Immunobiology of COVID-19: Mechanistic and therapeutic insights from animal models

The distribution of the immune system throughout the body complicates in vitro assessments of coronavirus disease 2019(COVID-19)immunobiology,often resulting in a lack of reproducibility when extrapolated to the whole organism.Consequently,developing animal models is imperative for a comprehensive understanding of the pathology and immunology of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.This review summarizes current progress related to COVID-19 animal models,including non-human primates(NHPs),mice,and hamsters,with a focus on their roles in exploring the mechanisms of immunopathology,immune protection,and long-term effects of SARS-CoV-2 infection,as well as their application in immunoprevention and immunotherapy of SARS-CoV-2 infection.Differences among these animal models and their specific applications are also highlighted,as no single model can fully encapsulate all aspects of COVID-19.To effectively address the challenges posed by COVID-19,it is essential to select appropriate animal models that can accurately replicate both fatal and non-fatal infections with varying courses and severities.Optimizing animal model libraries and associated research tools is key to resolving the global COVID-19 pandemic,serving as a robust resource for future emerging infectious diseases.

Parasites may exit immunocompromised northern pig-tailed macaques (Macaca leonina)infected with SIVmac239

Parasites can increase infection rates and pathogenicity in immunocompromised human immunodeficiency virus （HIV） patients. However, in vitro studies and epidemiological investigations also suggest that parasites might escape immunocompromised hosts during HIV infection Due to the lack of direct evidence from animal experiments, the effects of immunocompromised hosts parasitic infections on remain unclear. Here we detected 14 different parasites in six northern pig-tailed macaques （NPMs） before or at the 50th week of simian immunodeficiency virus （SIV） infection by ELISA. The NPMs all carried parasites before viral injection. At the 50th week after viral injection, the individuals with negative results in parasitic detection （i.e., 08247 and 08287） were characterized as the Parasites Exit （PE） group, with the other individuals （i.e., 09203, 09211, 10205, and 10225） characterized as the Parasites Remain （PR） group. Compared with the PR group, the NPMs in the PE group showed higher viral loads, lower CD4＋ T cells counts, and lower CD4/CD8 rates. Additionally, the PE group had higher immune activation and immune exhaustion of both CD4~ and CD8~ T cells. Pathological observation showed greater injury to the liver, cecum, colon, spleen, and mesenteric lymph nodes in the PE group This study showed more seriously compromised immunity in the PE group, strongly indicating that parasites might exit an immunocompromised host.

Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques

As of June 2020, Coronavirus Disease 2019(COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years,making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection. To examine the effect of age on death, we established a SARSCoV-2 infection model in Chinese rhesus macaques(Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b^+ and CD8^+ cells in lungs at one-week post infection(wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b^+ cells, and persistent infiltration of CD8^+ cells in the lungs at 2 wpi. In addition,peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

Superior intestinal integrity and limited microbial translocation are associated with lower immune activation in SIVmac239-infected northern pig-tailed macaques(Macaca leonina)

Microbial translocation is a cause of systemic immune activation in HIV/SIV infection. In the present study, we found a lower CD8+ T cell activation level in Macaca leonina (northern pig-tailed macaques, NPMs) than in Macaca mulatta (Chinese rhesus macaques, ChRMs) during SIVmac239 infection. Furthermore, the levels of plasma LPS-binding protein and soluble CD14 in NPMs were lower than those in ChRMs. Compared with ChRMs, SIV-infected NPMs had lower Chiu scores, representing relatively normal intestinal mucosa. In addition, no obvious damage to the ileum or colon epithelial barrier was observed in either infected or uninfected NPMs, which differed to that found in ChRMs. Furthermore, no significant microbial translocation (Escherichia coli) was detected in the colon or ileum of infected or uninfected NPMs, which again differed to that observed in ChRMs. In conclusion, NPMs retained superior intestinal integrity and limited microbial translocation during SIV infection, which may contribute to their lower immune activation compared with ChRMs.

