Whether CD147 serves as a functional receptor for SARS-CoV-2 infection is widely debated in the field of COVID-19 research. In this study, we generated two novel knock-in mouse models in the NSG background for: 1) hACE2, the putative receptor for SARS-CoV-2 entry (hACE2KI-NSG), and for 2) CD147, the potential accessory or co-receptor for coronavirus entry (hCD147KI-NSG). Next, we characterized the hCD147KI-NSG mice by flow cytometry and IHC and confirmed physiological distribution of the hCD147 protein across a variety of tissues in hCD147KI-NSG mice. Finally, we compared the ability of live SARS-CoV-2 virus to infect both knock-in models and observed significant amounts of viral RNA in lung tissues by sm-FISH and qRT-PCR.
Key to the development of successful and effective vaccines to SARS-CoV-2 infections and treatments for COVID-19 patients is the understanding SARS-CoV-2 infectivity and pathogenesis. The fundamental mechanism underlying SARS-CoV-2 entry remains poorly understood. Previous studies show that the spike proteins of SARS-CoV [4, 44] and SARS-CoV-2 [45–49] bind to hACE2, a well-recognized, functional receptor that mediates viral entry. A hACE2 transgenic (hACE2Tg) mouse model is being widely used [42, 43, 50, 51], which is clearly invaluable but with some limits (e.g., low expression of hACE2 in human lung, heart, and immune cells). Other models for studying SARS-CoV-2 infection in mice are currently being optimized, including mouse-adapted virus derivations [52, 53], immunocompromised or obese mice that lack interferon receptors , or utilize cats, ferrets, and hamsters [54, 55].
However, while these models have recapitulated some aspects of the COVID-19 disease course, such as lung inflammation , cytokine storm , viral neuroinvasion , and impaired lung function , a majority of them cannot fully explain other aspects of COVID-19 disease, such as increased thrombosis risk, increased risk for severe COVID-19 disease in diabetic patients , associations between predisposing risk factors, such as stroke and immunosuppression, and clinical sequelae of COVID-19 [61–63].
Coronaviruses are known to have a high diversity of entry receptors, which includes the newly proposed human CD147 (hCD147) as a receptor for SARS-CoV-2. CD147 is a transmembrane glycoprotein with multiple functions in normal lung, immune cells, and diseased tissues . Normal epithelial and fetal tissues have low expression of CD147, when measured by immunohistochemical analysis . However, CD147 is significantly upregulated in aggressive and chronic disease states, such as in cancers [66, 67], atherosclerosis , diabetes , ischemic stroke , and chronic lung obstruction diseases . Additionally, CD147 is strongly expressed on endothelial cells in the brain , gastrointestinal tract tissues , platelets , conjunctival tissues , kidney glomerular cells and podocytes , and cardiac pericytes [16, 77], where it could serve a more dominant role in SARS-CoV-2 infection and mediate COVID-19-related neurological disturbance, digestive tract vascular damage, increased thrombosis, conjunctivitis, acute kidney injury, and cardiovascular disruption, respectively. Intriguingly, recent studies show that CD147 plays a functional role in facilitating SARS-CoV and SARS-CoV-2 entry [78, 79], and antibodies against CD147 block the infection capabilities of SARS-CoV-2 . A humanized anti-CD147 antibody (Meplazumab) efficiently improves the recovery of COVID-19 patients with pneumonia with a favorable safety profile . However, the majority of studies related to CD147, and SARS-CoV-2 are focused on cell line-based in vitro assays and protein binding experiments and have yet-to-be verified in vivo [28, 81, 82]. Additionally, recent reports found no evidence of direct interaction between CD147 protein and the RBD domain of SARS-CoV-2 [13, 83]. Thus, it is imperative to verify a potential functional role of CD147 in a live mouse model.
The hCD147KI-NSG and hACE2KI-NSG mice developed in this study utilize the natural promoter for mouse Bsg and Ace2, respectively. The human keratin 18 promoter was used to overexpress hACE2 receptor in the epithelial cells of the most commonly used K18-hACE2-B6 model. Specifically, over-expression of human ACE2 was identified in airway epithelia and the epithelia lining the liver, kidney and gastrointestinal tract in K18-hACE2-B6 mice . However, expression was not observed in alveolar epithelial cells. Additionally, as multiple integrations were determined to exist on mouse chromosome 2, hemizygous mice are estimated to have between 8 full copies (or 12–30 partial copies) (commissioned analysis by The Jackson Laboratory) which is essential to establishing such a robust model for SARS-CoV-2 infection. Unlike the COVID-19 disease process in humans which has a global mortality rate of between 1–10% , a majority of SARS-CoV-2 infected K18-hACE2-B6 mice succumb to the disease process at an extremely rapid pace (as early as 6 days post-infection), making it difficult to study immunological responses .
CRISPR/Cas9 protein complexed with gRNA, along with the hCD147 or hACE2 plasmid constructs, were microinjected directly into NSG mouse zygotes, in order to generate a scarless knock-in of the human cDNA in frame within the first 22 amino acids of the mouse CD147 protein. Therefore, the human CD147 receptor and human ACE2 receptor are both expected to follow the same pattern of expression as the endogenous mouse receptor equivalents under normal conditions in hCD147KI-NSG and hACE2KI-NSG mice, respectively.
