Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin - the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

Assessment of neutralization of Micrurus venoms with a blend of anti-Micrurus tener and anti-ScNtx antibodies.

BACKGROUND: Micrurus venoms contain two main groups of toxic protein components: short-chain α-neurotoxins (SNtx) and phospholipases type A2 (PLA2). In North America, generally, the Micrurus venoms have low abundance of SNtx compared to that of PLA2s; however, both are highly toxic to mammals, and consequently both can play a major role in the envenomation processes. Concerning the commercial horse-derived antivenoms against Micrurus from the North America region, they contain a relatively large amount of antibodies against PLA2s, and a low content of antibodies against short chain α-neurotoxins. This is mainly due to the lower relative abundance of SNtxs, and also to its poor immunogenicity due to their size and nature. Hence, Micrurus antivenoms made in North America usually present low neutralizing capacity towards Micrurus venoms whose lethality depend largely on short chain α-neurotoxins, such as South American Micrurus species. METHODS: Horses were hyperimmunized with either the venom of M. tener (PLA2-predominant) or a recombinant short-chain consensus α-neurotoxin (ScNtx). Then, the combination of the two monospecific horse antibodies (anti-M. tener and anti-ScNtx) was used to test their efficacy against eleven Micrurus venoms. RESULTS: The blend of anti-M. tener and anti-ScNtx antibodies had a better capacity to neutralize the lethality of diverse species from North, Central and South American Micrurus venoms. The antibodies combination neutralized both the ScNtx and ten out of eleven Micrurus venom tested, and particularly, it neutralized the venoms of M. distans and M. laticollaris that were neither neutralized by monospecific anti-M. tener nor anti-ScNtx. CONCLUSIONS: These results provide a proof-of-principle for using recombinant immunogens to enrich poor or even non-neutralizing antisera against elapid venoms containing short chain α-neurotoxins to develop antivenoms with higher effectiveness and broader neutralizing capacity.

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.

Horse immunization with short-chain consensus α-neurotoxin generates antibodies against broad spectrum of elapid venomous species.

Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus, Dendroaspis, Naja, Walterinnesia, Ophiophagus and Hydrophis. These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.

Use of irradiated elapid and viperid venoms for antivenom production in small and large animals.

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD50 of 1.25 mg/kg in mice, and the elapid mixture had an LD50 of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LD50s of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.

Pressure-induced stromal keratopathy after laser in situ keratomileusis: Acute and late-onset presentations.

We present a series of 4 cases of pressure-induced stromal keratopathy after laser in situ keratomileusis (LASIK). Four patients (5 eyes) with previous LASIK presented for poor visual acuity and ocular pain because of ocular hypertension. At examination, all cases revealed corneal haze and a space filled with fluid between the surgical flap and the residual stroma. All cases were managed with topical hypotensive treatment and one of them was also treated with a valve drainage device. Topical steroids restriction was indicated in all cases. Intraocular pressure (IOP) was normalized in all cases with subsequent interface fluid resolution and significant improvement of vision in most cases. Early recognition and appropriate treatment for pressure-induced stromal keratopathy is essential to avoid complications associated with prolonged elevated IOP. It is extremely important to measure the IOP in the peripheral cornea because IOP in the central cornea can be incorrectly measured with the characteristic interface fluid developed in this entity.

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Anterior segment optical coherence tomography angle and vault analysis after toric and non-toric implantable collamer lens V4c implantation in patients with high myopia.

OBJECTIVE: To assess anterior segment changes, including iridocorneal angle and vault, after toric and non-toric implantable collamer lens (ICL) V4c (STAAR Surgical AG) implantation under different lighting conditions. METHODS: Longitudinal, prospective, case series. Patients with high myopia (>6 dioptres) underwent toric and non-toric ICL V4c implantation. Optical coherence tomography measurements were taken under different lighting conditions preoperatively and at 1 week and 1, 6 and 12 months of follow-up. RESULTS: Seventy-six eyes of 42 patients underwent ICL V4c implantation. Mean age was 27.4 years (±5.14 years, range 20 to 39 years). The average iridocorneal angle showed a statistically significant decrease (p<0.05) in all mesopic, scotopic and photopic conditions after 1 week of surgery compared with the preoperative measurements; in mesopic conditions, it decreased 14.1°, in photopic conditions 14.8° and scotopic conditions 13.2°. The angle measurement had a statistically significant change only in mesopic conditions (p=0.01) over the 1-year follow-up. The average vault under mesopic conditions was 0.661±0.21 mm at week 1. The vault measurement change was statistically significant over the 1-year follow-up in mesopic conditions (p=0.01). Refractive results showed a significant improvement in both uncorrected and corrected distance visual acuity (p<0.001). CONCLUSION: There is a significant reduction in the iridocorneal angle after ICL V4c implantation. Furthermore, there is a change under mesopic conditions in both the iridocorneal angle and vault during long-term follow-up.

