Genomic sequencing and neutralizing serological profiles during acute dengue infection: A 2017 cohort study in Nepal.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM + IgG + ; 7 were DENV IgM + IgG - , 2 were IgM - IgG + , and 7 were IgM - IgG - by specific ELISAs. Seven of the 21 NS1+ sera were RNA+ by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Serology analysis suggested that 12 (26%) and 19 (40%) of the 49 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort, and the RNA analyses further indicate that DENV2 was the likely dominant serotype circulating in Nepal in 2017. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

Transcriptome Analysis Reveals the Induction of Apoptosis-Related Genes by a Monoclonal Antibody against a New Epitope of CD99 on T-Acute Lymphoblastic Leukemia.

CD99 was demonstrated to be a potential target for antibody therapy on T-acute lymphoblastic leukemia (T-ALL). The ligation of CD99 by certain monoclonal antibodies (mAbs) induced T-ALL apoptosis. However, the molecular basis contributing to the apoptosis of T-ALL upon anti-CD99 mAb engagement remains elusive. In this study, using our generated anti-CD99 mAb clone MT99/3 (mAb MT99/3), mAb MT99/3 engagement strongly induced apoptosis of T-ALL cell lines, but not in non-malignant peripheral blood cells. By transcriptome analysis, upon mAb MT99/3 ligation, 13 apoptosis-related genes, including FOS, TNF, FASLG, BCL2A1, JUNB, SOCS1, IL27RA, PTPN6, PDGFA, NR4A1, SGK1, LPAR5 and LTB, were significantly upregulated. The epitope of CD99 recognized by mAb MT99/3 was then identified as the VDGENDDPRPP at residues 60-70 of CD99, which has never been reported. To the best of our knowledge, this is the first transcriptome data conducted in T-ALL with anti-CD99 mAb engagement. These findings provide new insights into CD99 implicated in the apoptosis of T-ALL. The identification of a new epitope and apoptosis-related genes that relate to the induction of apoptosis by mAb MT99/3 may serve as a new therapeutic target for T-ALL. The anti-CD99 mAb clone MT99/3 might be a candidate for further development of a therapeutic antibody for T-ALL therapy.

Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future.

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes type II.

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1ß. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Effect of adenosine and cordycepin on recombinant antibody production yields in two different Chinease hamster ovary cell lines.

In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO-DHFR- and GS-CHO knockout (GS-KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO-DHFR- cells but extended culture duration and significantly increased antibody titers in GS-KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS-KO CHO cells in medium- to large-scale batches. In case of cordycepin, a derivative of adenosine, CHO-DHFR- cells required higher concentration of cordycepin than GS-KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO-DHFR- cell cultures without glucose feeding.

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications.

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 µM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.

Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies.

The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, deleting only 1 GS gene could potentially result in the activation of other GS genes, consequently reducing the selection efficiency. Therefore, in this study, both GS genes identified on chromosome 5 (GS5) and 1 (GS1) of CHO-S and CHO-K1, were deleted using CRISPR/Cpf1. Both single and double GS-KO CHO-S and K1 showed robust glutamine-dependent growth. Next, the engineered CHO cells were tested for their efficiency of selection of stable producers of two therapeutic antibodies. Analysis of pool cultures and subclones after a single round of 25 µM methionine sulfoxinime (MSX) selection indicated that for CHO-K1 the double GS5,1-KO was more efficient as in the case of a single GS5-KO the GS1 gene was upregulated. In CHO-S, on the other hand, with an autologously lower level of expression of both variants of GS, a single GS5-KO was more robust and already enabled selection of high producers. In conclusion, CRISPR/Cpf1 can be efficiently used to knock out GS genes from CHO cells. The study also indicates that for the generation of host cell lines for efficient selection, the initial characterisation of expression levels of the target gene as well as the identification of potential escape mechanisms is important.

Effects of collagen, chitosan and mixture on fibroblast responses and angiogenic activities in 2D and 3D in vitro models.

Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube-like network formation and upregulated VE-cadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 µg/mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 µg/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary-like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p-Fak, p-Akt and Cdk5 whereas chitosan upregulated p-Fak and Cdk5 expressions. Comparing to the single treatments, p-Fak, p-Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen-chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.

Production and applications of fluorobody from redox-engineered Escherichia coli.

Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2-8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets. KEY POINTS: • E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody) • Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal • This platform can be used to produce fluorobodies for numerous purposes.

A Bench-Top Approach for Isolation of Single Antibody Producing Chinese Hamster Ovary (CHO) Cells Using a Microwell-Based Microfluidic Device.

