The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.
Various COVID-19 vaccine candidates are currently under clinical trial. However, no live attenuated vaccine has been developed yet, despite their generally high efficacy. Here, we established temperature-sensitive mutant strains of SARS-CoV-2, whose growth was significantly slower than that of the parent strain at 37˚C. One of the strains, A50-18, which presented mutations in nonstructural protein 14, did not replicate at all at 37˚C in vitro. In vivo experiments demonstrated that this strain replicated inefficiently in the lungs of Syrian hamsters, and intra-nasal inoculation induced sufficient anti-SARS-CoV-2-neutralizing antibodies to protect against wild type virus infection. These results suggest that the A50-18 strain could be a promising live attenuated vaccine candidate against SARS-CoV-2.
A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh‐7 and 293T, permitted the propagation of recombinant green fluorescent protein–expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh‐7 and 293T cells. We then confirmed that recombinant green fluorescent protein–expressing RSV was rescued in both Huh‐7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh‐7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II.
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