Synthetic peptide antigens derived from long-chain alpha-neurotoxins: Immunogenicity effect against elapid venoms.

Three-finger toxins (3FTXs), especially α-neurotoxins, are the most poorly neutralized elapid snake toxins by current antivenoms. In this work, the conserved structural similarity and motif arrangements of long-chain α-neurotoxins led us to design peptides with consensus sequences. Eight long-chain α-neurotoxins (also known as Type II) were used to generate a consensus sequence from which two peptides were chemically synthesized, LCP1 and LCP2. Rabbit sera raised against them were able to generate partially-neutralizing antibodies, which delayed mice mortality in neutralization assays against Naja haje, Dendrospis polylepis and Ophiophagus hannah venoms.

Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis.

BACKGROUND: The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. METHOD: Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). RESULTS: The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. CONCLUSION: HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.

Combined small-incision lenticule extraction and intrastromal corneal collagen crosslinking to treat mild keratoconus: Long-term follow-up.

PURPOSE: To report visual, refractive, and topographic outcomes of sequential, same-day small-incision lenticule extraction and intrastromal corneal collagen crosslinking (CXL) in eyes with mild keratoconus. SETTING: Institute of Ophthalmology Conde de Valenciana, Mexico City, Mexico. DESIGN: Prospective interventional case series. METHODS: Fifteen eyes with forme fruste keratoconus and/or irregular corneas, corrected distance visual acuity 20/40 or better, stable refraction of at least 1 year, age 18 years or older, and residual corneal thickness of greater tan 400 µm before performing collagen crosslinking were studied. Patients were treated with small-incision lenticule extraction followed by intrastromal injection of riboflavin inside the pocket. Ultraviolet A light with a wavelength of 370 nm to 3 mW/cm(2) was applied for 30 minutes. Follow-up was done at 1 day, at 1 week, and at 1, 3, 6, 12, 18, and 24 months. RESULTS: Eight patients were included in the study. The mean age was 29.5 years ± 5.5 (SD) (range 20 to 36 years). Twenty-four months of follow-up were completed in 13 eyes, and 12 months were completed in 2 eyes. Preoperative uncorrected distance visual acuity improved from 1.6 ± 0.3 LogMAR (Snellen 20/796) to postoperative 0.12 ± 0.20 LogMAR (Snellen 20/26) and was statistically significant (P < .001). Best-corrected distance visual acuity did not change significantly (P = .186), from 0.006 ± 0.02 LogMAR (Snellen 20/20) preoperatively to 0.04 ± 0.05 LogMAR (Snellen 20/21) postoperatively, and spherical equivalent improved from -4.3 ± 1.02 preoperatively to 0.2 ± 0.66 (P < .001). CONCLUSION: Although further follow-up and larger samples are needed to fully confirm these findings, the results suggest that combined small-incision lenticule extraction and intrastromal corneal collagen crosslinking are a promising treatment option for patients for whom conventional laser refractive surgery is contraindicated. FINANCIAL DISCLOSURE: Drs. Ramirez-Miranda and Navas are consultants to Carl Zeiss Meditec. No other author has a financial or proprietary interest in any material or method mentioned.

Dihydropyridines in MCRs. Tandem processes leading to modular tetrahydroquinoline systems with up to 6 diversity elements.

An efficient, modular method for the synthesis of highly substituted tetrahydroquinoline systems is described. The Lewis acid catalyzed interaction of dihydropyridines with glyoxalate and anilines affords the heterocyclic parent systems in good yields. Tandem one-pot processes allow the incorporation of additional components: a preliminary nucleophilic attack on pyridinium salts generates the reactive dihydropyridine in situ, and subsequent electrophilic reactions on the secondary amine complete the assembly of the final targets, which have up to 6 diversity points.