Safety assessments of recombinant DTaP vaccines developed in South Korea.

Purpose: Pertussis bacteria have many pathogenic and virulent antigens and severe adverse reactions have occurred when using inactivated whole-cell pertussis vaccines. Therefore, inactivated acellular pertussis (aP) vaccines and genetically detoxified recombinant pertussis (rP) vaccines are being developed. The aim of this study was to assess the safety profile of a novel rP vaccine under development in comparison to commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccines. Materials and Methods: The two positive control DTaP vaccines (two- and tri-components aP vaccines) and two experimental recombinant DTaP (rDTaP) vaccine (two- and tri-components aP vaccines adsorbed to either aluminum hydroxide or purified oat beta-glucan) were used. Temperature histamine sensitization test (HIST), indirect Chinese hamster ovary (CHO) cell cluster assay, mouse-weight-gain (MWG) test, leukocytosis promoting (LP) test, and intramuscular inflammatory cytokine assay of the injection site performed for safety assessments. Results: HIST results showed absence of residual pertussis toxin (PTx) in both control and experimental DTaP vaccine groups, whereas in groups immunized with tri-components vaccines, the experimental tri-components rDTaP absorbed to alum showed an ultra-small amount of 0.0066 IU/mL. CHO cell clustering was observed from 4 IU/mL in all groups. LP tests showed that neutrophils and lymphocytes were in the normal range in all groups immunized with the two components vaccine. However, in the tri-components control DTaP vaccine group, as well as two- and tri-components rDTaP with beta-glucan group, a higher monocyte count was observed 3 days after vaccination, although less than 2 times the normal range. In the MWG test, both groups showed changes less than 20% in body temperature and body weight before the after the final immunizations. Inflammatory cytokines within the muscle at the injection site on day 3 after intramuscular injection revealed no significant response in all groups. Conclusion: There were no findings associated with residual PTx, and no significant differences in both local and systemic adverse reactions in the novel rDTaP vaccine compared to existing available DTaP vaccines. The results suggest that the novel rDTaP vaccine is safe.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Protective efficacy of a bivalent H5 influenza vaccine candidate against both clades 2.3.2.1 and 2.3.4.4 high pathogenic avian influenza viruses in SPF chickens.

Worldwide, high pathogenic avian influenza viruses belonging to clades 2.3.4.4 and 2.3.2.1 have been circulating in both poultry and wild birds. Since 2018, Korea has built a national antigen bank to ensure preparedness in an emergency. In this study, we developed a bivalent vaccine candidate containing antigens derived from two reassortant KA435/2.3.2.1d and H35/2.3.4.4b strains for Korean national antigen bank. We evaluated its immunogenicity and protective efficacy in specific pathogen free chickens. The two vaccine strains, rgKA435-H9N2 PB2/2.3.2.1d and rgH35/2.3.4.4b, both of which were generated successfully by reverse genetics, were highly immunogenic (titres of haemagglutination inhibition: 8.3 and 8.4 log2, respectively) and showed good protective efficacy (100 and 147 50% protective dose, respectively) against lethal challenge with wild-type virus when delivered as a 1:1 mixture. Notably, the vaccine provided complete protection against viral shedding at a full dose (512 HAU) and a 1/10 dose (51.2 HAU), with no clinical signs, after challenge with H35/2.3.4.4b. The bivalent vaccine developed in this study may reduce the cost of vaccine production and could be used as a H5 subtype avian influenza vaccine candidate against two clades simultaneously.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line.

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.

Current situation and control strategies of H9N2 avian influenza in South Korea.

The H9N2 avian influenza (AI) has become endemic in poultry in many countries since the 1990s, which has caused considerable economic losses in the poultry industry. Considering the long history of the low pathogenicity H9N2 AI in many countries, once H9N2 AI is introduced, it is more difficult to eradicate than high pathogenicity AI. Various preventive measures and strategies, including vaccination and active national surveillance, have been used to control the Y439 lineage of H9N2 AI in South Korea, but it took a long time for the H9N2 virus to disappear from the fields. By contrast, the novel Y280 lineage of H9N2 AI was introduced in June 2020 and has spread nationwide. This study reviews the history, genetic and pathogenic characteristics, and control strategies for Korean H9N2 AI. This review may provide some clues for establishing control strategies for endemic AIV and a newly introduced Y280 lineage of H9N2 AI in South Korea.

