Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks

Characterization of the haemagglutinin properties of the H5N1 avian influenza virus that caused human infections in Cambodia

High pathogenicity avian influenza (HPAI) H5N1 is a subtype of the influenza A virus primarily found in birds. The subtype emerged in China in 1996 and has spread globally, causing significant morbidity and mortality in birds and humans. In Cambodia, a lethal case was reported in February 2023 involving an 11-year-old girl, marking the first human HPAI H5N1 infection in the country since 2014. This research examined the zoonotic potential of the human H5N1 isolate, A/Cambodia/NPH230032/2023 (KHM/23), by assessing its receptor binding, fusion pH, HA thermal stability, and antigenicity. Results showed that KHM/23 exhibits similar receptor binding and antigenicity as the early clade 2.3.2.1c HPAI H5N1 strain, and it does not bind to human-like receptors. Despite showing limited zoonotic risk, the increased thermal stability and reduced pH of fusion in KHM/23 indicate a potential threat to poultry, emphasizing the need for vigilant monitoring

Liquid crystal-amplified optofluidic biosensor for ultra-highly sensitive and stable protein assay

Protein assays show great importance in medical research and disease diagnoses. Liquid crystals (LCs), as a branch of sensitive materials, offer promising applicability in the field of biosensing. Herein, we developed an ultrasensitive biosensor for the detection of low-concentration protein molecules, employing LC-amplified optofluidic resonators. In this design, the orientation of LCs was disturbed by immobilized protein molecules through the reduction of the vertical anchoring force from the alignment layer. A biosensing platform based on the whispering-gallery mode (WGM) from the LC-amplified optofluidic resonator was developed and explored, in which the spectral wavelength shift was monitored as the sensing parameter. The microbubble structure provided a stable and reliable WGM resonator with a high Q factor for LCs. It is demonstrated that the wall thickness of the microbubble played a key role in enhancing the sensitivity of the LC-amplified WGM microcavity. It is also found that protein molecules coated on the internal surface of microbubble led to their interactions with laser beams and the orientation transition of LCs. Both effects amplified the target information and triggered a sensitive wavelength shift in WGM spectra. A detection limit of 1 fM for bovine serum albumin (BSA) was achieved to demonstrate the high-sensitivity of our sensing platform in protein assays. Compared to the detection using a conventional polarized optical microscope (POM), the sensitivity was improved by seven orders of magnitude. Furthermore, multiple types of proteins and specific biosensing were also investigated to verify the potential of LC-amplified optofluidic resonators in the biomolecular detection. Our studies indicate that LC-amplified optofluidic resonators offer a new solution for the ultrasensitive real-time biosensing and the characterization of biomolecular interactions

Arylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus contortus in vitro

Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n = 600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a whole-organism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ± 0.01 μM to 4.25 ± 0.82 μM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads. Keywords: Arylpyrrole, Fipronil, Haemonchus contortus, Anthelmintic, Drug discover

Targeting Haemagglutinin Antigen of Avian Influenza Virus to Chicken Immune Cell Receptors Dec205 and CD11c Induces Differential Immune-Potentiating Responses

Improving the immunogenicity and protective efficacy of vaccines is critical to reducing disease impacts. One strategy used to enhance the immunogenicity of vaccines is the selective delivery of protective antigens to the antigen presenting cells (APCs). In this study, we have developed a targeted antigen delivery vaccine (TADV) system by recombinantly fusing the ectodomain of hemagglutinin (HA) antigen of H9N2 influenza A virus to single chain fragment variable (scFv) antibodies specific for the receptors expressed on chicken APCs; Dec205 and CD11c. Vaccination of chickens with TADV containing recombinant H9HA Foldon-Dec205 scFv or H9HA Foldon-CD11c scFv proteins elicited faster (as early as day 6 post primary vaccination) and higher anti-H9HA IgM and IgY, haemagglutination inhibition, and virus neutralisation antibodies compared to the untargeted H9HA protein. Comparatively, CD11c scFv conjugated H9HA protein showed higher immunogenic potency compared to Dec205 scFv conjugated H9HA protein. The higher immune potentiating ability of CD11c scFv was also reflected in ex-vivo chicken splenocyte stimulation assay, whereby H9HA Foldon-CD11c scFv induced higher levels of cytokines (IFNγ, IL6, IL1β, and IL4) compared to H9HA Foldon-Dec205 scFv. Overall, the results conclude that TADV could be a better alternative to the currently available inactivated virus vaccines

Risk assessment of the newly emerged H7N9 avian influenza viruses

ABSTRACTSince the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and in ovo virus replication. We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry

CRISPR/Cas9 Editing of Duck Enteritis Virus Genome for the Construction of a Recombinant Vaccine Vector Expressing <i>ompH</i> Gene of <i>Pasteurella multocida</i> in Two Novel Insertion Sites

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector

Refractometric Sensitivity Enhancement of Weakly Tilted Fiber Bragg Grating Integrated with Black Phosphorus

The sensitivity enhancement of the weakly tilted fiber Bragg grating (WTFBG) integrated with black phosphorus (BP) was investigated via numerical simulations and experimental demonstrations. BP nanosheets were deposited twice on the cylindrical WTFBG surface using the in situ layer-by-layer (i-LbL) deposition technique. The resonance intensity of the deepest cladding mode located around 1552 nm of WTFBG had a 9.2 dB decrease after the BP deposition process. This allows for the application of the intensity-modulated refractive index (RI) sensor. The sensing platform was implemented on the use of the BP integrated with WTFBG (BP-WTFBG). The refractometric sensing was achieved with the sensitivity enhancement of the resonance intensity modulation of the deepest cladding mode for the BP-WTFBG. The sensitivities were 137.6 dB/RIU and 75.6 dB/RIU in the RI region of 1.33&ndash;1.35 and 1.35&ndash;1.38, respectively. This platform shows great potential applications for biochemical sensing because of its highly sensitive RI sensing ability around the biochemical sensing window