Venoms and antivenoms

Venoms of Naja sputatrix, Naja siamensis, Naja kaouthia (Thailand), Naja philippinensis, Naja oxiana, Naja atra, Naja naja (Sri Lanka, sample 1), Naja naja (India, sample 1 and 2), Naja melanoneuca, Naja nigricollis, Naja nubiae, Naja katiensis, Naja haje, Bungarus multicinctus and Bungarus caeruleus were purchased from Latoxan (Valence, France). Venoms of Naja sumatrana, Naja kaouthia (Malaysia), Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus and Bungarus flaviceps were pooled samples obtained from several adult individuals captured in Malaysia whilst Naja naja (Sri Lanka sample 2) was a pooled sample obtained from several adult individuals captured in Sri Lanka. After extraction, the venoms were instantly lyophilized. Two antivenoms were studied: (a) Neuro Polyvalent Snake Antivenom (NPAV) (Lyophilised; Batch no. 0030208; Exp. Date April 21st, 2013), a purified F(ab′)₂ obtained from serum of equines hyperimmunized against a mixture of four venoms: Naja kaouthia (Thai monocellate cobra), Ophiophagus hannah (king cobra), Bungarus candidus (Malayan krait) and Bungarus fasciatus (banded krait); (b) Naja kaouthia monovalent antivenom (NKMAV) (Full name: Cobra antivenin; Lyophilised; Batch no. 0090406; Exp. Date August 31st, 2014), a purified F(ab′)₂ obtained from serum of equines hyperimmunized specifically against the venom of Thai N. kaouthia. Both of these antivenoms are produced by Queen Saovabha Memorial Institute (QSMI), the Thai Red Cross Society from Bangkok, Thailand. For neutralization studies, both antivenoms were reconstituted in the same manner: 10 mL of normal saline was added to 1 vial of the freeze-dried antivenom. According to the attached fact sheet, 1 mL of the NPAV antivenom is able to neutralize the following amount of snake venoms: 0.6 mg each of N. kaouthia and B. fasciatus venoms, 0.4 mg of B. candidus and 0.8 mg of O. hannah venoms; while 1 mL of the NKMAV can neutralize 0.6 mg of N. kaouthia venom.

Animals

Albino mice (ICR strain, 20–25 g) and male Sprague Dawley rats (250–300 g) were supplied by the Laboratory Animal Centre, Faculty of Medicine, University of Malaya. The animals were handled according to the guidelines given by CIOMS on animal experimentation [8]. All experiments involving animals were approved by the Animal Care and Use Committee (ACUC) of the University of Malaya (Ethical clearance letter No. PM/03/03/2010/FSY(R)).

Determination of protein content

Protein content was determined by Bradford method [9]. All measurements were performed in triplicate. Bovine serum albumin (Sigma, USA) was use to generate a standard curve.

Chromatographic and electrophoretic profiling of the antivenom

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method of Studier [10], using the Bio-Rad broad-range prestained SDS-PAGE standards (6.5–200 kDa) and 15 µL of each antivenom sample (3 mg/mL) was loaded in the gel (12.5%). High performance gel filtration chromatography of the reconstituted antivenom (100 µL, 10 mg/mL) was performed using a Superdex 200 HR 10/30, 13 µm SEC 10×300 mm (GE Healthcare, Sweden). Elution buffer was 100 mM sodium phosphate, 0.15 M NaCl, pH 7.4 at a flow rate of 0.75 mL/min. Protein was monitored by absorbance measurement at 280 nm. The column was calibrated using the following protein standards obtained from Bio-Rad (BIO-RAD Gel filtration Standard): thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa).

Determination of venom lethality

The median lethal dose, LD₅₀, of the venom was determined by intravenous or intramuscular injection into ICR mice (20–25 g, n = 4). The survival ratio was recorded after 48 h to determine the LD₅₀.

