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Candida albicans is a major opportunistic pathogen, responsible for nearly half of clinical candidemia cases. The rising prevalence of azole-resistant Candida species represents a significant clinical challenge, underscoring the urgent need for alternative therapeutic strategies. Monoclonal antibody-based therapies have emerged as a promising and cost-effective approach to combating Candida infections. Agglutinin-like sequence protein 3 (Als3), a key cell surface protein of C. albicans , plays a pivotal role in adherence and biofilm formation, both of which are essential for its pathogenesis. In this study, recombinant Als3 protein was purified and utilized to immunize chickens, resulting in the production of Als3-specific immunoglobulin Y (IgY) antibodies. Two single-chain variable fragment (scFv) antibody libraries were subsequently constructed using phage display technology, yielding transformant counts of 5.3 × 10 7 and 2.8 × 10 7 , respectively. Phage-based enzyme-linked immunosorbent assay (ELISA) revealed enhanced signals following bio-panning, enabling the identification and sequence validation of three scFv antibodies. These scFv antibodies exhibited strong binding activities to Als3, as confirmed through ELISA and western blot analyses. Binding affinities were determined to be ~ 10⁻⁸ M via serial titration ELISA and competitive ELISA. Additionally, the selected scFv antibodies specifically recognized endogenous Als3 protein in C. albicans , as demonstrated by western blot and cell-based ELISA assays. In conclusion, this study successfully generated and characterized high-affinity scFv antibodies targeting Als3, which exhibited exceptional specificity and binding activity. These findings highlight their potential as promising immunotherapeutic candidates for the treatment of C. albicans infections. Key points • The Als3 protein of C. albicans is a critical biomarker and therapeutic target • Chicken-derived scFv antibodies against Als3 were developed via phage display • The scFv antibodies showed strong binding to endogenous Als3 in C. albicans

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. 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 docking profile were also characterized. 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. 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.

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.

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. 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. 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. 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.

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.

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.

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.

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.

Russell’s vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY) antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs) containing 3.4 × 107 and 5.5 × 107 transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

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 × 10 7 and OmpA-FL scFv-L 5.01 × 10 6 ) 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.