Prolonged viral shedding in an immunocompromised Korean patient infected with hMPXV, sub-lineage B.1.3, with acquired drug resistant mutations during tecovirimat treatment.

Following the worldwide surge in mpox (monkeypox) in 2022, cases have persisted in Asia, including South Korea, and sexual contact is presumed as the predominant mode of transmission, with a discernible surge in prevalence among immunocompromised patients. Drugs such as tecovirimat can result in drug-resistant mutations, presenting obstacles to treatment. This study aimed to ascertain the presence of tecovirimat-related resistant mutations through genomic analysis of the monkeypox virus isolated from a reported case involving prolonged viral shedding in South Korea. Here, tecovirimat-resistant mutations, previously identified in the B.1 clade, were observed in the B.1.3 clade, predominant in South Korea. These mutations exhibited diverse patterns across different samples from the same patient and reflected the varied distribution of viral subpopulations in different anatomical regions. The A290V and A288P mutant strains we isolated hold promise for elucidating these mechanisms, enabling a comprehensive analysis of viral pathogenesis, replication strategies, and host interactions. Our findings imply that acquired drug-resistant mutations, may present a challenge to individual patient treatment. Moreover, they have the potential to give rise to transmitted drug-resistant mutations, thereby imposing a burden on the public health system. Consequently, the meticulous genomic surveillance among immunocompromised patients, conducted in this research, assumes paramount importance.

Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Development and comparison of two H5N8 influenza A vaccine candidate strains.

Avian influenza viruses circulating in birds have caused outbreaks of infection in poultry and humans, thereby threatening public health. Recently, a highly pathogenic avian influenza (HPAI) virus (H5N8) of clade 2.3.4.4 emerged in Korea and other countries and caused multiple outbreaks in domestic and wild birds, with concerns for human infection. To combat HPAI viral infections, novel vaccines are likely to be the most effective approach. Therefore, in this study, we generated H5N8 vaccine candidate viruses based on a Korean isolate (A/broiler duck/Korea/Buan2/2014). The vaccine candidate viruses were 2:6 reassortants expressing the two surface glycoproteins of A/broiler duck/Korea/Buan2/2014 on an A/Puerto Rico/8/34 (PR8) backbone generated by using an eight-plasmid-based reverse genetics system with or without replacement of the multi-basic amino acid cleavage motif (MBCM, a crucial pathogenic factor in HPAI virus) with a bi-basic amino acid cleavage motif (BBCM) in their HA. An H5N8 vaccine candidate virus containing the BBCM showed attenuated pathogenesis in embryonated eggs and exhibited less virulence in the infected mice compared with the wild H5N8 virus containing an MBCM. Vaccination with an inactivated preparation of the vaccine candidate virus protected mice from lethal H5N8 viral challenge. This is the first report of the development and evaluation of H5N8 vaccine strains (with an MBCM or BBCM) of HA clade 2.3.4.4 as vaccine candidates. Our findings suggest that H5N8 strains with a BBCM instead of an MBCM might be considered for H5N8 vaccine seed virus development or as a reference vaccine against H5N8 viral strains.

Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection.

Infection with the highly pathogenic avian influenza H5N1 virus results in a high incidence of mortality in humans. Severe complications from infection are often associated with hypercytokinemia. However, current neuraminidase inhibitors (NAIs) have several limitations including the appearance of oseltamivir-resistant H5N1 virus and the inability to completely ameliorate hyper-immune responses. To overcome these limitations, we evaluated the anti-viral activity of mycophenolic mofetil (MMF) against A/Vietnam/1194/2004 (H5N1) virus infection using MDCK cells and mice. The IC50 of MMF (0.94 µM) was comparable to that of zanamivir (0.87 µM) in H5N1 virus-infected MDCK cells based on ELISA. Time-course assays demonstrated that MMF completely inhibited H5N1 viral mRNA replication and protein expression for approximately 8 h after the initiation of treatment. In addition, MMF treatment protected 100% of mice, and lung viral titers were substantially reduced. The anti-viral mechanism of MMF against H5N1 virus infection was further confirmed to depend on the inhibition of cellular inosine monophosphate dehydrogenase (IMPDH) by exogenous guanosine, which inhibits viral mRNA and protein expression. Moreover, IL-1ß, IFN-ß, IL-6, and IP-10 mRNA expression levels were significantly downregulated in MDCK cells with MMF treatment. These results indicated that MMF could represent a novel inhibitor of viral replication and a potent immunomodulator for the treatment of H5N1 virus infection.

