-Hu-1) by day 28 after immunization.³² Further, the PTM were found to have nAb titers similar to those found in convalescent human sera in an 80% plaque reduction neutralization test (PRINT₈₀).³² The vaccine, commercially known as HDT-301, has been approved for Phase I and II clinical trials.^76,128

The second vaccine, YF-S0, uses the YF17D live-attenuated yellow fever vaccine vector to express a noncleavable prefusion form of the SARS-CoV-2 S protein.⁹⁸ CM were vaccinated SC with one dose of YF-S0 and challenged 21 d later by the combined IT/IN route. Macaques were protected after viral challenge and no adverse clinical signs were observed.⁹⁸ Clinical trials are planned for the second quarter of 2021.⁶¹

Inactivated Virus Vaccines.

Inactivated virus vaccines have a long history of providing safe and effective protection. The first inactivated virus vaccine was influenza (1936), followed by polio (1955), and rabies (1980). Two inactivated SARS-CoV-2 vaccines have been tested in NHP.^80,85,117 The first, referred to as PiCoVacc, was used to immunize RM IM 3 times at days 0, 7, and 14 with either a high or medium dose. S-specific IgG and nAb were detected at week 2 with titers similar to convalescent COVID-19 patients.⁴¹ RM were challenged IT with SARS-CoV-2 one week after the third immunization. All control macaques had high copy numbers of gRNA (10^4-10⁶/mL) in the pharynx and lung tissues and developed severe interstitial pneumonia by 3 to 7 dpc. In contrast, vaccinated RM had mild histopathologic lesions at necropsy and no detectable virus in the respiratory tract of RM receiving high dose vaccination. RM receiving a medium vaccine dose had detectable virus, although viral loads were reduced by approximately 95%. No ADE developed. The second vaccine, BBIBP-CorV, was administered to CM and RM with a prime-boost strategy. The vaccine protected against IT challenge and was safe, with no virus or pneumonia detected in the lungs of vaccinated macaques 7 dpc. Both vaccines are in Phase III clinical trials.^76,128

DNA Vaccines.

DNA vaccines represent a third vaccine strategy being explored. Two DNA vaccines have been tested in macaques. The first, INO-4800, which encodes the SARS-CoV-2 S protein, was administered ID via electroporation to Chinese RM at 0 and 4 wks. After SARS-CoV-2 challenge (IT/IN route) at 4 wk after booster immunization, vaccinated macaques had lower viral loads in the lung and faster nasal clearance of virus.⁴² The second DNA vaccine tested encodes the full-length S protein and is referred to as SARS-CoV-2.S. RM were vaccinated IM, without adjuvant, at 0 and 3 wk and challenged with SARS-CoV-2 IT/IN 2 wk after the booster immunization. The immunization elicited nAb titers comparable to those found in the serum of convalescent humans. After viral challenge, sgRNA levels were significantly lower in BALF and nasal swabs as compared with sham controls.¹³⁹

mRNA Vaccines.

Two mRNA vaccines have recently been granted Emergency Use Authorization (EUA) due to the COVID-19 global public health emergency. EUA allows these vaccines to be used in certain people prior to the completion of safety and efficacy studies. On December 11, 2020, the U.S. FDA issued the first EUA for a COVID-19 vaccine.³⁷ The vaccine was BNT162, developed by Pfizer-BioNTech. Prior to approval for use in humans, the safety and efficacy of the vaccine, referred to as BNT162b2, was tested in RM. RM were vaccinated with BNT162b2 at 0 and 3 wk, followed by IT/IN SARS-CoV-2 challenge 55 d after the last immunization. Both high nAb titers and a strong T_H1 response were produced. After challenge, no vRNA was detected in BALF. vRNA detection was limited to 1 dpc in throat and nasal swabs.¹¹⁸ The second COVID-19 vaccine receiving U.S. FDA EUA is Moderna’s mRNA-1273 COVID-19 vaccine, approved on December 18, 2020.³⁶ During preclinical testing in RM, the vaccine induced antibody titers exceeding those in the serum of convalescent COVID-19 patients.¹⁵ mRNA-1273 produced significantly higher nAb titers than had been previously reported in NHP vaccinated with whole inactivated, DNA, or adenovirus vector vaccines, which all protected the lower airways of NHP after SARS-CoV-2 viral challenge.^{41,115,124,139} RM were immunized with either 10 or 100 µg of mRNA-1273 IM at 0 and 4 wk and challenged IT/IN with SARS-CoV-2 4 wk after the second vaccination. The vaccine-induced T_H1-biased CD4 T-cell responses, minimal T_H2 or CD8 T-cell responses, and no indication of ADE. sgRNA was undetectable in BALF by 2 dpc in 7 of 8 vaccinated RM in either the low or high-dose vaccine groups. No sgRNA was found in the nasal swabs of 8 of 8 RM receiving the 100-µg dose of the vaccine. Thus, mRNA-1273 provided rapid protection of the lower and upper airways with no lung pathology in RM.¹⁵

Protein Subunit Vaccines.

