Evolution of neutralizing antibodies through vaccination and breakthrough infections in the era of COVID-19 endemicity.

Despite a high vaccination rate, the COVID-19 pandemic continues with immune-evading Omicron variants. The success of additional antigenic stimulation through breakthrough infection (BI) and updated vaccination in overcoming antigenic imprinting needs to be determined. Participants in a long-term follow-up cohort of healthcare worker (HCW) vaccinee were categorized according to their infection/vaccination status. Anti-SARS-CoV-2 spike/nucleocapsid protein antibodies were measured, and plaque reduction neutralization tests (PRNTs) against wild-type (WT), BA.5, BN.1, and XBB.1.5 were conducted. The neutralization activity of intravenous immunoglobulin (IVIG) products was evaluated to assess the immune status of the general population. Ninety-five HCWs were evaluated and categorized into seven groups. The WT PRNT ND50 value was highest regardless of infection/vaccination status, and groups with recent antigenic stimulation showed high PRNT titers overall. Groups with double Omicron stimulation, either by BI plus BA.4/5 bivalent vaccination or repeated BI, exhibited significantly higher BA.5 and BN.1 PRNT to WT PRNT ratios than those with single Omicron stimulation. Overall group immunity was estimated to be boosted in January 2023, reflecting the effect of the BA.4/5 bivalent booster and additional BIs, but slightly declined in June 2023. A substantial increase in the antibody concentrations of IVIG products was noticed in 2022, and recently produced IVIG products exhibited a substantial level of cross-reactive neutralizing activity against emerging variants. Neutralizing activity against emerging variants could be enhanced by repeated antigenic stimulation via BI and/or updated vaccination. Overall group immunity was elevated accordingly, and IVIG products showed substantial activity against circulating strains.

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.

Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain H0001, Isolated from a Tularemia Patient in the Republic of Korea.

Francisella tularensis is the etiological agent of the zoonosis tularemia. Here, we report the draft genome sequence of F. tularensis subsp. holarctica H0001, which was isolated from a tularemia patient in the Republic of Korea.

Genomic evidence of SARS-CoV-2 reinfection in the Republic of Korea.

As the coronavirus disease 2019 (COVID-19) pandemic continues, reinfection is likely to become increasingly common. However, confirming COVID-19 reinfection is difficult because it requires whole-genome sequencing of both infections to identify the degrees of genetic differences. Since the first reported case of reinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Republic of Korea in April 2020, four additional cases were classified as suspected reinfection cases. We performed whole-genome sequencing of viral RNA extracted from swabs obtained at the initial infection and reinfection stages of these four suspected cases. The interval between initial infection and reinfection of all four suspected cases was more than 3 months. All four patients were young (10-29 years), and they displayed mild symptoms or were asymptomatic during the initial infection and reinfection episodes. The analysis of genome sequences combined with the epidemiological results revealed that only two of the four cases were confirmed as reinfection, and both were reinfected with the Epsilon variant. Due to the prolonged COVID-19 pandemic, the possibility of reinfections with SARS-CoV-2 variants is increasing, as reported in our study. Therefore, continuous monitoring of cases is necessary.

Genomic epidemiology reveals the reduction of the introduction and spread of SARS-CoV-2 after implementing control strategies in Republic of Korea, 2020.

Genomic epidemiology is a core component in investigating the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, the efficacy of control strategies in South Korea was evaluated using genomic epidemiology based on viral genome sequences of 2,065 SARS-CoV-2 cases identified in South Korea from January 2020 to December 2020. Phylogenetic analysis revealed that the majority of viruses introduced from inbound travelers did not further spread throughout South Korea; however, four distinct subgroups (KR.1-4, belonging to B.1.497, B.1, K.1 and B.41) of viruses caused local epidemics. After the introduction of enhanced social distancing, the viral population size and daily case numbers decreased, and KR.2-4 subgroups were extinguished from South Korea. Nevertheless, there was a subsequent increase in KR.1 subgroups after the downgrading of social distancing level. These results indicate that the international traveler quarantine system implemented in South Korea along with social distancing measures efficiently reduced the introduction and spread of SARS-CoV-2, but it was not completely controlled. An improvement of control strategies will be required to better control SARS-CoV-2, its variants, and future pandemic viruses.