Impact of influenza virus escape-mutations on influenza detection by the rapid influenza diagnostic test.

During the influenza pandemic of 2009-2010, rapid influenza diagnostic tests (RIDTs) were used to detect influenza viral infections because they are quick and simple to use. However, retrospective studies showed that RIDTs performed poorly when used to diagnose pandemic viral infections. Determining how amino acid sequence changes in pandemic or epidemic influenza viral antigens impact clinical value of RIDTs has not been possible, because the viral epitopes recognized by RIDTs have been not mapped. In this study, the effect of escape-variations or mutations in influenza viral antigens upon the sensitivity and specificity of an RIDT was investigated by characterizing the immunological properties of the antibodies used in the RIDT. Escape-mutants were generated by cultivating A/Korea/01/2009 in the presence of an excess of the same antibodies used in the RIDT. Escape-mutants not recognized by the RIDT were selected. Epitopes recognized by the RIDT were mapped by comparing the sequence and immunological analysis of the escape-variants and wild-type isolates. The RIDT antibodies recognized epitopes on the Sa antigenic site and in the F subdomain in hemagglutinin. Variants bearing mutations in these epitopes were not detected by the RIDT. The frequency of escape-variants emerging since the 2009-2010 pandemic was calculated as 1.27% using in silico surveillance of influenza sequence databases. These results suggest that mapping the relevant epitopes of RIDTs and making such information available to clinics would be helpful for determining whether RIDTs match newly emergent strains and subtypes prior to retrospective re-evaluation of the RIDTs using clinical specimens.

Immunogenicity and Protective Efficacy of Recombinant Protective Antigen Anthrax Vaccine (GC1109) in A/J Mice Model.

A recombinant protective antigen anthrax vaccine (GC1109) is being developed as a new-generation vaccine by the Korea Disease Control and Prevention Agency. In accordance with the ongoing step 2 of phase II clinical trials, the immunogenicity and protective efficacy of the booster dose of GC1109 were evaluated in A/J mice after 3 serial vaccinations at 4-week intervals. The results indicated that the booster dose significantly increased the production of anti-protective antigen (PA) IgG and toxin-neutralizing antibody (TNA) compared with those of the group without booster. An enhanced protective effect of the booster dose was not observed because the TNA titers of the group without booster were high enough to confer protection against spore challenge. Additionally, the correlation between TNA titers and probability of survival was determined for calculating the threshold TNA titer levels associated with protection. The threshold 50 % neutralization factor (NF50) of TNA showing 70 % probability of protection was 0.21 in A/J mice with 1,200 LD50 Sterne spores challenge. These results indicate that GC1109 is a promising candidate as a new-generation anthrax vaccine and that a booster dose might provide enhanced protection by producing toxin-neutralizing antibodies.

Complete Genome Sequence of Monkeypox Virus Strain MPXV-ROK-P1-2022 Isolated from the First Monkeypox Patient in the Republic of Korea.

We report the complete genome sequence of the monkeypox virus strain MPXV-ROK-P1-2022, isolated from the first patient diagnosed with monkeypox in the Republic of Korea in June 2022. The virus was fully sequenced on the Illumina MiSeq instrument, and the phylogenetic tree showed that the strain belongs to lineage B.1.1.

Isolation and identification of monkeypox virus MPXV-ROK-P1-2022 from the first case in the Republic of Korea.

OBJECTIVES: Monkeypox outbreaks in nonendemic countries have been reported since early May 2022. The first case of monkeypox in the Republic of Korea was confirmed in a patient who traveled to Europe in June 2022, and an attempt was made to isolate and identify the monkeypox virus (MPXV) from the patient's specimens. METHODS: Clinical specimens from the patient were inoculated in Vero E6 cells. The isolated virus was identified as MPXV by the observation of cytopathic effects on Vero E6 cells, transmission electron microscopy, conventional polymerase chain reaction (PCR), and sequencing of PCR products. RESULTS: Cytopathic effects were observed in Vero E6 cells that were inoculated with skin lesion swab eluates. After multiple passages from the primary culture, orthopoxvirus morphology was observed using transmission electron microscopy. In addition, both MPXV-specific (F3L and ATI) and orthopoxvirus-specific genes (A39R, B2R, and HA) were confirmed by conventional PCR and Sanger sequencing. CONCLUSION: These results indicate the successful isolation and identification of MPXV from the first patient in the Republic of Korea. The isolated virus was named MPXV-ROK-P1-2022.

