The saw-scaled viper Echis carinatus, one of the “Big Four” causes of snakebite in India, occurs from Sri Lanka to eastern Iraq. To investigate clinical reports regarding limited efficacy of Indian polyvalent antivenom (IPAV) against envenomation in Echis carinatus sochureki (ECS) in northwestern India, we obtained 22 snakes from three locations in Rajasthan and identified 148-174 toxin isoforms belonging to 21–25 toxin families in their venom using a bottom-up proteomics approach. All samples showed a high abundance of snake venom metalloproteinases (SVMPs), particularly SVMP class III. Other major components were Phospholipases A₂, L-amino-acid oxidases, snake venom serine proteases and Snaclecs. Variation in venom composition among locations in Rajasthan and compared to *E. c. carinatus *(ECC) from southern India were primarily due to differences in relative abundance of these toxin families. Recognition of all venom components by Indian poly-valent antivenom (IPAV) using second-generation antivenomics was poor at lower antivenom concentrations. Notably, SVMP classes II and III were poorly recognized at all venom to antivenom ratios in all ECS venoms and a plasma clotting assay revealed poor neutralization of procoagulant activity. This collaborative study highlights the need for the development of regional antivenoms to effectively treat snakebites in northwestern India.

Materials and Methods

Venom collection

A total of 22 ECS snakes were captured from the deserts of Barmer and Jaisalmer districts of Rajasthan (Figure 1), and subsequently released after venom extraction. Extraction of venom was done by gently massaging the glands, prompting the snakes to bite on the Parafilm membrane fastened onto a Nalgene beaker [35]. The venoms were collected from the beaker, placed into 1.5 ml microcentrifuge tubes and desiccated using silica beads in vacuum chambers. The dried samples were stored at -80 ℃ as soon as practicable. ECC-TN venom from Tamil Nadu, obtained from the Irula Snake-catchers Industrial Cooperative Society, venom from Tamil Nadu was procured from Irula Cooperative Society, Tamil Nadu.

 SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

Dried crude venom from 22 specimens of ECS (Table S1), were reconstituted in Milli-Q water (0.5 mg in 50 µl). Protein concentration in the samples was estimated using a Thermo Nanodrop Lite spectrophotometer and 20 µg of each sample was subjected to electrophoretic separation under reducing and non-reducing conditions using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A broad range marker (Takara catalog #3597A) was used as a reference. Bands were visualized by Coomassie brilliant blue R-250 staining, followed by destaining in methanol-acetic acid solution. To generate population-level venom profiles, based on the SDS results, samples from each location were pooled in equimolar amounts to form three regional groups (each within a ~25 km radius, see Figure 1): ECS-BA (Barmer, n=3), ECS-SA (Sam, n=13) and ECS-PO (Pokhran, n=6). Equal quantities of venom from each sample were used to ensure equimolar representation of toxins in each pooled sample; this also ensured sufficient quantity of venoms for further analyses. The pooled samples were lyophilized and stored at -20 °C until further use.

Reverse-phase high-performance liquid chromatography (RP-HPLC)

For protein fractionation, 800 µg of venom from each pool was subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) using a Thermo Ultimate 3000 UHPLC system equipped with Zorbax 300SB-C18 column (4.6 × 150 mm, 3.5 µm; Agilent Technologies, CA, USA). Prior to injection, samples were centrifuged at ~9,400 g for 10 minutes to remove particulates. The separation was carried out using a linear gradient of solvent A (0.1% TFA in water) and solvent B (100% acetonitrile), with some modifications, under the following elution conditions: 5% B for 5 minutes followed by 5–15% B for 5 minutes, 15–35% B for 10 minutes, 35–50% B for 35 minutes and 50–75% B for 10 minutes. The flow rate was maintained at 0.8 mL/min and protein elution was monitored at 215 nm [11,36]. Distinct chromatographic fractions were collected manually and vacuum-dried using Savant™ SpeedVac (ThermoFisher Scientific, Waltham, MA, USA). 