Updating the National Antigen Bank in Korea: Protective Efficacy of Synthetic Vaccine Candidates against H5Nx Highly Pathogenic Avian Influenza Viruses Belonging to Clades 2.3.2.1 and 2.3.4.4.

Since 2018, Korea has been building an avian influenza (AI) national antigen bank for emergency preparedness; this antigen bank is updated every 2 years. To update the vaccine strains in the antigen bank, we used reverse genetics technology to develop two vaccine candidates against avian influenza strains belonging to clades 2.3.2.1d and 2.3.4.4h, and then evaluated their immunogenicity and protective efficacy in SPF chickens challenged with H5 viruses. The two vaccine candidates, named rgCA2/2.3.2.1d and rgES3/2.3.4.4h, were highly immunogenic, with hemagglutination inhibition (HI) titers of 8.2−9.3 log2 against the vaccine strain, and 7.1−7.3 log2 against the lethal challenge viruses (in which the HA genes shared 97% and 95.4% homology with that of rgCA2/2.3.2.1d and rgES3/2.3.4.4h, respectively). A full dose of each vaccine candidate provided 100% protection against the challenge viruses, with a reduction in clinical symptoms and virus shedding. A 1/10 dose provided similar levels of protection, whereas a 1/100 dose resulted in mortality and virus shedding by 7 dpi. Moreover, immunity induced by the two vaccines was long lasting, with HI titers of >7 log2 against the vaccine strain remaining after 6 months. Thus, the two vaccine candidates show protective efficacy and can be used to update the AI national antigen bank.

Distribution of emm genotypes in group A streptococcus isolates of Korean children from 2012 to 2019.

OBJECTIVES: Changes in the epidemiology of group A streptococcus (GAS) infection is related to emm genotype. We studied the distribution of emm genotypes and their antibiotic susceptibility among Korean children. METHODS: Isolates from children with GAS infection between 2012 and 2019 were collected. emm typing and cluster analysis was performed according to the Centers for Disease Control emm cluster classification. Antimicrobial susceptibility was tested using the E-test and resistance genes were analyzed for macrolide resistant phenotypes. RESULTS: Among 169 GAS isolates, 115 were from children with scarlet fever. Among invasive isolates, emm1 (6/22, 27.3%), emm12 (4/22, 18.2%), and emm4 (4/22, 18.2%) were most common. In scarlet fever, although emm4 (38/115, 33.0%) was the most prevalent throughout the study period, emm4 was replaced by emm3 (28/90, 31.1%) during an outbreak in 2017-2018. Among all isolates, only 2 (1.2%) exhibited erythromycin resistance and harbored both ermA and ermB genes. CONCLUSIONS: In this analysis of GAS isolated from Korean children, emm1 was the most prevalent in invasive infection. In scarlet fever, emm4 was prevalent throughout the study period, with an increase in emm3 during 2017-2018. GAS isolates during 2012-2019 demonstrated low erythromycin resistance.

Emergence of clade 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza virus in South Korea during late 2021.

High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety.

For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3'-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.

Development of a recombinant H9N2 influenza vaccine candidate against the Y280 lineage field virus and its protective efficacy.

Since June 2020, a new H9N2 virus of the Y280 lineage has been epidemic in Korea. Initially, a Korean commercial vaccine against the Y280 and Y439 lineages of H9N2 was evaluated for use in SPF chickens. A single vaccination did not protect chickens against virus of the Y280 lineage, with no significant reduction in virus shedding and a 37.5% inhibition in virus recovery rate in cecal tonsil. rgHS314 was selected as a vaccine candidate, showing immunogenicity in SPF chickens, and was highly productive in eggs. Moreover, rgHS314 protected with high levels of protective immunity and significantly reduced virus shedding, with 100% and 83.3% inhibition of virus recovery in the cecal tonsil against homologous and heterologous challenge viruses, respectively. Taken together, these data suggest that a single vaccination with this recombinant vaccine candidate could elicit cross-reactive immune responses capable of protecting chickens against H9N2 viruses of the Y439 and Y280 lineages.