Genetically-modified monoclonal cell lines are currently used for monoclonal antibody (mAbs) production and drug development. The isolation of single transformed cells is the main hindrance in the generation of monoclonal lines. Although the conventional limiting dilution method is time-consuming, laborious, and skill-intensive, high-end approaches such as fluorescence-activated cell sorting (FACS) are less accessible to general laboratories. Here, we report a bench-top approach for isolating single Chinese hamster ovary (CHO) cells using an adapted version of a simple microwell-based microfluidic (MBM) device previously reported by our group. After loading the cell suspension to the device, the electrostatically trapped cells can be viewed under a microscope and transferred using a micropipette for further clone establishment. Compared to the conventional method, the invented approach provided a 4.7-fold increase in the number of single cells isolated per round of cell loading and demonstrated a 1.9-fold decrease in total performing time. Additionally, the percentage of correct single-cell identifications was significantly improved, especially in novice testers, suggesting a reduced skill barrier in performing the task. This novel approach could serve as a simple, affordable, efficient, and less skill-intensive alternative to the conventional single-cell isolation for monoclonal cell line establishment.

Targeting acute myeloid cell surface using a recombinant antibody isolated from whole-cell biopanning of a phage display human scFv antibody library.

To discover new therapeutic antibodies for treatment of acute myeloid leukemia (AML) without the requirement of a known antigen, a human single-chain variable fragment (scFv) library was used to isolate novel antibody fragments recognizing HL-60 AML cells. After three rounds of biopanning, scFv-expressing phages were selected to test for binding to the target cell by flow cytometry. The clone with highest binding specificity to HL-60 cells (designated y1HL63D6) was further investigated. Fluorescent staining indicated that y1HL63D6 scFv bound to a target located on the cell surface. Whole immunoglobulin, IgG-y1HL63D6 was then generated and tested for the binding against bone marrow mononuclear cells (BMMCs) from AML patients. Significantly higher fluorescent signals were observed for some patient samples when compared to normal BMMCs or non-AML patients' BMMCs. Next, the IgG-y1HL63D6 format was tested for antibody-dependent cell cytotoxicity (ADCC). The results demonstrated that IgG-y1HL63D6 but not the control antibody, trastuzumab, could mediate specific killing of HL-60 target cells. In conclusion, our results indicate that specific antibodies can be isolated by biopanning whole cells with a non-immunized human scFv antibody phage display library and that the isolated antibody against HL-60 cells showed therapeutic potential.

Determination of a distinguished interferon gamma epitope recognized by monoclonal antibody relating to autoantibody associated immunodeficiency.

Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27-40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27-L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27-L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency.

Immunoreactivity of humanized single-chain variable fragment against its functional epitope on domain 1 of CD147.

Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future.

Effect of morpholine and charge distribution of cyanine dyes on cell internalization and cytotoxicity.

To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and synthesized two novel Hcyanines based theranostic probes, IR794-Morph and IR794-Morph-Mpip, to enhance cancer cell internalization and targeting. In acidic conditions that resemble to tumour environment, both IR794 derivatives exhibited broad NIR absorption band (704‒794 nm) and fluorescence emission (798‒828 nm) that is suitable for deep seated tumour imaging. Moreover, in vitro study revealed that IR794-Morph-Mpip exhibited better cancer targetability towards various cancer cell lines under physiological and slightly acidic conditions compared to normal cells. IR794-Morph-Mpip was fast internalized into the cancer cells within the first 5 min and mostly localized in lysosomes and mitochondria. In addition, the internalized signal was brighter when the cells were in the hypoxic environment. Furthermore, cellular uptake mechanism of both IR794 dyes, investigated via flow cytometry, revealed that endocytosis through OATPs receptors and clathrin-mediated endocytosis were the main routes. Moreover, IR794-Morph-Mpip, displayed anti-cancer activity towards all tested cancer cell types with IC50 below 7 µM (at 6 h incubation), which is approximately three times lower than that of the normal cells. Therefore, increasing protonated cites in tumour environment of Hcyanines together with incorporating morpholine in the molecule can enhance structure-inherent targeting of these dyes.

Binding Characteristic of Various Antibody Formats Against Aflatoxins.

The application of recombinant antibodies for the analysis of foods and food contaminants is now a major focus, given their capacity to be engineered to tailor their specificity, enhance their stability, and modify their structural formats to fit the desired analytical platform. In this study, human scFv antibody fragments generated against aflatoxin B1 (AFB1) were selected as the model antibody to explore the effect of antibody formats on their binding activity and to evaluate their potential use as immunoreagents for food contaminant analysis. Four human scFv antibody fragments against aflatoxin B1 (AFB1), previously isolated and engineered by chain shuffling, were converted into various formats, that is, scFv-AP fusions, scFv-Fc, and whole IgG molecules. The result indicated that the effects of the antibody format on the binding property varied, depending on the sequence of scFv. For all of the scFv clones, the scFv-AP fusion format showed the highest sensitivity by competitive ELISA, while the effects on the binding activity after conversion to scFv-Fc or IgG format varied, depending on the amino acid sequence of the antibodies. The sAFH-3e3 antibodies that showed the best performance by competitive ELISA were selected for further investigation. The sAFH-3e3 was converted to the scFv-GFP format and tested by fluorescence-linked immunosorbent assay (FLISA), which showed that its binding property was equivalent to those of scFv-Fc and IgG formats. The potential applications of the sAFH-3e3 in a rapid test kit format based on ELISA (scFv-AP) and in a lateral flow immunochromatography assay (LFIA) (IgG) were demonstrated. A comparison of methods for the extraction of AFB1 from matrices for use with these assay formats indicated that PBS and TBST are better than 70% methanol.