In vitro neutralization of lethality of venoms

In vitro neutralization of lethality was conducted as described by Ramos-Cerrillo et al. [11]. Briefly, a challenge dose of the venom in 50 µL saline was pre-incubated at 37°C for 30 min with various dilutions of the reconstituted antivenom (NPAV or NKMAV) in normal saline, to give a total volume of 250 µL. The mixture was subsequently centrifuged at 10000× g before being injected into the caudal vein of the mice. The number of survival after 48 h was recorded. Generally, the challenge dose used was 5 LD₅₀. However, if 200 µL of the reconstituted antivenom (maximum permitted volume to inject into the mouse) failed to give full protection of the mice, a lower challenge dose of 2.5 LD₅₀ was used instead. The antivenom was considered ineffective when none of the animals injected with the pre-incubated mixture (containing 2.5 LD₅₀ challenge venom in 50 µL saline and 200 µL of the undiluted reconstituted antivenom) survived. Neutralizing potency of the antivenom was expressed as ED₅₀ (the amount of reconstituted antivenom in µL or the ratio of mg venom/mL reconstituted antivenom that gives 50% survival of the animals tested) as well as in term of ‘neutralization potency’ (P, the amount of venom that is completely neutralized by a unit volume of antivenom) calculated according to Morais et al. [12].

In vivo neutralization of lethality of venom

This was carried out by intramuscular injection of 5 LD₅₀ or 2.5 LD₅₀ of the venom into mice (n = 4) followed by intravenous injection of 200 µL of appropriately diluted reconstituted antivenom, 10 min later. The number of survival after 48 h was recorded.

Protective actions of the antivenom against N. sputatrix venom-induced cardiovascular depressant and neuromuscular blocking effects in anesthetized rats

The study was conducted on three groups of rats (n = 3, 250–300 g) anesthetized with intraperitoneal injection of urethane (1.4 g/kg, i.p.) to the point of loss of the eyelid reflex and the pedal withdrawal reflex on painful stimuli. The anesthetized animals were surgically prepared for the simultaneous measurement of blood pressure, heart rate, respiratory rate and muscle twitch tension. Data collection and analysis were conducted using PowerLab 4/30 data Acquisition system equipped with LabChart software (AD Instruments, Australia). Rats in group 1 (termed ‘saline/-’ group) were injected with 50 µL saline intramuscularly at 0 min and served as control; Rats in group 2 (termed ‘NsV/-’ group) were injected with 6 mg/kg N. sputatrix venom (dissolved in 50 µL saline) intramuscularly; and rats in group 3 (termed ‘NsV/NPAV’ group) were injected with the same dose of venom intramuscularly followed by intravenous administration of 3 mL of the reconstituted NPAV (1 mL each at 10 min, 30 min and 50 min post-injection of the venom). The volume of antivenom administered and the time points were chosen to ensure no disturbance of the blood pressure, heart rate and respiratory rate occurred.

Statistical analysis

LD₅₀ of the venoms and ED₅₀ of antivenoms are expressed as means with 95% confidence intervals (C.I.). LD₅₀, ED₅₀ (median effective dose) and the 95% confidence intervals (C.I.) were calculated using the probit analysis method of Finney [13] with the BioStat 2009 analysis software (AnalystSoft Inc.). The statistical analysis for pharmacological study was conducted using SPSS. The data (expressed as mean ± S.D.) were analyzed using one-way ANOVA, with Tukey's post hoc multiple-comparison test, with P<0.05 as significant.

Composition of the antivenoms

The protein contents of reconstituted NPAV and NKMAV were 20.3 mg/mL and 12.5 mg/mL, respectively. The SDS-PAGE patterns of NPAV (Neuro Polyvalent Snake Antivenom) and NKMAV (Naja kaouthia monovalent antivenom) indicated that there was no distinct band of high or low molecular weight proteins (Fig. 1). The same was also observed in the gel-filtration chromatographic profiles of the two antivenoms (Fig. 2). Based on ‘area-under-the-curve’ comparison, the quantity of F(ab′)₂ in both these antivenoms were comparable (91–96%) whilst the quantity of respective dimers and low molecular weight proteins in NKMAV were slightly higher than those in NPAV (Figure 2). No high molecular weight aggregates were detected in both of these antivenoms.

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Figure 1. SDS-PAGE of Neuro Polyvalent Snake Antivenom (NPAV) and Naja kaouthia monovalent antivenom (NKMAV).

