Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody.

Dengue virus (DENV), transmitted by mosquitoes, is classified into four serotypes (DENV1-4) and typically causes mild, self-limiting symptoms upon initial infection. However, secondary infection can lead to severe symptoms due to antibody-dependent enhancement (ADE). To address this, anti-DENV antibodies are being developed with the goal of neutralizing infection without ADE activity. Previous attempts using a 54_hG1 antibody from CHO-K1 mammalian cells resulted in ADE induction, increasing viral infection. This study aimed to express the D54 monoclonal antibody in Nicotiana benthamiana. The plant-produced antibody had a similar neutralizing profile to the previous 54_hG1 antibody. Notably, the ADE activities of the plant-derived antibody were successfully eliminated, with no sign of viral induction. These findings suggest that N. benthamiana could be a source of therapeutic DENV antibodies. The method offers several advantages, including lower ADE, cost-effectiveness, simple facility requirements, scalability, and potential industrial-scale production in GMP facilities.

Potential Protective Effect of Dengue NS1 Human Monoclonal Antibodies against Dengue and Zika Virus Infections.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1ß, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.

Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

Modulating biomolecular networks in cells with peptides and proteins has become a promising therapeutic strategy and effective biological tools. A simple and effective reagent that can bring functional proteins into cells can increase efficacy and allow more investigations. Here we show that the relatively non-toxic and non-immunogenic oxidized carbon black particles (OCBs) prepared from commercially available carbon black can deliver a 300 kDa protein directly into cells, without an involvement of a cellular endocytosis. Experiments with cell-sized liposomes indicate that OCBs directly interact with phospholipids and induce membrane leakages. Delivery of human monoclonal antibodies (HuMAbs, 150 kDa) with specific affinity towards dengue viruses (DENV) into DENV-infected Vero cells by OCBs results in HuMAbs distribution all over cells' interior and effective viral neutralization. An ability of OCBs to deliver big functional/therapeutic proteins into cells should open doors for more protein drug investigations and new levels of antibody therapies and biological studies.

Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand.

Chikungunya virus was isolated in Thailand, 2010.

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008-2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

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.

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.