Corrigendum to: Genomic epidemiology reveals the reduction of the introduction and spread of SARS-CoV-2 after implementing control strategies in Republic of Korea, 2020.

[This corrects the article DOI: 10.1093/ve/veab077.].

Early Laboratory Preparedness of the Korea Disease Control and Prevention Agency and Response to Unknown Pneumonia Outbreak from Wuhan, China, in January 2020.

We report the response process of the Laboratory Analysis Task Force (LATF) for Unknown Disease Outbreaks (UDOs) at the Korea Disease Control and Prevention Agency (KDCA) during January 2020 to coronavirus disease 2019 (COVID-19), which developed as a UDO in Korea. The advanced preparedness offered by the laboratory diagnostic algorithm for UDOs related to respiratory syndromes was critical for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enabled us to establish and expand the diagnostic capacity for COVID-19 on a national scale in a timely manner.

Genetic Characterization of Clostridium botulinum Isolated from the First Case of Infant Botulism in Korea.

Botulism is a neuroparalytic disease caused by a neurotoxin produced by Clostridium botulinum. This study aimed to genetically characterize C. botulinum strain isolated from the first case of infant botulism in Korea reported on June 17, 2019. We isolated C. botulinum strain CB-27 from a stool sample of the patient and analyzed the toxin types and toxin gene cluster compositions of the strain using a mouse bioassay, real-time PCR, and genome sequencing. Toxin gene cluster analysis showed that strain CB-27 possesses a C. botulinum neurotoxin type A harboring an unexpressed B gene. Although the nucleotide and amino acid sequences of toxin genes as well as the toxin gene cluster arrangements in strain CB-27 were identical to those of the known strain CDC_69094, the total nucleotide sequences of the toxin gene clusters of CB-27 differed from those of CDC_69094 by 0.47%, indicating genetic diversity of toxin gene clusters of CB-27 among other previously reported C. botulinum strains. To our knowledge, this is the first description of a C. botulinum strain with two separate toxin gene clusters in Korea.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103.

BACKGROUND: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. RESULTS: Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. CONCLUSIONS: We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

Genomic Surveillance of SARS-CoV-2: Distribution of Clades in the Republic of Korea in 2020.

Since a novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019, there has been a rapid global spread of the virus. Genomic surveillance was conducted on samples isolated from infected individuals to monitor the spread of genetic variants of SARS-CoV-2 in Korea. The Korea Disease Control and Prevention Agency performed whole genome sequencing of SARS-CoV-2 in Korea for 1 year (January 2020 to January 2021). A total of 2,488 SARS-CoV-2 cases were sequenced (including 648 cases from abroad). Initially, the prevalent clades of SARS-CoV-2 were the S and V clades, however, by March 2020, GH clade was the most dominant. Only international travelers were identified as having G or GR clades, and since the first variant 501Y.V1 was identified (from a traveler from the United Kingdom on December 22nd, 2020), a total of 27 variants of 501Y.V1, 501Y.V2, and 484K.V2 have been classified (as of January 25th, 2021). The results in this study indicated that quarantining of travelers entering Korea successfully prevented dissemination of the SARS-CoV-2 variants in Korea.

2019 Tabletop Exercise for Laboratory Diagnosis and Analyses of Unknown Disease Outbreaks by the Korea Centers for Disease Control and Prevention.

OBJECTIVES: The Korea Centers for Disease Control and Prevention has published "A Guideline for Unknown Disease Outbreaks (UDO)." The aim of this report was to introduce tabletop exercises (TTX) to prepare for UDO in the future. METHODS: The UDO Laboratory Analyses Task Force in Korea Centers for Disease Control and Prevention in April 2018, assigned unknown diseases into 5 syndromes, designed an algorithm for diagnosis, and made a panel list for diagnosis by exclusion. Using the guidelines and laboratory analyses for UDO, TTX were introduced. RESULTS: Since September 9th, 2018, the UDO Laboratory Analyses Task Force has been preparing TTX based on a scenario of an outbreak caused by a novel coronavirus. In December 2019, through TTX, individual missions, epidemiological investigations, sample treatments, diagnosis by exclusions, and next generation sequencing analysis were discussed, and a novel coronavirus was identified as the causal pathogen. CONCLUSION: Guideline and laboratory analyses for UDO successfully applied in TTX. Conclusions drawn from TTX could be applied effectively in the analyses for the initial response to COVID-19, an ongoing epidemic of 2019 - 2020. Therefore, TTX should continuously be conducted for the response and preparation against UDO.