Fifteen µL of the respective reconstituted antivenom (3 mg/ml) was loaded on the middle (NPAV) and right lane (NKMAV). Prestained molecular weight markers mixture was loaded on the left lane (M, molecular weight in kDa). The digested heavy chain (H′) and light chains (L) are indicated by black arrowheads.

https://doi.org/10.1371/journal.pntd.0001672.g001

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Figure 2. Elution profiles of (A) Neuro Polyvalent Snake Antivenom (NPAV) and (B) Naja kaouthia monovalent antivenom (NKMAV).

One mg of each sample was applied to Superdex 200 10/300 GL gel column. The arrows identify the peaks. The results of integration of the areas-under-the-curve are shown in the insert table on the upper left. LMWC stands for low molecular weight components.

https://doi.org/10.1371/journal.pntd.0001672.g002

In vitro neutralization of venom lethal effects by the antivenoms

The results of the in vitro neutralization of venom lethality by the NPAV and the monovalent N. kaouthia antivenom (only for selected venoms) are shown in Table 1 and 2, respectively. The results showed that NPAV was able to confer protection/cross-protection against the venoms of all krait as well as almost all the Afro-Asian cobra venoms examined (except for the African spitting cobra N. katiensis), although the neutralizing potency range varied from low to high. The NKMAV was able to neutralize the venoms of six Asian cobras (N. kaouthia [Thailand & Malaysia], N. sputatrix, N. sumatrana, N. siamensis and O. hannah) tested, with efficacy comparable to that of NPAV, but failed to neutralize the venoms of B. fasciatus and B. candidus. It is interesting to note that our results on the neutralization potentials of NPAV against N. kaouthia, B. fasciatus, B. candidus and O. hannah venoms are much higher than stated in the antivenom fact sheet provided, in particular against O. hannah venom. It should be noted, however, that the definition of neutralization potential used in the fact sheet has not been clearly stated.

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Table 1. In vitro neutralization of lethality of Afro-Asian cobra and krait venoms by NPAV.

https://doi.org/10.1371/journal.pntd.0001672.t001

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Table 2. In vitro and in vivo neutralization of cobra and krait venoms by NPAV and NKMAV.

https://doi.org/10.1371/journal.pntd.0001672.t002

In vivo neutralization of lethal effects of some Asian cobra and krait venoms

Table 3 shows the in vivo neutralization of lethality of four venoms from Asian cobras and krait (N. sputatrix, N. kaouthia (Thailand), N.kaouthia (Malaysia) and B. candidus) by NPAV. It is interesting to note that the i.m.LD₅₀ values determined were comparable to the corresponding i.v.LD₅₀ values. Our results showed that NPAV effectively neutralized all four venoms in vivo, with ED₅₀ comparable to the corresponding ED₅₀ in the in vitro neutralization assays.

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Table 3. In vivo neutralization of lethality of some cobra and krait venoms by NPAV.

https://doi.org/10.1371/journal.pntd.0001672.t003

The protective effects of NPAV on the N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in the anesthetized rats

Figure 3 shows that NPAV was able to fully protect against the N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in the anesthetized rats. The mean blood pressure (BP), heart rate, respiratory rate and muscle twitch tension of the control anesthetized rats (‘saline/-’ group) remained constant for at least 6 hours after the initial stabilization. Following an intramuscular injection of the venom at 4×LD₅₀ dose (6 mg/kg), however, there was an immediate small decrease (about 20%) in the BP, which remained constant thereafter for the next 90 min (Figure 3A). During this period, the heart rate remained essentially unaffected (Figure 3B). And then the BP and heart rate both began to fall precipitously from 90 min onward. On the other hand, the respiratory rate and muscle twitch tension were stable only for the first 60 min after venom administration. Both these parameters, however, began to decrease sharply thereafter (Figure 3C and 3D) and death occurred at 125–130 min after the venom injection.

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Figure 3. Effects of Neuro Polyvalent Snake Antivenom (NPAV) on the cardio-respiratory depressant and neuromuscular blocking effects.