Cross-protective efficacy of inactivated whole influenza vaccines against Korean Y280 and Y439 lineage H9N2 viruses in mice.

Since June 2020, the Y280 lineage H9N2 virus, which is distinct from the previously endemic Y439 lineage, has been circulating in poultry in Korea. In this study, we developed two whole inactivated vaccines, rgHS314 and vac564, against the Y280 and Y439 lineages, respectively, and evaluated their immunogenicity and protective efficacy against homologous or heterologous viral challenge in mice. Serum neutralizing antibody titers in the rgHS314-vaccinated group were higher (68 ± 8.4 10log2) than in the vac564-vaccinated group (18 ± 8.4 10log2). In homologous challenge, rgHS314 conferred 100% protection, with no severe clinical signs, no body weight loss, and no viral replication in any tissues tested except the nasal turbinate. Viral replication in the lungs at 1, 3, 5, and 7 days post-infection (dpi) was significantly lower than in the sham group (p < 0.01). By contrast, all mice in the sham group were dead by 8 dpi with severe clinical signs and weight loss. Likewise, vac564 conferred 100% protection with no weight loss and with significantly lower viral replication in the lung than in the sham group at 3 dpi (p < 0.01). However, both vaccines showed partial protection in heterologous challenge. Our results suggest that both the rgHS314 and vac564 vaccines could be candidate vaccines for further evaluation in humans.

The complete chloroplast genome of Castanopsis sieboldii (Makino) Hatus (Fagaceae).

Castanopsis sieboldii (Makino) Hatus is an evergreen tree that distributes in Eastern Asia including Islands of Korea and Japan. The chloroplast genome of C. sieboldii was successfully sequenced. Its length is 160,705 bp long (GC ratio is 36.8%) and has four subregions: 90,821 bp of large single copy (34.6%) and 19,014 bp of small single copy (30.8%) regions are separated by 25,075 bp of inverted repeat (42.8%) regions including 134 genes (89 protein-coding genes, eight rRNAs, and 37 tRNAs). Interspecific variations of Castanopsis are at a moderate level in comparison to those of the other genera. Phylogenetic trees show that C. sieboldii chloroplast genome was clustered with the other two Castanopsis species.

Single dose of multi-clade virus-like particle vaccine protects chickens against clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6-8.5 log2). Moreover, the multi-clade VLP showed high productivity (128-256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, respectively), and effective antibody response was maintained for 2-3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

Protection of layers and breeders against homologous or heterologous HPAIv by vaccines from Korean national antigen bank.

Korean government has selected and stocked five type antigens of two clades as Korean national antigen bank having high possibility of introduction to Korea. We aimed to evaluate the efficacy of the clade 2.3.2.1c and 2.3.4.4c H5Nx vaccines from the Korean avian influenza (AI) national antigen bank for emergency preparedness for their potency and protective efficacy against lethal homologous and heterologous viruses in layer and breeder chickens practically. The PD50 (dose of vaccine that protects 50% of chickens from viral challenge) of all vaccinated groups was >50, which was satisfied with minimum antigen requirement of OIE, and the PD50 levels of the two vaccines differed depending on strain and chicken breed. In homologous challenge, all vaccinated groups exhibited 100% survival with no clinical symptoms and high levels of pre-challenge protective immunity (7.2-8.5 log2), although they did not completely prevent virus shedding. On the other hand, against heterologous virus challenge, vaccinated animals exhibited 62.5-80% survival with lower antibody titers (2.3-3.4 log2) and a longer period of virus shedding (14 days post infection [dpi]). Our results suggest that the clade 2.3.2.1c and 2.3.4.4c H5Nx vaccines are good candidates for emergency vaccination of commercial chickens and support the idea that close genetic matching between vaccine and challenge virus provides the best protection.

