The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.

In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16.

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.

Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies.

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain-Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 106 and 4.5 × 106 transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV.

Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies.

Bacterial meningitis is a severe disease that is fatal to one-third of patients. The major cause of meningitis in neonates is Escherichia coli (E. coli) K1. This bacterium synthesizes an outer membrane protein A (OmpA) that is responsible for the adhesion to (and invasion of) endothelial cells. Thus, the OmpA protein represents a potential target for developing diagnostic and therapeutic agents for meningitis. In this study, we expressed recombinant OmpA proteins with various molecular weights in E. coli. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the molecular size of OmpA's full length (FL) and truncated proteins. OmpA-FL protein was purified for immunizing chickens to produce immunoglobulin yolk (IgY) antibodies. We applied phage display technology to construct antibody libraries (OmpA-FL scFv-S 1.1 × 107 and OmpA-FL scFv-L 5.01 × 106) to select specific anti-OmpA-FL scFv antibodies; these were characterized by their binding ability to recombinant or endogenous OmpA using ELISA, immunofluorescent staining, and confirmed with immunoblotting. We found 12 monoclonal antibodies that react to OmpA fragments; seven scFvs recognize fragments spanning amino acid (aa) residues 1-346, aa 1-287, aa 1-167, and aa 60-192, while five scFvs recognize fragments spanning aa 1-346 and aa 1-287 only. Two fragments (aa 246-346 and aa 287-346) were not recognized with any of the 12 scFvs. Together, the data suggest three antigenic epitopes (60 aa-160 aa, 161 aa-167 aa, 193 aa-245 aa) recognized by monoclonal antibodies. These scFv antibodies show strong reactivity against OmpA proteins. We believe that antibodies show promising diagnostic agents for E. coli K1 meningitis.

Production and Characterization of Neutralizing Antibodies against Bungarus multicinctus Snake Venom.

The venom of the banded krait (Bungarus multicinctus), one of the major venomous species in Taiwan, contains neurotoxic venom proteins (B. multicinctus proteins) that pose a serious medical problem in tropical and subtropical countries. Even though horse-derived serum is an efficient therapy against snake venom, it is associated with a high cost and side effects. Therefore, developing a more cost-effective alternative treatment option is highly envisaged. In this study, chickens were immunized with B. multicinctus proteins, and polyclonal immunoglobulin Y (IgY) antibodies were purified from eggs. IgY showed a binding activity to B. multicinctus proteins that was similar to horse antivenin, and its titer in chickens lasted for at least 6 months. We constructed two antibody libraries by phage display antibody technology, which contain 1.0 × 107 and 2.9 × 108 transformants, respectively. After biopanning, a phage-based enzyme-linked immunosorbent assay (ELISA) indicated that specific clones were enriched. Thirty randomly selected clones expressing monoclonal single-chain variable-fragment (scFv) antibodies were classified into four groups with a short linker and two with a long linker. These selected scFv antibodies showed specific binding activities to B. multicinctus proteins but not to the venomous proteins of other snakes. Most importantly, polyclonal IgY demonstrated a similar neutralization efficiency as did horse-derived antivenin in mice that were injected with a minimum lethal dosage (MLD) of venom proteins. A mixture of several monoclonal anti-B. multicinctus scFv antibodies was also able to partially inhibit the lethal effect on mice. We profoundly believe that IgY and scFv antibodies can be applied in developing diagnostic agents for wound exudates and as an alternative treatment for snakebite envenomation in the future.IMPORTANCE Snake envenomation is one of the global medical issues of concern. Horse-derived antivenin is an effective way to treat snakebites, but it is costly and occasionally causes severe side effects. In this study, we first generated and characterized IgY antibodies with neutralization activity in chickens. Subsequently, we generated a panel of monoclonal scFv antibodies using phage display antibody technology. A mixture of scFv antibodies was able to partially inhibit the lethal effect in mice that were injected with lethal dosages of venom proteins and prolong their survival time. We believe that chicken-derived IgY and scFv antibodies have great potential for the development of diagnostic agents for wound exudates and therapeutic agents against snake envenomation in the future.

Antibodies against Venom of the Snake Deinagkistrodon acutus.

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.

Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom.

Trimeresurus mucrosquamatus (TM) is one of majorities of snake envenomation with necrotic and hemorrhagic toxin in Taiwan. In this study, chickens were used as an alternative animal model for immunization with TM venom. Using phage display technology to process four rounds of panning, selected single chain variable fragments (scFv) could specifically recognize TM venom proteins, which were later identified as a group of homogeneous venom serine protease. The specific scFv antibodies showed various inhibitory effects on sheep RBC lysis induced by TM venom using an indirect hemolytic assay in vitro. In addition, the survival times of mice were extended to certain degrees when treated with these scFv antibodies individually or in a combination. To elucidate the inhibitory mechanism, we used molecular modeling to build up the serine protease structure to simulate the possible interactions with scFv antibodies. The results suggested that the CDR-loop of the scFv antibodies (3S10 or 4S1) might bind at the 99-loop of venom serine protease so as to affect substrate access due to the partial collapse of the subsite S2 and the partial movement of the subsite S4. It is hoped these chicken-derived antibodies could be applied to develop diagnostic and therapeutic agents against snakebites.

Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae.

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

Generation and characterization of avian single chain variable fragment against human Alpha-Enolase.

Overexpression of human alpha-enolase (hEno1)has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Correction: The development of surgical risk score and evaluation of necrotizing soft tissue infection in 161 Naja atra envenomed patients.

[This corrects the article DOI: 10.1371/journal.pntd.0010066.].

Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells.

The Taiwanese cobra, Naja atra, is a clinically significant species of snake observed in the wild in Taiwan. Victims bitten by N. atra usually experience severe pain and local tissue necrosis. Although antivenom is available for treatment of cobra envenomation, its neutralization potency against cobra-induced necrosis is weak, with more than 60% of cobra envenoming patients developing tissue necrosis after antivenom administration. The present study found that cytotoxin (CTX) is a key component of N. atra venom responsible for cytotoxicity against myoblast cells. Anti-CTX IgY was generated in hens, and the spleens of these hens were used to construct libraries for the development of single chain variable fragments (scFv). Two anti-CTX scFv, S1 and 2S7, were selected using phage display technology and biopanning. Both polyclonal IgY and monoclonal scFv S1 reacted specifically with CTX in cobra venom. In a cell model assay, the CTX-induced cytolytic effect was inhibited only by monoclonal scFv S1, not by polyclonal IgY. Moreover, the neutralization potency of scFv S1 was about 3.8 mg/mg, approximately three times higher than that of conventional freeze-dried neurotoxic antivenom (FNAV). Collectively, these results suggest that scFv S1 can effectively neutralize CTX-induced cytotoxicity and, when combined with currently available antivenom, can improve the potency of the latter, thereby preventing tissue damage induced by cobra envenoming.

The development of surgical risk score and evaluation of necrotizing soft tissue infection in 161 Naja atra envenomed patients.

BACKGROUND: Naja atra bites cause wound necrosis, secondary infection, and necrotizing soft tissue infection (NSTI) requiring repetitive surgeries. Little information is known about the predictors for surgery after these bites. MATERIALS AND METHODS: We retrospectively evaluated 161 patients envenomed by N. atra, 80 of whom underwent surgery because of wound necrosis and infection. We compared the patients' variables between surgical and non-surgical groups. To construct a surgical risk score, we converted the regression coefficients of the significant factors in the multivariate logistic regression into integers. We also examined the deep tissue cultures and pathological findings of the debrided tissue. RESULTS: A lower limb as the bite site, a ≥3 swelling grade, bullae or blister formation, gastrointestinal (GI) effects, and fever were significantly associated with surgery in the multivariate logistic regression analysis. The surgical risk scores for these variables were 1, 1, 2, 1, and 2, respectively. At a ≥3-point cutoff value, the model has 71.8% sensitivity and 88.5% specificity for predicting surgery, with an area under the receiver operating characteristic curve of 0.88. The histopathological examinations of the debrided tissues supported the diagnosis of snakebite-induced NSTI. Twelve bacterial species were isolated during the initial surgery and eleven during subsequent surgeries. DISCUSSION AND CONCLUSIONS: From the clinical perspective, swelling, bullae or blister formation, GI effects, and fever appeared quickly after the bite and before surgery. The predictive value of these factors for surgery was acceptable, with a ≥3-point risk score. The common laboratory parameters did not always predict the outcomes of N. atra bites without proper wound examination. Our study supported the diagnosis of NSTI and demonstrated the changes in bacteriology during the surgeries, which can have therapeutic implications for N. atra bites.

Clinical manifestations and treatments of Protobothrops mucrosquamatus bite and associated factors for wound necrosis and subsequent debridement and finger or toe amputation surgery.