Our data supports the hypothesis that human CD147 plays an accessory role in SARS-CoV-2 infection. We confirmed strong expression of the human CD147 protein in the bronchiolar airway cells in hCD147KI-NSG mice compared to lung parenchymal cells. This protein distribution pattern may help explain why newer variants can replicate faster and more efficiently in the bronchus compared to lower airway regions . Importantly, we observed increased viral presence measured by sm-FISH and qRT-PCR in the lungs of hCD147KI-NSG and hACE2KI-NSG mice, compared to WT-NSG littermates at both two days and 7 days after infection. While the overall degree of infection in hACE2KI-NSG mice was considerably more severe than that in hCD147KI-NSG mice, we observed a significant body weight drop, higher SARS-CoV-2 RNA level, and detected multiple SARS-CoV-2 infected cells in the lungs of both hCD147KI-NSG and hACE2KI-NSG mice for up to seven days post-infection, compared to WT-NSG mice. We also suspect that hCD147KI-NSG and hACE2KI-NSG mice may develop increased leukocytosis in the lungs shortly after SARS-CoV-2 infection. Unexpectedly, we did not observe a similar correlation in RBD levels in hCD147KI-NSG mice. This could be explained by three possible reasons: 1) sm-FISH and IHC may have different levels of sensitivity because sm-FISH detects the RNA expression level of SARS-CoV-2, which may represent an earlier window timepoint for detection, compared with IHC detection as it detects the protein. 2) this may be due to a divergence between viral RNA replication and viral protein production/clearance (or regulation) during the recovery phase seven-day post-infection, 3) CD147 may play a role in late phase cell-to-cell infection (Fig. 5), but not as a functional receptor (such as ACE2) to mediate viral entry, evidenced by the lack of a significant difference in viral load between infected WT-NSG and in hCD147KI-NSG mice measured by qRT-PCR at the early infection (Day 2).
Additionally, CD147 is an important immune-modulator  making it difficult to dissect mechanisms in infection models utilizing humanized immunocompetent mice. NSG mice, on the other hand, are immunocompromised and allow for unbiased evaluation of SARS-CoV-2 infectivity without immunological interference.
The limitations of the current study include small sample sizes and lack of exact molecular mechanisms. Future experiments will be able to compare the natural course of SARS-CoV-2 infection in immunocompromised hCD147KI-NSG and immunocompetent hCD147KI-B6 mice and will further elucidate the role of the human CD147 receptor during the SARS-CoV-2-induced immune response. While our results do not provide clarity on the mechanism by which this receptor enhances viral entry or exacerbates viral replication and persistence, we confirm here that this model can set the stage for future inquiry and examination.
The hCD147KI model offers several strengths to the scientific community as it will better capture other nuances of the COVID-19 disease. (1) This model will allow researchers to study hemodynamic instability and increased thrombosis risk following COVID-19 infection as the hCD147 protein is expressed in circulating erythrocytes. (2) The NSG background will allow scientists to study how adoptive transfer can either dampen or exacerbate COVID-19-induced cytokine storm that is often seen in severe disease. Additionally, as the NSG background has been used to study diabetes , our model will allow further studies into the role of diabetes in COVID-19. (3) This model can be crossed with other mouse models to determine whether a combination of human CD147 and other viral entry-related receptors (e.g., ACE2, TMPRSS2) can exacerbate clinical disease as well via interactions between the two receptors to facilitate cell-to-cell transmission in different tissues or different time-points. If such a phenomenon is observed, the exact function of CD147 as an accessory receptor to the dominant viral entry receptor, ACE2, can be interrogated and properly confirmed in vivo (Fig. 5). (4) As the human CD147 protein is expressed at physiological levels in these mice, this model will better recapitulate true physiological conditions and expression patterns normally observed in mice and humans. Even if CD147 is later determined to play a relatively minor role compared to ACE2 in SARS-CoV-2 viral entry, this mouse model may prove to be invaluable for understanding how the virus globally impacts CD147-positive cells and tissues in the in vivo setting and how therapies may modulate COVID-19 disease via this receptor. (5) These models can be used to test the infectivity and pathogenesis of the emergence of variants of SARS-CoV-2, such as B.1.1.7 [alpha], B.1.351 [beta], B.1.617.2 [delta], P.1 (20J501Y.V3) [gamma], and B.1.621 [mu], and B.1.1.529 [omicron] to additional co-receptors, given recently reported studies showing mutation-driven extension of host range to common laboratory mice  and increased dependence on other accessory receptors other than ACE2 . (6) As CD147 is expressed on many tissues and cell types outside of the respiratory system, if in fact this receptor is implicated in viral entry and dissemination, the hCD147KI-NSG model could help shed light onto the nature of chronic COVID-19 syndrome, also known as long COVID. (7) The hCD147KI-NSG background with hACE2 mouse model can be used to test novel immunotherapies (e.g., CAR-NK) to prevent and/or treat new mutants of SARS-CoV-2 in the future. In summary, the newly generated hCD147KI-NSG mouse model can be used as a platform where direct clinical implications for vaccine and therapeutic strategies can be evaluated in preparation for future global pandemics.