Evaluation of the Immune Response of a Candidate Phage-Based Vaccine against Rhipicephalus microplus (Cattle Tick)

Cattle tick (Rhipicephalus microplus) represents a severe problem causing substantial economic losses, estimated in billions of dollars annually. Currently, chemical acaricides represent the most widely used control method. However, several problems such as resistance have been described. Phage-based vaccines represent a fast and low-cost tool for antigen delivery. In this regard, the objective of the present work was to develop a candidate phage-based vaccine displaying a cattle tick antigen (Bm86-derived Sbm7462 antigen) on the surface of bacteriophage M13. Phage ELISA and dot blotting analysis confirmed the display of the antigen. Vaccine immunogenicity was evaluated using a bovine monocyte-derived dendritic cell-based ex vivo assay and a murine in vivo assay. The ex vivo model showed the maturation of dendritic cells after being pulsed with the phage-based vaccine. The humoral response was confirmed in the in vivo assay. These results demonstrated the capacity of the phage-based vaccine to induce both humoral and cellular immune-specific responses. Importantly, this is the first report describing a control method for cattle ticks using a candidate phage-based vaccine. Further studies to evaluate the immunogenicity in a bovine model are needed. The current approach represents a promising alternative to control cattle tick infestations.

Development of Antibody Detection ELISA Based on Immunoreactive Toxins and Toxin-Derived Peptides to Evaluate the Neutralization Potency of Equine Plasma against Naja atra in Taiwan

Naja atra, also known as Taiwanese cobra, is one of the most prevalent venomous snakes in Taiwan. Clinically, freeze-dried neurotoxic antivenom (FNAV) produced from horses by Taiwan Centers for Disease Control (CDC) has been the only approved treatment for N. atra envenoming for the last few decades. During antivenom production, large numbers of mice are used in the in vivo assay to determine whether the neutralization potency of hyperimmunized equines is satisfactory for large-scale harvesting. However, this in vivo assay is extremely laborious, expensive, and significantly impairs animal welfare. In the present study, we aimed to develop an in vitro ELISA-based system that could serve as an alternative assay to evaluate the neutralization potency of plasma from hyperimmunized equines. We initially obtained 51 plasma samples with known (high or low) neutralization potency assessed in vivo from 9 hyperimmunized equines and subsequently determined their antibody titers against the five major protein components of N. atra venom (neurotoxin (NTX), phospholipase A2 (PLA2), cytotoxin (CTX), cysteine-rich secretory protein (CRISP), and snake venom metalloproteinase (SVMP)) via ELISA. The antibody titer against NTX was the most effective in discriminating between high and low potency plasma samples. To identify the specific epitope(s) of NTX recognized by neutralization potency-related antibodies, 17 consecutive NTX-derived pentadecapeptides were synthesized and used as antigens to probe the 51 equine plasma samples. Among the 17 peptides, immunoreactive signals for three consecutive peptides (NTX1-8, NTX1-9, and NTX1-10) were significantly higher in the high potency relative to low potency equine plasma groups (p < 0.0001). Our ELISA system based on NTX1-10 peptide (RWRDHRGYRTERGCG) encompassing residues 28–42 of NTX displayed optimal sensitivity (96.88%) and specificity (89.47%) for differentiating between high- and low-potency plasma samples (area under the receiver operating characteristic curve (AUC) = 0.95). The collective data clearly indicate that the antibody titer against NTX protein or derived peptides can be used to efficiently discriminate between high and low neutralization potency of plasma samples from venom-immunized horses. This newly developed antibody detection ELISA based on NTX or its peptide derivatives has good potential to complement or replace the in vivo rodent assay for determining whether the neutralization potency of equine plasma is satisfactory for large-scale harvesting in the antivenom production process against N. atra.

Menthyl esterification allows chiral resolution for the synthesis of artificial glutamate analogs

Herein, we report the enantiospecific synthesis of two artificial glutamate analogs designed based on IKM-159, an antagonist selective to the AMPA-type ionotropic glutamate receptor. The synthesis features the chiral resolution of the carboxylic acid intermediate by the esterification with ʟ-menthol, followed by a configurational analysis by NMR, conformational calculation, and X-ray crystallography. A mice in vivo assay showed that (2R)-MC-27, with a six-membered oxacycle, is neuroactive, whereas the (2S)-counterpart is inactive. It was also found that TKM-38, with an eight-membered azacycle, is neuronally inactive, showing that the activity is controlled by the ring C.

The loopometer: a quantitative in vivo assay for DNA-looping proteins

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.