INTRODUCTION: Protobothrops mucrosquamatus bite induces wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and acute renal failure. The severity of the hematological derangements and associated factors for wound necrosis and subsequent surgery and the appropriate management of these conditions have not been well characterized. Although severe renal failure requiring hemodialysis has been reported following P. mucrosquamatus bite, the culprit snake may be erroneously classified. MATERIALS AND METHODS: A total of 186 patients with P. mucrosquamatus bites were retrospectively evaluated. They were categorized into group 1 (patients receiving debridement or finger/toe amputation) and group 2 (all other patients) to identify the associated factors for surgery. Characteristic data were compared between groups 1 and 2 and between definite and suspected cases. RESULTS: No differences were observed between definite and suspected cases in terms of symptomatology and management. Of the 186 patients, 7 (3.8%) were asymptomatic, 179 (96.2%) experienced tissue swelling and pain, and 107 (57.5%) had local ecchymosis. Coagulopathy, thrombocytopenia, and renal impairment were found in 13 (7%), 19 (10.2%), and 7 (3.8%) patients, respectively. None of the patients required transfusion therapy or hemodialysis. Furthermore, no systemic bleeding or death occurred. Antivenom was administered to all 179 envenomed patients at a median of 1.5 h post-bite. The median total dose of the specific antivenom was 5.5 vials. In multivariate logistic regression analysis, finger as the bite site, bullae and blister formation, and wound infection were significantly associated with wound necrosis; whereas finger as the bite site and bullae and blister formation were related to debridement or finger/toe amputation. DISCUSSION AND CONCLUSIONS: Protobothrops mucrosquamatus envenomation mainly exerts effects on local tissue. Systemic effects are uncommon and generally nonsevere and transient after the treatment with the specific antivenom. We speculated that severe renal failure requiring hemodialysis is not a typical finding of P. mucrosquamatus envenomation. Patients with finger as the bite site and bullae or blister formation should be carefully examined for wound necrosis, secondary infection, and subsequent surgery. Further evaluations of the efficacy of antivenom against local tissue effects and the effect of selective antibiotics in the management of bite wound infection are urgently required. Although the antivenom manufacturer suggested a skin test prior to use, we believed that it could be omitted because it does not accurately predict the allergic responses.

Analysis of the Necrosis-Inducing Components of the Venom of Naja atra and Assessment of the Neutralization Ability of Freeze-Dried Antivenom.

Patients bitten by Naja atra who are treated with bivalent freeze-dried neurotoxic antivenom in Taiwan have an improved survival rate but develop necrotic wound changes. The World Health Organization (WHO) has suggested using the minimum necrotizing dose (MND) of venom as a method of evaluating the neutralization effect of antivenom. The aim of this study was to evaluate the effectiveness of antivenom for the prevention of necrosis based on the MND and clarify which component of the venom of N. atra induces necrosis. The neurotoxins (NTXs) were removed from the crude venom (deNTXs), and different concentrations of deNTXs were injected intradermally into the dorsal skin of mice. After three days, the necrotic lesion diameter was found to be approximately 5 mm, and the MND was calculated. A reduction in the necrotic diameter of 50% was used to identify the MND50. Furthermore, both phospholipase A2 (PLA2) and cytotoxins (CTXs) were separately removed from the deNTXs to identify the major necrosis-inducing factor, and the necrotic lesions were scored. All mice injected with deNTXs survived for three days and developed necrotic wounds. The MND of the deNTXs for mice was 0.494 ± 0.029 µg/g, that of the deNTXs-dePLA2 (major component retained: CTXs) was 0.294 ± 0.05 µg/g, and that of the deNTX-deCTX (major component retained: PLA2) venom was greater than 1.25 µg/g. These values show that CTX is the major factor inducing necrosis. These results suggest that the use of the deNTXs is necessary to enable the mice to survive long enough to develop venom-induced cytolytic effects. CTXs play a major role in N. atra-related necrosis. However, the MND50 could not be identified in this study, which meant that the antivenom did not neutralize venom-induced necrosis.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan.

BACKGROUND: The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. METHODS: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. RESULTS: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. CONCLUSION: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.

Envenomation by Trimeresurus stejnegeri stejnegeri: clinical manifestations, treatment and associated factors for wound necrosis.

BACKGROUND: Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. MATERIALS: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. RESULTS: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. CONCLUSIONS: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.

OmpA is the critical component for Escherichia coli invasion-induced astrocyte activation.

Escherichia coli is the major Gram-negative bacterial pathogen in neonatal meningitis. Outer membrane protein A (OmpA) is a conserved major protein in the E. coli outer membrane and is involved in several host-cell interactions. To characterize the role of OmpA in the invasion of astrocytes by E. coli, we investigated OmpA-positive and OmpA-negative E. coli strains. Outer membrane protein A E44, E105, and E109 strains adhered to and invaded C6 glioma cells 10- to 15-fold more efficiently than OmpA-negative strains. Actin rearrangement, protein tyrosine kinase, and phosphoinositide 3-kinase activation were required for OmpA-mediated invasion by E. coli. In vitro infection of C6 cells and intracerebral injection into mice of the E44 strain induced expression of the astrocyte differentiation marker glial fibrillary acidic protein and the inflammatory mediators cyclooxygenase 2 and nitric oxide synthase 2. After intracerebral infection with E44, all C57BL/6 mice died within 36hours, whereas 80% of mice injected with E44 premixed with recombinant OmpA protein survived. Astrocyte activation and neutrophil infiltration were reduced in brain tissue sections in the mice given OmpA. Taken together, these data suggest that OmpA-mediated invasion plays an important role in the early stage of E.coli-induced brain damage, and that it may have therapeutic use in E. coli meningitis.