Successful salvage therapy with inhaled zanamivir in a patient with peramivir-resistant pandemic influenza A (H1N1) 2009 virus.

We describe a patient with multiple myeloma who developed a pandemic influenza A (H1N1) 2009 virus infection during chemotherapy. The clinical condition of the patient worsened and viral shedding persisted despite 10 days of peramivir treatment; however viral shedding ceased and the patient recovered completely with inhaled zanamivir.

Oseltamivir-resistant pandemic (H1N1) 2009 virus, South Korea.

To identify oseltamivir resistance, we analyzed neuraminidase H275Y mutations in samples from 10 patients infected with pandemic (H1N1) 2009 virus in South Korea who had influenza that was refractory to antiviral treatment with this drug. A neuraminidase I117M mutation that might influence oseltamivir susceptibility was detected in sequential specimens from 1 patient.

Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.

Lactobacillus casei suppresses experimental arthritis by down-regulating T helper 1 effector functions.

Although the beneficial effects of probiotics on wide variety of diseases have been shown, little is known about how probiotics modulate the immune system. In this study we elucidated the underlying mechanisms how Lactobacillus casei (L. casei) protects against rheumatoid arthritis (RA) progression by investigating the effector functions of CD4(+) T cells. Oral administration of L. casei suppressed collagen-induced arthritis (CIA) and reduced paw swelling, lymphocyte infiltration and destruction of cartilage tissue. L. casei administration reduced type II collagen (CII)-reactive proinflammatory molecules (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha and Cox-2) by CD4(+) T cells. L. casei administration also reduced translocation of NF-kappaB into nucleus and CII-reactive Th1-type IgG isotypes IgG2a and IgG2b, while up-regulating immunoregulatory IL-10 levels. Our results suggest that oral administration of L. casei suppresses the type II collagen-reactive effector function of Th1-type cellular and humoral immune responses in arthritic inflammation.

Lactobacillus casei potentiates induction of oral tolerance in experimental arthritis.

Probiotics have been shown to exert beneficial effects on modulation of diverse diseases. However, no information is available for the effect of probiotics in the induction of oral tolerance in autoimmune diseases. The main purpose of this study was to elucidate whether Lactobacillus casei (L. casei) affect the induction of oral tolerance in experimental rheumatoid arthritis (RA). Type II collagen (CII) alone or together with L. casei was orally administered into collagen-induced arthritis (CIA) rats, and its effects on the clinical and histopathological aspects of RA were investigated. Co-administration of L. casei with CII more effectively suppressed clinical symptoms, paw swelling, lymphocyte infiltration and destruction of cartilage tissues of experimental arthritis than the rats treated with CII alone. The enhanced therapeutic efficacy was associated with an increase in anti-inflammatory cytokines (IL-10 and TGF-beta) while decreasing pro-inflammatory cytokines (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma and TNF-alpha). Co-administration of L. casei with CII more effectively suppressed CII-reactive T cell proliferation and the levels of Th1-type IgG isotypes (IgG2a and IgG2b), while up-regulating Foxp3 expression levels and the population of Foxp3(+) CD4(+) T cells. Our study provides evidence that L. casei could potentiate antigen-specific oral tolerance and suppress Th1-type immune responses of arthritic inflammation.

Defect in TCR-CD3zeta signaling mediates T cell hypo-responsiveness in mesenteric lymph node.

Mesenteric lymph node (MLN) in gut-associated lymphoid tissue plays obligatory roles in the induction of oral tolerance and ignorance to commensals. However, little is known about its immunological characteristics. In this study, we investigated the hypo-responsiveness of MLN CD4(+) T cells, comparing them with spleen CD4(+) T cells. MLN CD4(+) T cells were hypo-proliferative and expressed low levels of Th1-type cytokines in response to antigen or CD3/T cell receptor (TCR) stimulation. The hypo-responsiveness of MLN CD4(+) T cells is linked neither with changes in the regulatory T cell population (CD4(+)CD25(+), CD4(+)Foxp3(+)) nor the apoptotic population. Rather, MLN CD4(+) T cells showed deformity of T cell:APC conjugation and reduced expression of TCR signaling molecules such as CD3zeta, PLC-gamma1, PKC-theta, Zap70, with reduced phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). Among the alterations in TCR signaling molecules, defective CD3zeta expression is the most evident, and reversal of the anergic state by CD3/CD28 costimulation restored CD3zeta expression levels. Collectively, we suggest that reduced CD3zeta expression and defects in TCR signaling mediate the anergy state of MLN CD4(+) T cells, which play a critical role in maintenance of mucosal tolerance in gut-associated lymphoid tissue.