Determination of Chinese hamster ovary (CHO) cell densities and antibody titers from small volumes of cell culture supernatants using multivariate analysis and partial least squares regression of UV-Vis spectra.

Antibody titer and viable cell density (VCD) are two important parameters that need to be closely monitored during the process of cell line development and manufacturing of therapeutic antibodies. Typically, determination of each parameter requires 10-100 µL of supernatant sample, which is not suitable for small scale cultivation. In this study, we demonstrated that as low as 2 µL of culture supernatants were sufficient for the analysis using UV-Vis spectrum assisted with partial least squares (PLS) model. The results indicated that the optimal PLS models could be used to predict antibody titer and VCD with the linear relationship between reference values and predicted values at R2 values ranging from 0.8 to > 0.9 in supernatant samples obtained from four different single clones and in polyclones that were cultured in various selection stringencies. Then, the percentage of cell viability and productivity were predicted from a set of samples of polyclones. The results indicated that while all predicted % cell viability were very similar to the actual value at RSEP value of 6.7 and R2 of 0.8908, the predicted productivity from 14 of 18 samples were closed to the reference measurements at RSEP value of 22.4 and R2 of 0.8522. These results indicated that UV-Vis combined with PLS has potential to be used for monitoring antibody titer, VCD, and % cell viability for both online and off-line therapeutic production process. The process of multivariate analysis and partial least squares regression of UV-Vis spectrum for the determination of CHO cell densities and antibody titers obtained from small volume of cell culture supernatant samples.

Repeated exposure to dengue virus elicits robust cross neutralizing antibodies against Zika virus in residents of Northeastern Thailand.

Zika virus (ZIKV) and dengue virus (DENV) are antigenically related mosquito-borne flaviviruses. ZIKV is becoming increasingly prevalent in DENV-endemic regions, raising the possibility that pre-existing immunity to one virus could modulate the response to a heterologous virus, although whether this would be beneficial or detrimental is unclear. Here, we analyzed sera from residents of a DENV-endemic region of Thailand to determine the prevalence of DENV-elicited antibodies capable of cross-neutralizing ZIKV. Sixty-one participants who were asymptomatic and unselected for viral serostatus were enrolled. Among them, 52 and 51 were seropositive for IgG antibody against DENV or ZIKV E proteins (ELISA assay), respectively. Notably, 44.23% (23/52) of DENV seropositive participants had serological evidence of multiple exposures to DENV, and these subjects had strikingly higher titers and broader reactivities of neutralizing antibodies (NAbs) against ZIKV and DENV heterotypes compared with participants with serological evidence of a single DENV infection (25/52, 48.1%). In total, 17 of the 61 participants (27.9%) had NAbs against ZIKV and all four DENV serotypes, and an additional 9 (14.8%) had NAbs against ZIKV and DENV1, 2, and 3. NAbs against DENV2 were the most prevalent (44/61, 72.1%) followed by DENV3 (38/61, 62.3%) and DENV1 (36/61, 59.0%). Of note, anti-ZIKV NAbs were more prevalent than anti-DENV4 NAbs (27/61, 44.3% and 21/61, 34.4%, respectively). Primary ZIKV infection was detected in two participants, confirming that ZIKV co-circulates in this region. Thus, residents of DENV-endemic regions with repeated exposure to DENV have higher titers of NAbs against ZIKV than individuals with only a single DENV exposure.

Development and characterization of human single chain antibody against Iranian Macrovipera lebetina snake venom.

Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera lebetina is one of the most dangerous snakes in Iran. Envenoming by this snake can lead to respiratory distress, heart attack, bleeding, and death. The specific treatment available is immunized equine serum, which has several side effects like serum sickness. Nowadays, single-chain fragment variable antibodies (scFvs) are one of the fast growing classes of monoclonal antibodies, which are suggested for treatment of envenoming. This study aimed to achieve a fully human scFv antibody against M. lebetina venom from human non-immune library. In this study, scFvs against M. lebetina venom were isolated by phage display technique. Using three rounds of biopanning, two specific scFvs (C37 and C69) with the highest affinity were selected. The selected scFvs purified by nickel affinity chromatography. The specific binding of purified antibodies were confirmed by enzyme-linked immunosorbent assay. The LD50 as well as HD50 concentration of the crude venom were obtained to be 45 µg and 120 µg/ml, respectively. C69 neutralized 48% of the hemolysis activity of M. lebetina venom and C37 survived 66% of mice after 115 min of envenoming. Taken together, the results indicate the potential of human non-immune libraries for selection of functional antibodies against M. lebetina venom.