Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world because of severe symptoms and relatively high mortality. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate the efficacy of prophylactics and therapeutics in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in cynomolgus macaques until 28 days after virus inoculation in the present study. Cynomolgus macaques showed body temperature rises after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening clinical signs of disease corresponding that approximately 80% of human patients did not show a critical disease in COVID-19. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 N protein were detected on day 14 in the macaque that showed viral pneumonia. On the other hand, in the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14 prior to an increase in the number of T-lymphocytes that produced IL-2. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild clinical signs of disease and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development. Author SummarySevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate their efficacy in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in a non-human primate model until 28 days after virus inoculation. Cynomolgus macaques showed a fever after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening symptoms. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 protein were detected on day 14 in the macaque that showed viral pneumonia. In the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild symptoms and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development.

Repeatability of mechanomyogram(MMG) from voluntary isometric contraction of biceps brachii muscles / 体力科学

The repeatability of the mechanomyogram (MMG) must be carefully determined before the signal can be generally used as a reliable tool in the fields such as physical fitness, sports and rehabilitation. In the present investigation, both the between-trial and between-day repeatability of the MMG was examined in the time and frequency domain and was compared with that of the more established electromyogram (EMG) . Isometric contraction trials of the biceps brachii muscles at different force levels of 10% to 90% MVC were held until a force plateau lasting 5 s was completed. The testing session consisting of 9 submaximal trials was repeated 6 times on the same day for estimation of the variation between trials. The same testing session was also performed 8 times over 3 weeks with a 2-day rest interval to examine the day-to-day variation. The coefficient of variation (CV) between-trials as well as between-days of the root mean squared (rms) MMG, mean frequency and median frequency did not demonstrate any significant differences among the force levels. The combined CV of the rms MMG over all the force levels was approximately 9 % between trials and 22% between days. The mean frequency and the median frequency presented the CV between-trials and between-days of approximately 6% and 10%, respectively. The different CV between the time and frequency domain parameters of the MMG may result from different contributing factors in the motor unit activation pattern. The results of this study suggest that the repeatability of the time domain parameter of the MMG is similar to that of the more established EMG, whereas the frequency parameters are somewhat less repeatable than those of the EMG.

REPEATABILITY OF MECHANOMYOGRAM (MMG) FROM VOLUNTARY ISOMETRIC CONTRACTION OF BICEPS BRACHII MUSCLES / 体力科学

The repeatability of the mechanomyogram (MMG) must be carefully determined before the signal can be generally used as a reliable tool in the fields such as physical fitness, sports and rehabilitation. In the present investigation, both the between-trial and between-day repeatability of the MMG was examined in the time and frequency domain and was compared with that of the more established electromyogram (EMG) . Isometric contraction trials of the biceps brachii muscles at different force levels of 10% to 90% MVC were held until a force plateau lasting 5 s was completed. The testing session consisting of 9 submaximal trials was repeated 6 times on the same day for estimation of the variation between trials. The same testing session was also performed 8 times over 3 weeks with a 2-day rest interval to examine the day-to-day variation. The coefficient of variation (CV) between-trials as well as between-days of the root mean squared (rms) MMG, mean frequency and median frequency did not demonstrate any significant differences among the force levels. The combined CV of the rms MMG over all the force levels was approximately 9 % between trials and 22% between days. The mean frequency and the median frequency presented the CV between-trials and between-days of approximately 6% and 10%, respectively. The different CV between the time and frequency domain parameters of the MMG may result from different contributing factors in the motor unit activation pattern. The results of this study suggest that the repeatability of the time domain parameter of the MMG is similar to that of the more established EMG, whereas the frequency parameters are somewhat less repeatable than those of the EMG.