Single-nucleus transcriptomic profiling of multiple organs in a rhesus macaque model of SARS-CoV-2 infection

Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000single-nucleus transcriptomes of the lung, liver,kidney, and cerebral cortex in rhesus macaques(Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multiorgan dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019(COVID-19).Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway,which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy(an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection,which may facilitate the development of therapeutic interventions for COVID-19.

Northern pig-tailed macaques(Macaca leonina)infected with SARS-CoV-2 show rapid viral clearance and persistent immune response

Coronavirus disease 2019(COVID-19),which is caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2),has become an unprecedented global health emergency.At present,SARS-CoV-2-infected nonhuman primates are considered the gold standard animal model for COVID-19 research.Here,we showed that northern pig-tailed macaques(Macaca leonina,NPMs)supported SARS-CoV-2 replication.Furthermore,compared with rhesus macaques,NPMs showed rapid viral clearance in lung tissues,nose swabs,throat swabs,and rectal swabs,which may be due to higher expression of interferon(IFN)-αin lung tissue.However,the rapid viral clearance was not associated with good outcome.In the second week post infection,NPMs developed persistent or even more severe inflammation and body injury compared with rhesus macaques.These results suggest that viral clearance may have no relationship with COVID-19 progression and SARS-CoV-2-infected NPMs could be considered as a critically ill animal model in COVID-19 research.

Successful implementation of intestinal resection and anastomosis in non-human primates suggests the possibility of longitudinal intestinal research

DEAR EDITOR,Intestinal biopsy is a basic experimental method for studying pathological changes in the intestinal tract during human immunodeficiency virus(HIV)infection.In this study,jejunal resection and anastomosis were successfully performed in 12 Chinese rhesus macaques(Macaca mulatta).

Monkeypox,wild animals,and potential big problem

A new outbreak of monkeypox virus(MPV)is currently occurring in several regions of the world.Since the beginning of May 2022,3040 cases have been reported to the World Health Organization(WHO)globally.Outside of its usual“endemic”base in Africa,the virus has so far been detected in 47 countries,including in Europe,America,Oceania,and Asia,where monkeypox is uncommon or previously unreported.On 25 June 2022,the WHO Emergency Committee declared that the outbreak does not currently constitute a Public Health Emergency of International Concern,but unanimously acknowledged the emergency nature of the event and that controlling further spread will require a vigorous response.Recently,new cases of MPV infection have been confirmed in Korea,Singapore,and Taiwan,China.Thus,a better understanding of MPV and enhanced surveillance are essential for preventing disease recurrence.

Author Correction: Successful implementation of intestinal resection and anastomosis in non-human primates suggests the possibility of longitudinal intestinal research

After the publication of Wang et al.(2020),we realized that there were some inappropriate statements in the content.Hereby,we correct them and apologize for any confusion this may have caused.

Particulate matter exposure exacerbates susceptibility to SARS-CoV-2 infection in humanized ACE2 mice

The global outbreak of coronavirus disease 2019(COVID-19),which is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),as of 8 May 2021,has surpassed 150700000 infections and 3279000 deaths worldwide.Evidence indicates that SARS-CoV-2 RNA can be detected on particulate matter(PM),and COVID-19 cases are correlated with levels of air pollutants.However,the mechanisms of PM involvement in the spread of SARS-CoV-2 remain poorly understood.Here,we found that PM exposure increased the expression level of angiotensin-converting enzyme 2(ACE2)and transmembrane serine protease 2(TMPRSS2)in several epithelial cells and increased the adsorption of the SARS-CoV-2 spike protein.Instillation of PM in a hACE2 mouse model significantly increased the expression of ACE2 and Tmprss2 and viral replication in the lungs.Furthermore,PM exacerbated the pulmonary lesions caused by SARS-CoV-2 infection in the hACE2 mice.In conclusion,our study demonstrated that PM is an epidemiological factor of COVID-19,emphasizing the necessity of wearing anti-PM masks to cope with this global pandemic.

