Longitudinal virological changes and underlying pathogenesis in hospitalized COVID-19 patients in Guangzhou,China

Science China Life Sciences - Prolonged viral RNA shedding and recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19) patients have been...     

A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein

The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.     

T Cell Responses Are Required for Protection from Clinical Disease and for Virus Clearance in Severe Acute Respiratory Syndrome Coronavirus-Infected Mice

A dysregulated innate immune response and exuberant cytokine/chemokine expression are believed to be critical factors in the pathogenesis of severe acute respiratory syndrome (SARS), caused by a coronavirus (SARS-CoV). However, we recently showed that inefficient immune activation and a poor virus-specific T cell response underlie severe disease in SARS-CoV-infected mice. Here, we extend these results to show that virus-specific T cells, in the absence of activation of the innate immune response, were sufficient to significantly enhance survival and diminish clinical disease. We demonstrated that T cells are responsible for virus clearance, as intravenous adoptive transfer of SARS-CoV-immune splenocytes or in vitro-generated T cells to SCID or BALB/c mice enhanced survival and reduced virus titers in the lung. Enhancement of the number of virus-specific CD8 T cells by immunization with SARS-CoV peptide-pulsed dendritic cells also resulted in a robust T cell response, earlier virus clearance, and increased survival. These studies are the first to show that T cells play a crucial role in SARS-CoV clearance and that a suboptimal T cell response contributes to the pathological changes observed in SARS. They also provide a new approach to SARS vaccine design.Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in over 8,000 cases of respiratory disease, with 10% mortality, in 2002 and 2003 (18). Patients with severe disease developed acute lung injury (ALI), concomitant with neutrophilia, lymphopenia, and prolonged expression of several proinflammatory cytokines (4, 17, 18, 32). Virus was cleared slowly from these patients and could be detected in respiratory secretions for as long as 21 days postinfection (p.i.) (14, 17).SARS has not recurred to a significant extent since 2003, so most studies have used animal infections to investigate the mechanism of severe disease. These studies have been facilitated by the isolation and characterization of SARS-CoV variants that cause severe respiratory disease in mice or rats after adaptation to these hosts by serial passage (15, 16, 22). One strain, isolated after 15 passages through the lungs of BALB/c mice, caused respiratory disease in young BALB/c mice (MA15 virus) (22). As in humans with severe disease, cytokine expression is elevated and prolonged in animals with severe disease, whether caused by the human Urbani or rodent-adapted strains of SARS-CoV, and is accompanied by delayed kinetics of virus clearance (1, 5, 15, 23, 26, 35). This elevated expression of proinflammatory mediators is believed to be a major contributory factor in the development of pneumonia and, subsequently, ALI.These studies did not address the role of the antivirus T cell response in disease. T cell responses are critical for virus clearance and protection from clinical disease in mice infected with other coronaviruses, such as mouse hepatitis virus (MHV) (31), or other pulmonary pathogens, such as influenza A virus or Sendai virus (7, 29). Although some studies using the Urbani strain showed that virus clearance was T cell independent (5, 34), mice infected with this strain develop only mild pneumonitis, so that the requirement for a virus-specific T cell response for protection in SARS-CoV-infected animals remains unclear.Young BALB/c mice infected with MA15 virus develop severe respiratory disease, with substantial mortality, dependent upon virus dosage (22). We recently showed that the pulmonary immune response is inefficiently activated in these mice at early times after infection, resulting in a barely detectable antivirus T cell response in mice with a lethal infection (35). Survival correlated with the development of a SARS-CoV-specific CD4 and CD8 T cell response. Poor activation of the immune response could be reversed by depletion of inhibitory alveolar macrophages with clodronate, a drug that depletes phagocytic cells (28, 30), or by treatment with poly(I-C) or CpG, both of which activate macrophages and dendritic cells (DCs). Disease could also be ameliorated by adoptive transfer of activated DCs, which are able to traffic to the lung draining lymph nodes (DLN) and prime the antivirus T cell response. These interventions effect two changes in the host immune response: they result in activation of the innate immune system and facilitate the development of a robust virus-specific T cell response. From these results, we could not determine the relative importance of these two limbs of the immune response in protection. Here, we examined whether a robust antivirus T cell response was sufficient to protect mice in the absence of interventions that activated the innate immune response. Our results indicate that virus-specific T cells are necessary and sufficient for virus clearance and for protection from clinical disease in MA15 virus-infected mice.     

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Is Required for Optimal Replication

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several structural proteins, including the spike, nucleocapsid, membrane, and envelope proteins (13). Integrated between and within these structural proteins are eight accessory proteins (6, 8, 10, 15, 16, 18, 21-27). Our laboratory showed previously that one of these SARS-CoV-specific accessory proteins, encoded by ORF6, showed a clearly recognizable phenotype when introduced into a heterologous attenuated murine coronavirus, mouse hepatitis virus (MHV) strain J2.2-V-1 (rJ2.2.6). rJ2.2.6 grew more rapidly and to higher titers in tissue culture cells and in the murine central nervous system than control viruses, and the presence of p6 increased mortality in mice from 10 to 20% to 80% (7, 19, 20). However, the absence of p6 did not diminish SARS-CoV growth in tissue culture cells when cells were infected with 1 PFU/cell (31). In addition to a role in enhancing virus replication, when expressed in the context of a SARS-CoV infection or by transfection, p6 blocked interferon (IFN)-induced STAT1 nuclear translocation by retention of the nuclear import adaptor molecule karyopherin alpha 2 in the cytoplasm, indicating a role in thwarting innate immune effectors (5, 11). In contrast, p6 did not significantly diminish IFN sensitivity when expressed in the context of rJ2.2 (20).The results described above were puzzling, because p6 seemed to be required for the optimal replication of a heterologous coronavirus but not for that of SARS-CoV. Thus, the objective of this study was to determine whether p6 could enhance SARS-CoV replication in tissue culture cells under any conditions. For this purpose, we examined its function by comparing the growth of a recombinant SARS-CoV (rSARS-CoV) in which p6 was deleted (rSARS-CoVΔ6) with that of wild-type rSARS-CoV at a range of multiplicities of infection (MOIs). Normal mice infected with SARS-CoV readily cleared the infection, making it difficult to detect a role for p6 in vivo. However, mice that are transgenic for expression of the human receptor angiotensin-converting enzyme 2 (hACE2) are exquisitely sensitive to infection with SARS-CoV and are useful for identifying an in vivo role for p6 (14).     

Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease.     

Virus-Specific Regulatory T Cells Ameliorate Encephalitis by Repressing Effector T Cell Functions from Priming to Effector Stages

Several studies have demonstrated the presence of pathogen-specific Foxp3⁺ CD4 regulatory T cells (Treg) in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg) and conventional CD4 T cells (M133 Tconv) in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN). Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.