In solution digestion

For proteomic analysis, a total of 10 µg of venom from each peak fraction was subjected to in-solution trypsin digestion. The proteins were denatured using 50 μl of 6 M Urea in 50 mM Tris-HCl, pH 8.5. To reduce the disulphide bonds in the proteins, 2 μL of 200 mM dithiothreitol (DTT) in 50 mM Tris-HCl [AM3] was added to the peak fractions followed by 1 h incubation at room temperature. After incubation, 10 μL of 200 mM iodoacetamide (IAA) in 50 mM Tris-HCl[AM4] [AS5]  was added to acetylate the reduced cysteine residues, and the sample was then incubated in the dark for 1 h. Any unreacted IAA was then quenched by adding 10 µL of 200 mM DTT, followed by another 1 h incubation in the dark. 350 μl of 1mM CaCl2 in 50mM Tris-Cl was then added to reduce the urea concentration. Following the addition of trypsin solution at a ratio of 1:30 (w/w trypsin: protein), they were incubated at 37 °C for 16-20 h.  Formic acid was added to lower the pH to 2-4 to stop trypsin action before desalting [11]. Samples were desalted using C-18 Ziptips (Thermo Fisher Scientific, Waltham, MA, USA) and digested peptides were eluted in 70% ACN, 0.1% TFA. Eluted peptides were vacuum-dried and reconstituted in 5% Acetonitrile (ACN) for nano-LC-MS analysis.

MS/MS data analysis

Samples were analysed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA; hardware ID- 001999D89836) in conjunction with a nanoflow HPLC system (Easy-nLC 1200 Thermo Scientific). The samples were loaded onto Easy Spray Column PepMap ™ RSLC C18 (3 µm,100 Å, 75 µm x 15 cm) and spectra were generated in positive ion mode. A 45 min linear gradient of solution A (5% acetonitrile in-water containing 0.1% formic acid) and solution B (90% ACN in 0.1% formic acid) at the flow rate of 300 nl/min was applied for eluting peptides as follows: 5–25% B for 25 min followed by 25–60% for 12 min, 60–90% for 5 min,  and a final 3 min wash at 90% B. Eluted peptides were analyzed in data-dependent acquisition (DDA) mode with MS scans (range: m/z 400–1750) at a resolution of 70,000. Charge exclusion criteria were applied to ions with +1, +6 to +8, >+8. MS/MS of the top 10 precursor ions with isolation time of 15 milliseconds was achieved through higher-energy collisional dissociation (HCD) at 30% normalized collision energy. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200, within a scan range extending from 200 to 2000 m/z.

Spectra were analyzed using Proteome Discoverer (PD) v2.2 with the SEQUEST-HT algorithm against the Viperidae database (NCBI TaxID: 8689; 105,645 nr sequences) and a keratin contaminant database (15,154 nr sequences). For enhanced accuracy in protein identification, search parameters included a precursor mass tolerance of 10 ppm and fragment tolerance of 0.02 Da. Carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and N-terminal acetylation were considered variables. Percolator (PD) [AM6] was used to regulate the false discovery rates (FDR) of peptides and proteins at thresholds of 1% (strict) and 5% (relaxed). Entries corresponding to contaminant or low/moderate FDR confidence were eliminated, and high confidence proteins supported by a minimum of two unique peptides were taken as valid hits. For each protein, the abundances of associated PSMs were summed to estimate the toxin abundance. Toxin families were assigned to identified entries according to NCBI annotations, which are curated based on the sequence and protein superfamily characteristics. The relative abundances of toxin family were calculated using equations 1 and 2 essentially integrating precursor ion intensities with corresponding RP-HPLC peak areas at 215 nm (calculated using Chromeleon 7 software). The HPLC fractions were annotated by identifying toxin families contributing >20% of the total abundance within a peak, thereby reflecting the major components.