Immunological characterization of monoclonal antibodies used in rapid influenza diagnostic test for detection of the 2009 pandemic influenza A(H1N1)pdm09 infection.

Since the 2009 pandemic, monoclonal antibodies (mAbs) for rapid influenza diagnostic tests (RIDT) have been developed for specific diagnostics of pandemic viral infection. Most of the mAbs were poorly characterized because of urgency during the pandemic. Further characterization of the mAbs for RIDTs would be beneficial for understanding the immunological properties of the pandemic virus and utilizing the mAbs for other research purposes. In this study, it was confirmed that two mAbs (I38 and D383) in an RIDT for H1N1pdm09 diagnostics were able to detect H1N1pdm09 virus through enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Also, the two mAbs exhibited reactivity to hemagglutinins (HAs) of both the H1N1pdm09 and 1918 H1N1 viruses; therefore, the RIDT using the mAbs could detect HAs of H1N1pdm09 and also HAs of 1918 H1N1-like strains. In an extension to our previous study, the epitopes (Sa antigenic site and the interface area of F' and vestigial esterase subdomains on the HA1 domain of HA of H1N1pdm09) recognized by the mAbs were corroborated in depth by IFA with escape-mutants from the mAbs and mapping of the epitopes on the crystal structure of human H1N1 viral HAs. Collectively, these results imply that the mAbs for the RIDT may be suitable for use in studying the immunological properties of H1N1pdm09 viruses and that the Sa antigenic site and the interface area between F' and vestigial esterase subdomains on influenza viral HA recognized by the mAbs are immunologically conserved regions between H1N1pdm09 and 1918 H1N1.

STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy.

Aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to be associated with uncontrolled cell proliferation and suppression of host-immune surveillance. Conversely, silencing STAT3 can have the dual effects of inhibiting cancer cell proliferation and inducing anti-tumor immune responses. Here, we report on the effects of STAT3 silencing on suicide gene therapy with thymidine kinase (tk). STAT3 silencing by siRNA inhibited the proliferation of AGS human gastric cancer cells through G1 cell cycle arrest, decreased levels of immune-suppressive cytokines, and increased levels of immune-activating cytokines. CT26 mouse colon adenocarcinoma cells, in which STAT3 expression was knocked-down by a STAT3 shRNA-containing lentivirus, grew more slowly in syngenic model Balb/c mice than control CT26 cells. Moreover, we found that STAT3 silencing augmented the efficacy of suicide gene therapy in CT26 cell xenografted mice. When we administrated adenoviruses harboring the herpes simplex virus thymidine kinase gene (Ad5.CMV.HSV.tk) into STAT3-silenced CT26 cell tumors, extensive apoptosis was observed and there was a significant reduction in the size of CT26 cell tumors. STAT3 silencing also enhanced the recruitment and cytotoxic activity of CD3(+)CD8(+) T-cells, and changed the cytokine expression pattern of CT26 cell tumors, reflecting augmentation of anti-cancer immune responses. We conclude that combining suicide gene therapy with STAT3 silencing can result in enhanced anti-cancer effects.

A novel splicing variant of mouse interleukin (IL)-24 antagonizes IL-24-induced apoptosis.

Alternative splicing of mRNA enables functionally diverse protein isoforms to be expressed from a single gene, allowing transcriptome diversification. Interleukin (IL)-24/MDA-7 is a member of the IL-10 gene family, and FISP (IL-4-induced secreted protein), its murine homologue, is selectively expressed and secreted by T helper 2 lymphocytes. A novel splice variant of mouse IL-24/FISP, designated FISP-sp, lacks 29 nucleotides from the 5'-end of exon 4 of FISP. The level of FISP-sp expression is 10% of the level of total primary FISP transcription. Unlike FISP, FISP-sp does not induce growth inhibition and apoptosis. FISP-sp is exclusively localized in endoplasmic reticulum, and its expression is up-regulated by endoplasmic reticulum stress. Our results suggest that the novel splicing variant FISP-sp dimerizes with FISP and blocks its secretion and inhibits FISP-induced apoptosis in vivo.