Zika virus infection induces glycometabolic disorder in northern pig-tailed macaques

Dear Editor,The Zika virus(ZIKV)was first identified in Africa in 1947 but remained relatively obscure for nearly 70 years.However,from 2014 to 2016,ZIKV was introduced into Brazil from the Pacific Islands,leading to its rapid dissemination throughout the Americas(Fauci and Morens,2016).This mosquito-borne virus is distinguished by its capacity to cause congenital defects,as evidenced by the microcephaly epidemic in Brazil,and has been declared a Public Health Emergency of International Concern(Gulland,2016).Yuan et al.(2017)discovered that a specific mutation in the prM protein of American ZIKV strains increased infectivity in human and mouse neural progenitor cells.Despite reports of varying rates of microcephaly at different sites in Brazil,evidence from surveillance and observational studies indicates multifactorial etiologies beyond viral mutations(Barbeito-Andrés et al.,2018).

Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2.Here we report that nelfinavir,an FDA approved drug for the treatment of HIV infection,is effective against SARS-CoV-2 and COVID-19.Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2(IC50=8.26μM),while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93μM(EC50).In comparison with vehicle-treated animals,rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals.At necropsy,nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude.A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center,which were randomized(1:1)to nelfinavir and control groups,showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days(9.0 vs.14.5 days,P=0.055)and the duration of fever time by 3.8 days(2.8 vs.6.6 days,P=0.014)in mild/moderate COVID-19 patients.The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients,together with its well-established good safety profile in almost all ages and during pregnancy,indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

Tissue distribution of SARS-CoV-2 in non-human primates after combined intratracheal and intranasal inoculation

Dear Editor,As of 5 July,2020,approximately 20,162,000 confirmed cases of coronavirus disease 2019(COVID-19),which is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),have been reported globally,including an estimated 740,000 deaths.Although the clinical manifestations of COVID-19 are heterogeneous,viral infection and pathological changes in the respiratory system,especially the lungs,still represent common characteristics of COVID-19.The nose,served as the entrance to the respiratory system,seems to produce absent or minimal symptoms of nasal congestion or rhinorrhea during SRAS-CoV-2 infection,while other viral rhinitis(i.e.,adenovirus,rhinovirus,influenza virus,etc.)are characterized by typical nasal symptoms,including nasal congestion,rhinorrhea,and sneezing.Recent studies suggest that,despite a lack of other nasal symptoms,anosmia is an important sign of early-stage COVID-19(Lechien et al.,2020).

Pro-inflammatory microenvironment and systemic accumulation of CXCR3+cell exacerbate lung pathology of old rhesus macaques infected with SARS-CoV-2

Understanding the pathological features of severe acute respiratory syn drome coronavirus 2(SARS-CoV-2)infect io n in an animal model is crucial for the treatment of coronavirus disease 2019(COVID-19).Here,we compared imnnunopathological changes in young and old rhesus macaques(RMs)before and after SARS-CoV-2 infection at the tissue level.Quantitative analysis of multiplex immunofluoresce nee staining images of formali n-fixed paraffi n-embedded(FFPE)sections showed that SARS-CoV-2 infectio n specifically induced elevated levels of apoptosis,autophagy,and nuclear factor kappa-B(NF-kB)activation of angiotensirv convert!ng enzyme 2(ACE2)+cells,and increased interferon a(IFN-a)-and interleukin 6(IL-6)-secreting cells and C-X-C motif chemokine receptor 3(CXCR3)+cells in lung tissue of old RMs.This pathological pattern,which may be related to the age-related pro-inflammatory microenvironment in both lungs and spleens,was significantly correlated with the systemic accumulation of CXCR3+cells in lungs,spleens,and peripheral blood.Furthermore,the ratio of CXCR3+to T-box protein expression in T cell(T-bet)+(CXCR3+/T-bet+ratio)in CD8+cells may be used as a predictor of severe COVID-19.These findings uncovered the impact of aging on the immunopathology of early SARS-CoV-2 infection and demonstrated the potential application of CXCR3+cells in predicting severe COVID-19.