Changes in percentage (%) of (A) mean blood pressure, (B) heart rate, (C) respiratory rate and (D) muscle twitch tension, upon intramuscular injection of N. sputatrix venom (6 mg/kg) at 0 min in ‘NsV/-’ group (n = 3; green circle) and ‘NsV/NPAV’ group (n = 3; blue square ); and intramuscular injection of saline in ‘saline/-’ group (n = 3; red diamond) , which served as the control group. The ‘NsV/NPAV’ group received intravenous injection of 1 mL NPAV each at 10, 30 and 50 min (3 mL in total) post venom injection. *P<0.05 by one-way ANOVA between the ‘NsV/-’ group and ‘NsV/NPAV’ group, within the range indicated by the curly bracket. #P<0.05 by one-way ANOVA between the ‘saline/-’ group and ‘NsV/NPAV’ group, within the range indicated by the curly bracket. The animals in ‘NsV/-’ group died within 125–130 min upon venom injection. All values are expressed as mean ± S.D.

https://doi.org/10.1371/journal.pntd.0001672.g003

In a parallel series of experiment conducted to examine the ability of NPAV to protect against the cardiovascular depressant and neuromuscular blocking effects of N. sputatrix venom, 1 mL each of NPAV was administered via the left jugular vein, at 10, 30 and 50 min, respectively, following the i.m. injection of the venom. The results showed that the administration of the antivenom effectively reversed the cardio-respiratory depressant and neuromuscular blocking effects induced by the N. sputatrix venom. The heart rate and muscle twitch tension of the antivenom-treated animals (‘NsV/NPAV’ group) were restored to the same levels as the ‘saline/-’ control group (P>0.05, not significantly different between ‘saline/-’ group and ‘NsV/NPAV’ group by one-way ANOVA), whereas the blood pressure and respiratory rate were restored to 80% of the control group (P<0.05 between the two groups) and remained at that level throughout the monitoring period.

Expression of neutralization capabilities of antivenom

In this report, the neutralization capabilities of the antivenoms are expressed in three different ways: the commonly used median effective dose ED₅₀ in µL antivenom, ED₅₀ in mg/mL and potency, P, as defined by Morais et al. [12]. For ED₅₀, expressing the value in term of mg venom neutralized per mL antivenom is a more realistic assessment of the neutralization capabilities of the antivenom than in term µL antivenom or number of mouse LD₅₀, because the LD₅₀'s of the various cobra venoms differ substantially. For example, expressed in term of µL antivenom, the ED_50's of NPAV against N. kaouthia (Malaysia) venom and N. philippinensis venom are comparable, but when expressed in term of mg/mL, NPAV was obviously much more effective against N. kaouthia (Malaysia) venom then against N. philippinensis venom.

Because of the high lethality of certain venoms, a 2.5 LD₅₀ instead of the standard 5 LD₅₀ was used as the challenge dose. Since the ED₅₀ value of an antivenom is highly dependent on the challenge dose, ED₅₀ obtained from the two different challenge doses cannot be compared directly. As such, we also expressed neutralization capability of the antivenom in terms of P (potency), which is the mass of venom that is completely neutralized per unit volume of antivenom, as defined by Morais et al. [12]. Potency gives a better estimate of the relative efficacy of antivenoms than comparing ED₅₀ values when different challenge doses were used in the determination of ED₅₀, as P is theoretically independent of the amount of challenge doses. Nevertheless, since the relationship between antivenom neutralizing capability versus venom challenge dose is not necessarily linear due to the complexity of venom and antibodies composition, comparing the P values of antivenom when using different challenge doses does have its limitation.

