Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Evaluation of the fusion partner cell line SPYMEG for obtaining human monoclonal antibodies against influenza B virus.

Influenza B virus has been known to infect humans and other animals, including seals. Vaccination efficacy varies across seasons. Human monoclonal antibodies (mAbs) can be useful for developing novel vaccines, guided by epitope analysis, and can be used therapeutically. Hybridoma technology has been used to make mAbs. Here we evaluated SPYMEG as a fusion partner cell line for human mAb generation specific to influenza B hemagglutinin (HA). SPYMEG is a human/murine myeloma partner cell line that has previously been used to generate human mAbs that recognize the HA of influenza A and B viruses. Peripheral blood mononuclear cells were obtained from 16 volunteers, previously vaccinated with the 2014-2015 trivalent seasonal influenza vaccine, and were fused with SPYMEG to yield hybridomas. The resulting hybridomas were screened for antigen-specific antibody secretion and cloned by limiting dilution. We obtained 32 stable clones secreting anti-influenza B HA human IgG, although most of these clones were obtained from one volunteer (SeaV-29) who had a robust immune response. We conclude that SPYMEG is a good fusion partner cell line, although cloning by limiting dilution may lead to significant loss of hybridomas.

Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test.

Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay.

Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito­borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave­excited inductively­coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody­integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV­infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1­4 onto the beads was confirmed using reverse transcription­polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody­integration, represents a significant improvement in the detection of DENV.

Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection.

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcγ receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2-4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcγ receptors, was introduced into D23-1G7C2-IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcγRI and FcγRII-bearing THP-1 cells. By contrast, in FcγRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-α/ß/γ receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Jurkat cell proliferation is suppressed by Chlamydia (Chlamydophila) pneumoniae infection accompanied with attenuation of phosphorylation at Thr389 of host cellular p70S6K.

Chlamydia (Chlamydophila) pneumoniae infects T lymphocytes and multiplies within them. Our previous studies have indicated that C. pneumoniae infection suppresses proliferation of peripheral blood mononuclear cells stimulated with Staphylococcus-enterotoxin B; however, the mechanism of suppression was unclear. In this study, we explored the molecular mechanism involved in C. pneumoniae infection by using human acute T cell leukemia cell line, Jurkat E6-1. Proliferation of Jurkat cells was suppressed in an m.o.i.-dependent manner by C. pneumoniae infection. The suppression by the infection was particularly evident during the initial 24h of the infection, and down modulation of cyclin D3 protein levels were observed at the same time period by immunoblot analysis. The suppression of the Jurkat cell proliferation and the down modulation of cyclin D3 protein level were only induced by viable C. pneumoniae infection, not by exposure to UV-killed or heat-killed C. pneumoniae. Phosphorylations at Thr308 and Ser473 of AKT were induced by C. pneumoniae infection; however, phosphorylation at Thr389 of the downstream kinase, p70S6K was inhibited by unidentified mechanism associated with C. pneumoniae infection. Taking into account that G1 arrest of the C. pneumoniae infected Jurkat cells were not observed and that p70S6K is one of the most important regulators of protein synthesis, it was suggested that the suppression of Jurkat cell proliferation by C. pneumoniae was at least in part mediated by down modulation of protein synthesis through attenuation of Thr389 phosphorylation of p70S6K.

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1.

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.

Sequence analysis of East Asian cagA of Helicobacter pylori isolated from asymptomatic healthy Japanese and Thai individuals.

CagA, especially East Asian type, is one of the most important virulence factors of Helicobacter pylori, which is believed to contribute to the gastric cancer development. There is extreme sequence heterogeneity on 3' region of cagA gene, demonstrated by the sequence analysis of cagA of H. pylori strains isolated from gastric disease patients. However, whether such heterogeneity of the cagA gene sequence is related to the pathogenicity of H. pylori in the gastric cancer development is not certain. Therefore, in this study, the 3' region of cagA sequences isolated from asymptomatic healthy individuals in Japan and Thailand, which show high and low gastric cancer prevalence, respectively, were analyzed and compared with those from patients with gastric cancer. The CagA sequences analysis in 21 and 12 H. pylori DNA samples obtained from Japanese and Thai individuals, respectively, by the molecular phylogenetic method showed that the sequences were more conserved in the Thai individuals (concordance rates among Thai sequences, 93.9-100%) than in the Japanese individuals (concordance rates among Japanese sequences, 82.8-100%) as shown by unrooted neighbor-joining (N-J) consensus trees constructed with the sequence between Asn869 and Ala967 in CagA. CagA sequences in gastric cancer patients were obtained from published data; analysis of these sequences revealed that CagA sequences from almost all Thai individuals were concentrated in one branch. In contrast, CagA sequences from Japanese individuals were uniformly distributed throughout the N-J consensus tree. These results suggest that the difference in the CagA sequences between asymptomatic healthy Japanese and Thai individuals may be linked to the incidence of gastric cancer in Japan and Thailand.