Affinity chromatography for measuring the immunorecognition of IPAV

All 22 ECS venom samples from three locations Sam Kanoi[AM7] , Barmer, Pokhran were grouped as ECS-SA, ECS-BA, and ECS-PO respectively. Third-generation antivenomics was performed using IPAV from Bharat Serums and Vaccines Limited (BSV) as an affinity ligand (Batch No. A18224036). This particular antivenom was chosen as it is routinely supplied in Rajasthan for snakebite treatment. The antivenom was dialyzed overnight at 4 ℃, lyophilized, and reconstituted in coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3). We assessed the immunorecognition at six antivenom to venom ratios (20:1, 40:1, 60:1, 80:1, 100:1 and 120:1). For each ratio, a single Micro Bio-Spin column (Bio-Rad, USA) with 35.4 ± 0.1 mg of CNBr-activated Sepharose™ 4B beads (Cytiva) was used. The beads were activated with 10 Matrix Bed Volume (MBV) of 1mM cold HCl. Columns were then equilibrated with 10 MBV of coupling buffer (0.2M NaHCO3, 0.5M NaCl, pH 8.3). Eight mg of IPAV dissolved in coupling buffer was added to the equilibrated column and incubated at room temperature for 4 h. Post IPAV incubation, the flow-through was collected to estimate the unbound antivenom and to calculate antivenom coupling yield (ACY = Initial IPAV amount loaded – Amount of IPAV in flow through). The remaining active groups were blocked using 2 MBV of blocking buffer (0.1M Tris-Cl, pH 8.5), incubated for 4 h at room temperature followed by six rounds of alternative washes with 7 MBV of low pH (0.1M CH3COONa, 0.5M NaCl, pH 4) and high pH (0.1M Tris-Cl, pH 8.5) buffer. The columns were then equilibrated with 5 MBV of binding buffer (1X PBS, pH 7.4), and incubated with six different venom amounts. The toxin retention by antivenom was measured by comparing NR and R fractions for different amounts of incubated venom proteins (ECS-BA 53.61μg - 307.6μg, ECS-PO 55μg – 328μg and ECS-SA 50.8μg - 331.65μg) with the control venom profile. For control, beads were incubated without antivenom in coupling buffer and the highest venom amounts for all three locations were passed through it. The venom toxins that are not bound to the antivenom were collected as flow-through post-incubation and referred to as non-retained (NR) fractions. The bound toxins were further eluted using 10 MBV of the elution buffer (0.1M Glycine-Cl, pH 2.7) and brought to neutral pH immediately using 1M Tris-Cl, pH 9 to yield retained (R) fractions. All fractions were dried using Savant SpeedVac™ Vacuum Concentrators and reconstituted in 120 μl of Milli-Q water. Further, 100 μl of these fractions were loaded for reverse-phase HPLC as described in section 2.3. The remaining aliquots of NR and R fractions were subjected to in-solution trypsin digestion and MS/MS as described previously.  Mass spectrometry data were used to identify the most abundant toxins at the lowest antivenom-to-venom ratio (20:1), and their relative abundances were compared across increasing ratios to track sequential neutralization.

Neutralization of procoagulant activity of venoms

A preliminary coagulation assay was performed following Bhatia et al. [12]. Blood was collected in heparinized tubes from two healthy donors. Platelet poor plasma (PPP) was extracted through a standard procedure [37, 38].  The plasma clotting of venoms was tested for five dilutions 15, 10, 5, 2.5, 1.25, and 0.6 μg against 200 μl of heparinized PPP from two donors separately. The clotting time (CT) was measured in triplicate. Minimum clotting dose (MCD) of venoms was estimated at 60 s. All analyses were performed using GraphPad Prism (version 8.0.2). Different dilutions of antivenoms - 150:1, 120:1, 90:1, 60:1, 30:1, 15:1, 7.5:1, 3.25:1, and 1.6:1 were added to a challenge dose of venom equivalent to 2MCD and incubated at 37°C for 30 minutes. The fold change in the CT was estimated by dividing clotting time for VAV by clotting time for the challenge dose:

Effective dose was estimated as the AV ratio at which clotting time increased by five-fold.

Statistical analysis

ECC-TN venom was designated as the reference control group, since the IPAV is made using venoms from this source . Minimum clotting dose (MCD) and effective dose (ED) values of venoms from ECS-PO, ECS-BA, and ECS-SA were expressed as mean ± SEM and compared against ECC-TN venom. For each parameter, a one-way ANOVA was used to test for overall differences among groups. When significant, Dunnett’s post-hoc test was applied to determine which venoms differed significantly from the ECC-TN (p<0.05).