Protein subunit vaccines are the primary vaccine platform being tested in human clinical trials, making up 30% of all candidate COVID-19 vaccines. This vaccine technology is the basis of several currently approved human vaccines (Hepatitis B Virus and Human Papillomavirus).⁸⁵ The NVX-CoV2373 vaccine, currently in Phase III clinical trials, is a recombinant nanoparticle vaccine containing the prefusion, stabilized, full-length, wild-type SARS-CoV-2 S glycoprotein.^44,112 NVX-CoV2373 safety and immunogenicity was initially tested in baboons.¹¹² Baboons were vaccinated IM with NVX-CoV2373 with the adjuvant of saponin-based Matrix-M at 0 and 21 d. The vaccine generated a T_H1 dominant B- and T-cell response, SARS-CoV-2 nAb, and ACE2 receptor blocking Ab. In a separate experiment, the safety and efficacy of NVX-CoV2373 was tested in CM.⁴⁴ CM were immunized IM with NVX-CoV2373 with Matrix-M adjuvant at days 0 and 21 and then challenged IT/IN with SARS-CoV-2 on day 35. No detectable sgRNA was found in BALF or nasal swabs by 4 dpc. CM euthanized on day 42 had little to no lung inflammation as compared with placebo controls. These results indicate that NVX-CoV2373 has the potential to block viral replication and pulmonary disease. In another study, RM were immunized IM at days 0 and 7 with a protein subunit vaccine containing the SARS-CoV-2 RBD (residues 319 to 545 of the S protein) with the adjuvant aluminum hydroxide.¹³⁶ Immunization was followed by SARS-CoV-2 challenge on day 28, and RM were euthanized 7 d later. All vaccinated RM had nAb. Neither gRNA nor sgRNA were detectable by qPCR in lung tissue, throat, or anal swabs. In addition, vaccinated RM had no significant gross or microscopic pulmonary pathology, while placebo controls developed interstitial pneumonia. Based on these results, the vaccine is considered safe and effective and has entered Phase I clinical trials. In another study, 2 PTM were vaccinated with 250 µg of RBD-12GS-153 to 50 at 0 and 4 wk. The vaccine is a self-assembling nanoparticle that displays 60 SARS-CoV-2 spike RBDs. This vaccine is expected to limit the potential for emergence of escape mutants because it elicits antibodies to multiple distinct RBD epitopes. The vaccine-induced nAb titers in the PTM were 10-fold higher than with prefusion stabilized S protein vaccines, despite vaccination with a 5-fold lower dose.¹²⁰

Conclusion

NHP have been instrumental in SARS-CoV-2 disease pathogenesis, vaccine, and therapeutic studies. They are permissive to infection and are physiologically, genetically, and immunologically more closely related to humans than small animal models.⁶⁸ Further, the ACE2 receptor for SAR-CoV-2 binding in catarrhines (apes and Asian and African monkeys) is identical to the human ACE2 receptor.^24,74 Like most infected humans, macaques infected with SARS-CoV-2 develop mild to moderate respiratory disease with full recovery. However, disease features that are not completely expressed in current NHP models include ARDS, coagulopathy, systemic sequelae, and mortality.³¹

After SARS-CoV-2 infection, AGM and macaques exhibit disease parameters (Tables 1 through 3) like those reported in mild to moderate human infections. Clinical signs are mild and can include fever, weight loss, lethargy, and changes in respiratory rate or effort. Proinflammatory cytokines and chemokines are often upregulated, with a reversal in the lymphocyte to neutrophil ratio in the CBC.^{2,7,27,34,35,51,53,58,60,67,79,106,107,134,142} Most animals develop interstitial pneumonia, which can be detected on thoracic radiographs; however, PET/CT is a more sensitive imaging modality.^{6,27,45,51,53,67,79,96,101,106,107,134,140} Common gross and histopathologic findings include lung consolidation, edema, multifocal to diffuse hemorrhage, hilar/mediastinal lymphadenopathy, and interstitial to bronchopneumonia. Infrequent thrombus formation was observed, with no overt coagulopathy reported.^2,51,101 These findings depended on the duration of the infection at which the necropsies were performed. RM have been the most widely used NHP model for SARS-CoV-2 investigations, followed by CM and AGM. A comparison in disease susceptibility between RM and CM reported RM to be the most susceptible.⁶⁷ Baboons had a similar disease course after infection; however, marmosets were reported to be somewhat resistant to infection.^67,106,112 Advanced age is an important comorbidity for severe COVID-19 disease in humans. In one study, aged RM developed higher Ab titers and more interstitial pattern on radiographs than did juvenile RM.^67,107 However, 2 aged AGM developed ARDS and had to be euthanized prior to study endpoint; no other studies have reported ARDS in SARS-CoV-2 infected NHP.⁶

Therapeutics tested in NHPs include anti-inflammatory drugs, antivirals, convalescent plasma transfusion, and monoclonal antibodies (see Table 7). HCQ had no beneficial effect on viral replication, shedding, disease progression, or outcome.^71,94 Baricitinib reduced pathology and inflammation but did not impact viral replication.⁴⁸ Conversely, the anti-inflammatory Dalbavancin significantly reduced viral replication and histopathologic injuries.¹²² RM treated with the antiviral Remdesivir, had reduced viral replication and lung damage as compared with controls.¹³¹ Overall, nAb therapies effectively reduced viral replication, with some currently in clinical trials.^{5,54,56,104,124,146} Several vaccine platforms that have been tested in NHPs are now in clinical trials (see Table 6). RM were key to the successful preclinical testing of 2 mRNA vaccines (BioNTech/Pfizer and Moderna) that recently received U.S. FDA emergency use authorization for human use.^15,118

To summarize, studies in NHPs have accelerated our understanding of SARS-CoV-2 disease pathogenesis, facilitating the rapid development of several therapies and vaccines currently in human clinical trials. The NHP SARS-CoV-2 model will continue to be important in ending the COVID-19 pandemic.