Infection of less virulent Helicobacter pylori strains in asymptomatic healthy individuals in Thailand as a potential contributing factor to the Asian enigma.

In Thailand, gastric cancer incidence is considerably low despite the high prevalence of Helicobacter pylori infection. We investigated the prevalence of H. pylori infection and the genotypes of cagA by using 179 stool specimens obtained from asymptomatic Thai individuals. In this study, the prevalence of H. pylori infection was 43.6%, and the detection rate of cagA-positive strains was 43.5%. In addition, the proportion of the highly virulent East-Asian type of cagA was 7.2%. These results indicate that the low prevalence of cagA-positive H. pylori strain as well as the low prevalence of East-Asian genotype cagA-positive strains may contribute to the low gastric cancer incidence.

A method for assessment of Helicobacter pylori genotype using stool specimens.

Helicobacter pylori infection has been regarded as a major factor associated with the development of gastric diseases. The characterization of infected H. pylori in asymptomatic individuals is important for the prediction of the onset of such diseases. However, because of the difficulty in obtaining gastric biopsy samples, H. pylori in healthy subjects have not been studied sufficiently. Therefore, we tested a noninvasive method for the characterization of H. pylori using stool specimens. This method involved H. pylori antigen detection in stool specimens by immunochromatography; confirmation of H. pylori DNA by real-time PCR that involved the detection of its 16S rRNA gene in the DNA extracted from stool specimens; and nested PCR with genotype-specific primer pairs. A total of 80 samples obtained from asymptomatic subjects were assessed using this method. The results showed that the prevalence of H. pylori in asymptomatic Japanese individuals was 37.5%. The detection rate of the virulence factor gene cagA was 18.8%. Furthermore, all the detected cagA belonged to the highly virulent East-Asian type. These data suggest that the method used in this study is valuable for studying the molecular epidemiology of H. pylori infection in asymptomatic people.

Assessment of East Asian-type cagA-positive Helicobacter pylori using stool specimens from asymptomatic healthy Japanese individuals.

Recent investigations have suggested that CagA, a virulence factor of Helicobacter pylori and known to have multiple genotypes, plays a critical role in the development of stomach cancer. However, the prevalence of cagA-positive H. pylori strains and the cagA genotypes have not been well studied in healthy individuals because of the difficulty in collecting gastric specimens. In the present study, we assessed the prevalence of infection with H. pylori, particularly the strains with the East Asian cagA genotype (which is more potent in causing gastric diseases), among healthy asymptomatic Japanese individuals by a noninvasive method using stool specimens. The H. pylori antigen was detected in 40.3 % of healthy asymptomatic adult individuals (n=186) enrolled in the study. For the detection and genotyping of the cagA gene, DNA was extracted from the stool specimens of these individuals and analysed by PCR. We detected the East Asian cagA genotype in the DNA samples of a significantly high number (63.1 %) of healthy asymptomatic Japanese individuals. These results indicate that a significant number of asymptomatic healthy Japanese individuals were infected with highly virulent H. pylori.

[Analysis of Helicobacter pylori infection in a healthy Panamanian population].

Infection with Helicobacter pylori carrying a cagA virulence gene is a potential risk factor for gastric cancer. Since obtaining clinical isolates from healthy subjects is difficult, epidemiological analysis of cagA-positive H. pylori in healthy populations is not well studied. To assess H. pylori infection in a healthy population, we applied coprodiagnostic analysis to determine H. pylori infection prevalence. H. pylori antigen in stool specimens was detected by immunochrmoatograpy in 54.1% of participants. The average age in the H. pylori-positive group was higher than in the negative group. The alcohol intake group showed a significantly higher H. pylori prevalence. The genotype of cagA was assessed by polymerase chain reaction after bacterial DNA extraction from H. pylori-positive stool specimens. Only 20.0% of participants in sample in which H. pylori DNA was detected were infected with cagA-positive H. pylori, but all cagA-positive H. pylori was of the East-Asian genotype. These results indicate that a significant number of healthy adults in Panama are infected with H. pylori, but the majority of bacteria are less virulent. Alcohol intake may be a risk factor for H. pylori infection.