Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice

As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir, and molnupiravir and their combinations in SARS-CoV-2-infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20-40% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival times up to 80% compared to those with nirmatrelvir (P= 0.0001) and molnupiravir (P= 0.0001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P= 0.0001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P= 0.0001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy, demonstrating the inefficient therapeutic effect of this combination. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients. IMPORTANCESince SARS-CoV-2 spread rapidly with the emergence of new variants of concerns, it is necessary to develop effective treatment strategies to treat elderly individuals and those with comorbidities. Antiviral therapy using a combination of drugs is more effective in eradicating viruses and will undoubtedly improve the clinical outcome and survival probability of hospitalized SARS-CoV-2 patients. In the current study, we observed three FDA-approved antivirals nirmatrelvir, remdesivir, and molnupiravir have therapeutic significance with moderate survival for their monotherapies against SARS-CoV-2 infected K18-hACE2 mouse model. The combination of nirmatrelvir and molnupiravir showed significant antiviral activity and a higher survival rate of approximately 80%, providing in vivo evidence of the potential utility of this combination. In contrast, nirmatrelvir and remdesivir combination showed less antiviral potency and emphasized the ineffective significance with less survival. The current study suggests that the nirmatrelvir and molnupiravir combination is an effective drug regimen strategy in treating SARS-CoV-2 patients.

Mosaic receptor-binding domain nanoparticles induce protective immunity against SARS-CoV-2 challenges

Recurrent spillovers of - and {beta}-coronaviruses (CoV) such as acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, SARS-CoV-2, and possibly human CoV (NL63, 229E, OC43, and HKU1) have caused serious morbidity and mortality worldwide. Six receptor binding domains (RBDs) derived from - and {beta}-CoV that are considered to have originated from animals and cross-infected humans were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric subunits, PCNA1, PCNA2, and PCNA3. These were used to form a scaffold-based mosaic multivalent antigen, 6RBD-np. Electron microscopic and atomic force microscopic images show a ring-shaped disk with six protruding RBDs, like jewels in a crown, with a size of 40 nm. Prime-boost immunizations with 6RBD-np in BALB/c mice elicited strong, dose-dependent antibody responses. In human angiotensin converting enzyme 2-transgenic mice, the same immunization induced full-protection against SARS-CoV-2 wild type and Delta challenges, resulting in a 100% survival rate. The mosaic 6RBD-np provides a potential platform for developing a pan-CoV vaccine against newly emerging SARS-CoV-2 variants and future CoV spillovers. SignificanceDespite the arsenal of COVID-19 vaccines, hospitalization and mortality associated with SARS-CoV-2 (acute respiratory syndrome coronavirus 2) variants remain high. There is an urgent need to develop next-generation COVID vaccines that provide broad protection against diseases by current and newly emerging SARS-CoV-2 variants. In this study, six receptor binding domains (RBDs) derived from - and {beta}-CoV were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric scaffolds. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens. The prime-boost immunization in BALB/c and human angiotensin converting enzyme 2-transgenic mice with the 6RBD-np elicited strong, dose-dependent antibody responses and induced full-protection against both the SARS-CoV-2 wild type (WT) and Delta challenges. This study provides proof-of-concept that the mosaic 6RBD-np induces 100% protection against SARS-CoV-2 WT and Delta. It provides the potential of co-displaying heterologous antigens for novel vaccine designs, which can be deployed countering future pandemics.