Sales and immunogenicity of commercial vaccines to H9N2 low pathogenic avian influenza virus in Korea from 2007 to 2017.

The present study was conducted to monitor sales activity and immunogenicity of commercial H9N2 vaccines produced in Korea from 2007 to 2017. Recorded sales of H9N2 vaccine were around 671 million doses, with 10 million doses sold in 2007, rising to a peak of 93 million doses in 2016, with a slight fall in 2017. Multivalent combined vaccines made up around 90% of all vaccine sales, and around 30% of all vaccines were distributed by regional governments for free. The regional vaccination rate was the highest in Gyeonggi and Chungnam, respectively with proportional to the population of layer and breeder chickens. There have been no cases of field infection since 2009. The mean antibody titer was 5.82 log2 across the study period. Our results suggest that continuous genetic monitoring of H9N2 viruses circulating in the field and updating the vaccine seed strain periodically are necessary in order to control H9N2 outbreaks.

Protective efficacy of vaccines of the Korea national antigen bank against the homologous H5Nx clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

The occurrence of severe outbreaks of highly pathogenic avian influenza in Korea led to establishment of a national antigen bank for emergency preparedness. Here, we developed five vaccines for this bank (clade 2.3.2.1C, clade 2.3.4.4A, B, C, and D) by reverse genetics, inactivated them with formalin, and evaluated the protective efficacy and potency of serial dilutions against lethal homologous challenge in specific-pathogen-free chickens. After vaccination with one dose, each vaccine resulted in 100% survival, with no clinical symptoms, or lack of detectable virus shedding, and high levels of pre-challenge protective immunity (8.4-10.2 log2). After vaccination with one-tenth of the full dose, protection was similar to that with the full dose. After vaccination with one-hundredth of the initial dose, survival was 20-80%, and all vaccines showed virus shedding. Four vaccines (excluding clade 2.3.2.1C) had satisfactory potency. In antibody-persistence tests, all vaccines maintained long-lasting protective immunity. Our results suggest that inactivated reverse-genetics vaccines genetically matched to outbreak viruses provide adequate protection after a single vaccination.

Changes in clinical features in Henoch-Schönlein purpura during three decades: an observational study at a single hospital in Korea.

OBJECTIVE: It is unknown whether epidemiological or clinical characteristics of Henoch-Schönlein purpura (HSP) have changed over time. This study aimed at evaluating the epidemiological and clinical changes of HSP during 3 decades. METHODS: We retrospectively analyzed the data of 515 children with HSP (0-15 years of age) between 1987 and 2015. We compared the two HSP patient groups: those admitted from 1987 to 1996 (group A, 238 cases) and those admitted from 2006 to 2015 (group B, 98 cases), apart a decade. RESULTS: In total 515 patients, the mean age was 6.5 ± 3.0 years and the male-to-female ratio was 1.2:1 (278:237). The age distribution showed a peak at age 5 with a bell-shaped distribution pattern. The annual number of cases varied in each year with a trend of reduced cases in the recent decade. There were less cases during the summer season. Purpura, gastrointestinal involvement, joint involvement, and renal involvement were found in 100%, 56%, 38%, and 18% of the patients, respectively. In comparison between the two groups, there were similar findings in mean age, age distribution, and seasonal distribution. However, the hospitalization stay was longer, and the proportion of recurrent cases (14 cases vs. 0 case) and proteinuria (15% vs. 3%) were higher in the group A than in the group B. CONCLUSIONS: Long-term epidemiologic features of HSP were similar to those in other countries. Clinical manifestations of HSP showed a trend towards a less severe clinical phenotype over time in Deajeon, Korea. Key Points • It is unknown whether epidemiological and clinical traits of Henoch-Schönlein purpura (HSP) have changed over time. • We reported that clinical manifestations of HSP have changed to milder phenotype through a long-term observation of three decades at a single hospital in Daejeon, South Korea. • Clinical phenotype of infection-related diseases, including HSP, may be changed over time, and the etiology and the reason of clinical changes over time remain to be solved.