Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19

Authors: Chu et al.,

Link to paper: https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa410/5818134

Journal/ Pre-Print: Clinical Infectious Diseases

Tags: Immunology/Immunity, Clinical/ Diagnostics, Inflammation, Molecular Biology

Research Highlights 

1. SARS-CoV-2 demonstrates more successful infection of ex vivo human lung tissue vs SARS-CoV

2. SARS-CoV-2 and SARS-CoV infect similar cell types in human lungs

3. SARS-CoV-2 infection results in reduced IFN and proinflammatory responses vs SARS-CoV

Summary 

This study used human lung tissue to compare replication efficiency, cell tropism and immune activation profile during SARS-CoV and SARS-CoV-2 infection. Immunofluorescence and plaque formation analysis showed that within 48 hours post infection (hpi), SARS-CoV-2 achieved a more profound increase in viral titre in comparison to SARS-CoV. Immunohistochemistry showed there was no difference in the cell types that SARS-CoV and SARS-CoV-2 infected. qPCR of infected tissues showed that SARS-CoV-2 induced a markedly dampened IFN response alongside reduced expression of selected pro-inflammatory cytokines and chemokines 48 hpi. This data suggests that SARS-CoV-2, despite more robustly infecting pulmonary tissue, does not induce inflammation to the extent achieved by SARS-CoV. This may explain the differences seen between SARS-CoV and SARS-CoV-2 infection outcome.

Impact for SARS-CoV2/COVID19 research efforts

Understand the immune response to SARS-CoV2/COVID19

qPCR analysis of ex vivo lung tissues infected with SARS-CoV and SARS-CoV-2 details the differences in the immune response generated to SARS-CoV-2 vs SARS-CoV

Understand the virology and/or cell biology of SARS-CoV2/COVID19

The study details the potential tropism of SARS-CoV-2 vs SARS-CoV in the lung

Clinical symptoms and pathogenesis of SARS-Cov2/COVID19

Differences in viral burden and immune response generation obtained following SARS-CoV-2 vs SARS-CoV may explain the differences in infection outcome between the 2 viruses. 

Study Type

· Ex vivo study (human lung cell culture)

Strengths and limitations of the paper

Novelty: The study is the first to quantitatively compare the ex vivo replication, cell tropism and immune activation profiles of SARS-CoV-2 and SARS-CoV using human lung tissue.

Standing in the field: The genuine low induction of IFN agrees with most of the previous studies; Some previous studies suggest a normal or even high pro-inflammatory cytokine response despite the low IFN activation, which is contradicted by the finding of this study.

Appropriate statistics: n>=4 is used in most experiments. Area under curve (AUC) analysis not preferable, repeated measures ANOVA type analysis more appropriate. In addition, if to conduct statistics on AUC, t test should have been paired as infections done on samples originating from the same donor. In general, use of parametric analysis not appropriate, due to n=4 and thus cannot test for normality distribution. SD bars sporadically missing in fig 2A and authors do not detail where the replication originates from to calculate SD. In figs 4 & 5, authors show pooled technical + biological replicates – without indicating which data point represents which replicate type. This statistical analysis is thus not appropriate, as authors have increased n from 4, which is the number of biological replicates present.

Viral model used:

SARS-CoV-2 HKU-001a (MT230904)

SARS-CoV GZ50 (AY304495)

Translatability: Not very translatable. More about viral pathogenesis and immune activation than any clinical aspect. Could potentially provide insight into infection control and treatment strategies for COVID-19.

Main limitations:

- No information regarding sample origin past patient sex and age. This includes when samples were acquired. If this was during SARS-CoV-2 pandemic, this raises problems regarding donor SARS-CoV-2 positivity status

- Mock infection as a control not mentioned anywhere in paper aside from single figure legend + figure plots

- Different antibodies are used in immunofluorescent quantification without including a control to compare the antibody staining efficiency, meaning that the difference in fluorescent level may be due to the difference between antibodies.

- Limited sample size (n = 6 or 4)

- Poor image quality in figures

- Problems with statistical analysis and data presentation

- Infection time course stops at 48h post infection. Doesn’t allow for the establishment of effects after 48 hours. E.g. does this increase in SARS-CoV-2 load at 48 hours lead to the development of an exacerbated response later in infection?

- Did not attempt to explain the drops in virus titre in some samples in Fig 2A. (Donor 2 and Donor 4)

- No supplementary data despite being referred to in the text

- As discussed, human lung tissues are short-lasting and can only represent local but not systemic host immune response.