The significance of avian influenza virus mouse-adaptation and its application in characterizing the efficacy of new vaccines and therapeutic agents

Due to the increased frequency of interspecies transmission of avian influenza viruses, studies designed to identify the molecular determinants that could lead to an expansion of the host range have been increased. A variety of mouse-based mammalian-adaptation studies of avian influenza viruses have provided insight into the genetic alterations of various avian influenza subtypes that may contribute to the generation of a pandemic virus. To date, the studies have focused on avian influenza subtypes H5, H6, H7, H9, and H10 which have recently caused human infection. Although mice cannot fully reflect the course of human infection with avian influenza, these mouse studies can be a useful method for investigating potential mammalian adaptive markers against newly emerging avian influenza viruses. In addition, due to the lack of appropriate vaccines against the diverse emerging influenza viruses, the generation of mouse-adapted lethal variants could contribute to the development of effective vaccines or therapeutic agents. Within this review, we will summarize studies that have demonstrated adaptations of avian influenza viruses that result in an altered pathogenicity in mice which may suggest the potential application of mouse-lethal strains in the development of influenza vaccines and/or therapeutics in preclinical studies.

Phylogenetic Analysis of the 56-kDa Type-Specific Protein Genes of Orientia tsutsugamushi in Central Korea

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.

Phylogenetic Analysis of the 56-kDa Type-Specific Protein Genes of Orientia tsutsugamushi in Central Korea

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.

Clinical characteristics of acute lower respiratory tract infections due to 13 respiratory viruses detected by multiplex PCR in children / 소아과

PURPOSE: This study was performed to investigate the epidemiologic and clinical features of 13 respiratory viruses in children with acute lower respiratory tract infections (ALRIs). METHODS: Nasopharyngeal aspirates were prospectively obtained from 325 children aged 15 years or less from May 2008 to April 2009 and were tested for the presence of 13 respiratory viruses by multiplex real-time-polymerase chain reaction (RT-PCR). RESULTS: Viruses were identified in 270 children (83.1%). Co-infections with > or =2 viruses were observed in 71 patients (26.3%). Respiratory syncytial virus (RSV) was the most common virus detected (33.2%), followed by human rhinovirus (hRV) (19.1%), influenza virus (Flu A) (16.9%), human metapneumovirus (hMPV) (15.4%), parainfluenza viruses (PIVs) (8.3%), human bocavirus (hBoV) (8.0%), adenovirus (ADV) (5.8%), and human coronavirus (hCoV) (2.2%). Clinical diagnoses of viral ALRIs were bronchiolitis (37.5%), pneumonia (34.5%), asthma exacerbation (20.9%), and croup (7.1%). Clinical diagnoses of viral bronchiolitis and pneumonia were frequently demonstrated in patients who tested positive for RSV, hRV, hMPV, or Flu A. Flu A and hRV were most commonly identified in children older than 3 years and were the 2 leading causes of asthma exacerbation. hRV C was detected in 14 (4.3%) children, who were significantly older than those infected with hRV A (mean+/-SD, 4.1+/-3.5 years vs. 1.7+/-2.3 years; P=0.009). hBoV was usually detected in young children (2.3+/-3.4 years) with bronchiolitis and pneumonia. CONCLUSION: This study described the features of ALRI associated with 13 respiratory viruses in Korean children. Additional investigations are required to define the roles of newly identified viruses in children with ALRIs.

Genetic Characteristics and Phylogenetic Analysis of Influenza Type B Viruses Isolated from Nasopharyngeal Suction Samples of Korean Patients

To investigate the genetic characteristics of human influenza type B viruses circulating in Chungbuk province, Korea, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Twelve out of thirty-six isolates were identified as type B influenza virus by RT-PCR and sequencing analysis. Interestingly, genetic characterization of type B viruses isolated in this study revealed that all type B influenza viruses were the Yamagata lineages, a vaccine strains of southern hemisphere during 2007~2008, rather than the Victoria lineage of northern hemisphere during 2007~2008. Furthermore, there were a total of twelve unique mutations (HA: H40Y, D/G230S, V252M and K272R and NA: P3H, P/T/S42Q, N59S) occurred in our type B isolates. These results suggest that relative high prevalence of type B viruses in Korea during 2007~2008 season might be due to the wrong vaccine strains selection. Taken together, the results of this study demonstrate continuous evolutions of human type B viruses by antigenic drift and also highlight the need to closely monitoring of influenza viruses to aid the early detection of potentially pandemic strains as well as underscore the need for new therapeutics.