Evaluation of the protective efficacy of recombinant protective antigen vaccine (GC1109)-immunized human sera using passive immunization in a mouse model.

The protective efficacy of human sera from vaccinated individuals with a new recombinant protective antigen anthrax vaccine (GC1109) against lethal spore challenge was evaluated in a mouse model. Eighteen human sera were selected from the vaccinated individuals based on their toxin neutralizing assay (TNA) titer (ED50 of 55 to 668). The selected sera were diluted and passively transferred to A/J mice and the mice were subsequently challenged with 100 × LD50 of Bacillus anthracis Sterne spores. The correlation between the survival rate of passively immunized mice and the TNA ED50 of transferred sera was presented (r = 0.873, P-value < 0.001). The estimated TNA titer for 50% survival rate against lethal challenge was 197 (95% confidence interval of 149 and 260). The result suggest that GC1109 is protective against exposure to B. anthracis and the TNA titer of vaccinated serum can be an indicator for protective efficacy.

Development of a double-antibody sandwich ELISA for sensitive detection of Yersinia pestis.

We developed a biotin-streptavidin-based sandwich ELISA for the sensitive and specific detection of Yersinia pestis. In this assay, the F1 capsular protein and Y. pestis were captured by anti-F1 mouse monoclonal antibody followed by detection with biotinylated-anti-F1 rabbit polyclonal antibody and HRP-conjugated streptavidin. The developed F1 ELISA could detect not only the F1 protein up to 29 and 17 pg/ml but also Y. pestis up to 177.8 and 129.2 CFU/ml in PBS buffer and human serum, respectively. In addition, the F1 ELISA did not show any cross-reactivity with various proteins and bacterial strains.

SERS-Based Immunoassays for the Detection of Botulinum Toxins A and B Using Magnetic Beads.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h.

Immunogenicity and safety of a novel recombinant protective antigen anthrax vaccine (GC1109), a randomized, single-blind, placebo controlled phase II clinical study.

BACKGROUND: The demand on effective and safe anthrax vaccine is increasing as a part of the preparedness for possible bioterrorism in the future. We performed a randomized, single-blind, placebo controlled phase II clinical study to evaluate the immunogenicity and safety of a novel recombinant protective antigen (rPA) anthrax vaccine, GC1109, in healthy adult volunteers. METHODS: Participants were randomized to experiment groups (0.3 mL, 0.5 mL, and 1.0 mL of GC1109) or placebo group (normal saline 0.5 mL) in 2:2:2:1 ratio. They received respective vaccines intramuscularly at 0, 4 and 8 weeks. Immunogenicity was evaluated by seroconversion rate and geometric mean titer (GMT) of lethal toxin neutralizing assay (TNA) and anti-PA IgG by ELISA. Safety was assessed by laboratory tests, and solicited and unsolicited adverse events on diary cards. RESULTS: 30, 29, 30 participants were randomized to 0.3, 0.5, and 1.0 mL of GC1109 groups, respectively, while 15 to placebo group. 92 participants received all three doses. In per-protocol analysis, TNA GMTs at week 12 were 296.5, 285.2, and 433.2 in the three groups, respectively. Seroconversion rates measured by ELISA were 100% at week 12 in the three groups. Local and systemic vaccine-related adverse events were frequent; however, most of them were mild, and no serious events were observed. CONCLUSIONS: A new rPA anthrax vaccine GC1109 was immunogenic after three doses of intramuscular administration, and was well-tolerated.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Muramyl dipeptide potentiates a Bacillus anthracis poly-γ-d-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells.

Poly-γ-d-glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti-phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll-like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell-wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF-α, IL-6, MCP-1, and MIP1-α, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA-induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2- or nucleotide-binding oligomerization domain (NOD) 2-knockout DCs. Additionally, MDP augmented PGA-induced MAP kinases and NF-κB activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF-κB inhibitors attenuated MDP enhancement of PGA-induced cytokine production. In addition, co-culture of splenocytes and PGA/MDP-matured DCs induced higher expression of IL-2 and IFN-γ compared to that of splenocytes and PGA-matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF-κB pathways, subsequently leading to lymphocyte activation.