Promising application of monoclonal antibody against chikungunya virus E1-antigen across genotypes in immunochromatographic rapid diagnostic tests.

BACKGROUND: Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. METHODS: Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. RESULTS: The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0-95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. CONCLUSIONS: Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).

Inhibition of N-myristoyltransferase1 affects dengue virus replication.

Dengue virus (DENV) causes dengue fever, a self-limiting disease that could be fatal due to serious complications. No specific treatment is currently available and the preventative vaccine is only partially protective. To develop a potential drug target for dengue fever, we need to understand its biology and pathogenesis thoroughly. N-myristoyltransferase (NMT) is an N-terminal protein lipidation enzyme that catalyzes the covalent cotranslational attachment of fatty acids to the amino-terminal glycine residue of a number of proteins, leading to the modulation of various signaling molecules. In this study, we investigated the interaction of dengue viral proteins with host NMT and its subsequent effect on DENV. Our bioinformatics, molecular docking, and far-western blotting analyses demonstrated the interaction of viral envelope protein (E) with NMT. The gene expression of NMT was strongly elevated in a dependent manner during the viral replication phase in dendritic cells. Moreover, NMT gene silencing significantly inhibited DENV replication in dendritic cells. Further studies investigating the target cell types of other host factors are suggested.

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.

A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody.

Dengue virus (DENV) is an arthropod-borne human pathogen that represents a severe public health threat in both endemic and non-endemic regions. So far, there is no licensed vaccine or specific drugs available for dengue fever. A fifteen-amino-acid-long peptide that includes the NGR motif was chemically synthesized and conjugated with keyhole limpet hemocyanin. A standard immunization protocol was followed for the production of polyclonal antibodies by immunizing rabbits against the synthetic peptide. The immune response elicited high-titer polyclonal antibodies with the reactivity of the anti-peptide antibody against both synthetic peptide and four serotypes of DENV confirmed by DOT-ELISA. Neutralizing activity of anti-peptide antibody was found to be cross-reactive and effective resulting in 60% reduction of infectivity at 1:200 dilution in all four serotypes of DENV. Our findings have the potential to further improve our understanding of virus-host interactions and provide new insights into neutralizing antibodies and could also be used as a drug target.

Molecular genotyping of human papillomavirus l1 gene in low-risk and high-risk populations in Bangkok.

BACKGROUND: Human papillomavirus (HPV) infections in Thailand are a public health concern, but information on HPV infection in sex workers and men who have sex with men (MSM) is limited. The aim of this study was to measure the prevalence and genotype distribution of HPV among low- and high-risk, HIV-negative populations. METHODS: A total of 300 participants were categorized as general women, female sex workers, MSM, and MSM sex workers. Human papillomavirus infections were identified by the Papanicolaou test and nested polymerase chain reaction. A phylogenetic analysis of partial HPV L1 genes was performed. RESULTS: Abnormal cytology was found in 5% of general women, 10% of female sex workers, 24% of MSM, and 28% of MSM sex workers. Human papillomavirus was detected in 9% of general women, 13% of female sex workers, and 30% in both MSM and the MSM sex workers. The prevalence of HPV high-risk genotypes was significantly higher in female sex workers and MSM, whereas low-risk genotypes and genital warts were significantly higher in MSM sex workers. Significantly more patients with genital warts and cervical intraepithelial neoplasia I/anal intraepithelial neoplasia I harbored low-risk genotypes, whereas those with cervical intraepithelial neoplasia II/anal intraepithelial neoplasia II harbored high-risk genotypes. CONCLUSIONS: High- and low-risk HPV genotypes persist in high-risk groups in Bangkok. Some genotypes infecting at-risk populations are not vaccine preventable. These findings may help to elucidate the prevalence of HPV infections in Thailand and serve as the basis for additional investigations into risk factors for these populations.

T helper (Th) 1 and Th2 cytokine expression profile in dengue and malaria infection using magnetic bead-based bio-plex assay.

Dengue and malaria infections are two very common vector-borne diseases annually affecting millions of people around the world. Both diseases show a variety of clinical presentations, ranging from mild symptoms of dengue fever (DF) to severe dengue hemorrhagic fever (DHF) in dengue infection, and low and high parasitemia in malaria infection. T helper (Th)1 and Th2 cytokine expressions in mild and severe forms of dengue virus type-2 (DENV-2) and Plasmodium falciparum infection, were compared to normal human sera using high throughput magnetic bead-based Bio-Plex assay. A significant elevation of Th1 and Th2 cytokines expression [interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha] was detected in DENV-2 and P. falciparum malaria infections compared with normal controls (p < 0.05). DENV-2 infection showed a slight higher expression of Th1 and Th2 cytokines in DHF than DF, except for IL-13. In P. falciparum infection, high parasitemia showed a significantly higher expression of IL-4, IL-10, GM-CSF, and TNF-alpha (p < 0.05). Both DENV-2 and P. falciparum malaria infections manifested high IL-10 expression, greatest among the cytokines examined, and in the severe forms of infection. The results of this study should lead to a better understanding of pathogenesis of dengue infection and P. falciparum malaria.

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.

Impairment of CD4+CD25+ regulatory T cells in C4-deficient mice.

OBJECTIVE: To investigate the association between deficiencies of early components in the classical complement pathway and the development of SLE. METHODS: Forty inbred C57BL/6J mice and 40 knockout C4 complement gene (C4KO) mice, which included 10 mice in each age group (2, 4, 6, and 8 months) were used. The enumeration of CD4+CD25+ Tregs frequencies in bone marrow, spleen and peripheral blood from both normal and C4KO groups were performed by flow cytometry. The expression levels of Foxp3 and TGF-beta in the same tested tissues were measured using real time PCR. The antinuclear antibodies (ANA) were semi-quantitatively measured using ELISA. RESULTS: We report decreased frequencies of CD4+CD25+ Tregs and reduced expression levels of Foxp3 and TGF-beta, which efficiently program the development and function of Tregs, in lymphoid tissues and peripheral blood of C4KO mice. In this study, C4KO mice have higher titers of ANA than those of normal mice. Higher frequencies of mice positive for ANA are also found in older mice. CONCLUSIONS: The deficiency of the C4 gene induces the decreased numbers of Tregs that further increase the production of ANA resulting in the development of an autoimmune disorder. The outcomes of our study help us to understand the association between the deficiency of C4 in the classical complement pathway and development of autoimmune disorder via the role of Tregs.