Comparison of monovalent antivenoms versus polyvalent antivenoms

The relative merit of monovalent antivenoms versus polyvalent antivenoms has been the subject of much discussion [14] and there are some authors who suggested that monovalent antivenoms are generally more potent than polyvalent antivenoms and less likely to cause adverse reactions as it may involve administration of a lower quantity of antivenom IgG that with a polyvalent antivenom. Several studies have shown, however, that this is not necessarily true [14], [15]. Our results here demonstrated that the quality and neutralization capabilities of polyvalent antivenom are not necessary inferior to that of monovalent antivenoms. Here we compared the protein composition of the N. kaouthia monovalent antivenom(NKMAV) and Neuro Polyvalent Snake Antivenom (NPAV), as well as the in vitro neutralization potency of the two antivenoms against venoms from five common Asiatic cobras and O. hannah (Table 2). The results show that both antivenoms are devoid of high molecular weight aggregates, the compounds that are usually associated with adverse reactions. The protein contents of the two antivenoms are both relatively low (20.3 mg/mL and 12.5 mg/mL respectively, for NPAV and NKMAV). The neutralization potencies of NKMAV and NPAV against N. kaouthia (Thailand) venom (the venom used to raise both antivenoms) are essentially the same. This, however, is not surprising since according to the manufacturer, monovalent antivenoms (including NKMAV) were later added to the purified polyvalent antivenom to ensure the neutralizing potency of the NPAV was comparable with the neutralizing potencies of the respective monovalent antivenoms. What is interesting is that, the neutralization potencies of both antivenoms against four Asiatic cobra venoms tested are comparable. NPAV, however, is much more potent than NKMAV in neutralizing O. hannah venom. This is to be expected as O. hannah venom was included in the immunogen mixture used in raising NPAV. These observations are in accordance with the conclusion drawn by Raweerith and Ratanabanangkoon [15].

Neutralization potency of NPAV against venoms of Malaysian elapids

NPAV was raised using the four common elapid venoms in Thailand: N. kaouthia (Thailand), O. hannah, B. candidus and B. fasciatus. Our results showed that the polyvalent antivenom could effectively neutralize venoms of the same four species that originated from Malaysia. It is interesting that the polyvalent antivenom could effectively neutralize venom of N. kaouthia from Malaysia too. According to Wüster and Thorpe [16], the composition of the venom of Thai N. kaouthia is substantially different from that of the Malaysian N. kaouthia, the former appears to be more neurotoxic, while the latter more necrotic. It was suggested that this difference in the toxin composition may result in antivenom incompatibility. Our results, however, showed that while the two N. kaouthia venoms did differ substantially in their venom i.v. LD₅₀, the polyvalent antivenom that was raised from Thai N. kaouthia venom (together with other Thai elapid venoms) could effectively neutralize the venom of Malaysian N. kaouthia, albeit with a moderately lower ED₅₀. The same cross-neutralization potency was also observed with the monovalent N. kaouthia antivenom.

In addition to the four elapid venoms mentioned, NPAV also effectively neutralized the venoms of the other Malaysian kraits B. flaviceps and B. fasciatus, as well as the medically important Equatorial spitting cobra N. sumatrana. Thus, the results of our in vitro neutralization studies suggest that NPAV, which is prepared from Thai elapid venoms, can be useful in the treatment of elapid envenoming in Malaysia, since the polyvalent antivenom can effectively neutralize venoms of all medically important elapids in Malaysia.

Neutralization potencies of NPAV against other Asiatic cobra venoms

Our results showed that NPAV was effective (P>0.5 mg/mL) against other Asiatic cobra venoms, including the venoms of the three Southeast Asian spitting cobras N. siamensis, N. sumatrana and N. sputatrix, as well as the venoms of the Central Asia cobra N. oxiana and Chinese cobra, N. atra. Earlier report claimed that Chinese cobra N. atra venom was poorly neutralized by other commercial cobra antivenoms [17]. NPAV, however, was only weakly effective against the venom of N. philippinensis, the highly neurotoxic (lethal) Philippine cobra. The low neutralization potency of 0.11 mg/mL would mean that more than 50 vials of the antivenom may be required for the victim, as the amount of venom injected during a cobra bite can be more than 50 mg (dry weight). Indian polyvalent antivenom (raised from N. naja venom) has also been reported to show poor neutralizing ability against N. philippinensis venom.

NPAV was only moderately effective against N. naja venoms from the Indian subcontinent with the exception of one Sri Lankan N. naja venom sample (mixtures collected from several adult N. naja). The variation suggests geographical differences in N. naja venom.

Thus, our results suggest that NPAV is effective (P>0.5 mg/mL) in neutralizing the venoms of Asiatic cobras from Southeast Asia (except the Philippines cobra), Central Asia and China, but only moderately effective against venoms from the cobras from the Indian subcontinent. These observations can be used as the basis for the design of a polyvalent antivenom with a broader spectrum of cross-neutralization. For example, if venoms of N. philippinensis, N. naja and B caeruleus were added to the immunogen mixture to raise NPAV, the resulting polyvalent antivenom may well be an effective Pan-Asian polyvalent cobra and krait antivenoms.

Neutralization potencies of NPAV against African cobra venoms

Five common African cobra venoms, including venoms from three spitting cobras (N. nigricollis, N. nubiae and N. katiensis) were selected for this study. NPAV could neutralize effectively the venoms from N. melanoleuca, but is moderately effective against that of the spitting cobras N. nigricollis and N. nubiae, and weakly or not effective against venom of the highly lethal N. haje and the spitting cobra N. katiensis. We have not carried out a thorough study of the neutralization potency of NPAV against African cobra venoms, as many of the venoms are not available to us. Nevertheless, the results showed that there are still substantial cross-neutralizations between major venom toxins of the Asiatic and the African Naja, despite the fact that the Asian group lies in a more distant branch from the African group in the phylogenetic dendrogram [18].

Neutralization potency of NPAV against krait venoms

It is interesting to note that the NPAV could effectively neutralize the venoms of the three Southeast Asian kraits (B. fasciatus, B. candidus and B. flaviceps), but only moderately or weakly against the venoms of the other two kraits (B. multicinctus and B. caeruleus). The results indicate that krait venoms share enough common antigens among them to enable the NPAV raised against B. fasciatus and B. candidus to neutralize all 5 krait venoms tested. However, the low to moderate potency of NPAV against B. multicinctus and B. caeruleus venoms indicates significant differences in antigenicity of some of the venom toxins.

In vivo neutralization potency of NPAV versus in vitro neutralization potency

For all four elapid venoms tested, the i.m LD₅₀ values are comparable to the i.v. LD₅₀, indicating that the main venom toxins could diffuse effectively from muscle to circulation, presumably because these toxins are mainly low molecular weight proteins (phospholipases A₂ and the three-finger toxins). Also, the neutralization capability (as measured by ED₅₀) of NPAV in the in vivo assay is comparable to that of in vitro assay, suggesting that neutralization potential of antivenom against elapid snakes measured by the usual in vitro neutralization assay does provide a good indication of its effectiveness in the in vivo situation.

The protective effects of NPAV against cardiovascular depressant and neuromuscular blocking effects of N. sputatrix venom in anesthetised rats

To further examine the in vivo neutralization capability of the NPAV, we examined its ability to protect against N. sputatrix cobra venom-induced cardio-respiratory depressant and neuromuscular blocking effects in anesthetised rats. The antivenom (3 mL in total) was administered in 3 separate injections to minimize disturbing the cardio-respiratory parameters. It is well established that the major lethal toxins of N. sputatrix venom consist of polypeptide neurotoxins, polypeptide cardiotoxins and phospholipases A₂ [19]. The venom-induced cardio-respiratory depressant effect was likely due to the combined actions of polypeptide cardiotoxins and phospholipases A₂ whereas the neuromuscular blocking effect was likely due mainly to the action of the venom polypeptide neurotoxins, although venom phospholipases A₂ may also play a role. Thus, the ability of NPAV to effectively reverse the N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in the rats demonstrated that the antivenom did contain specific antibodies that could effectively neutralize the major lethal toxins of N. sputatrix venom.

Conclusions

In conclusion, the in vitro and in vivo neutralization studies indicated that Neuro Polyvalent Snake Antivenom (NPAV) effectively neutralized venoms from many Southeast Asian Naja, Bungarus and Ophiophagus hannah but less effective against the venoms of Naja from the India subcontinent and Africa, as well as the Asiatic N. philippinensis. This cross-neutralization information can be used as the basis for the design of broader-spectrum polyvalent cobra antivenom. The abilities of NPAV to protect against N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects confirmed that the antivenom effectively neutralized the major lethal toxins of Naja venoms. The antivenom may find potential application in the treatment of elapid bites in Southeast Asia, especially Malaysia, a neighboring nation of Thailand. Nevertheless, the applicability of NPAV in the treatment of cobra and krait envenomations in Southeast Asia needs to be confirmed by clinical trials, as it is known that antivenom that has been proved effective in murine model is not necessarily effective in treating human victims [20].