All four sea snake venoms exhibited highly potent lethality in mice (
Table 1), especially for the two common species in Malaysia: Both
H. schistosus and
H. curtus venoms possess LD
50 < 0.1 µg/g. This indicates that the bites from these species, though they appear to be under-reported, should be taken very seriously by local health authorities. There are no significant differences (
p > 0.05) noted in LD
50 values between the three routes for envenoming (intravenous, intramuscular and subcutaneous), indicating that the sea snake venoms share a near-complete systemic absorption from the subcutaneous site of a snake bite. This is likely because the principal lethal toxins of sea snake venoms consist mainly of low molecular mass toxins (α-neurotoxins and phospholipases A
2) [
17] that are able to cross barrier membranes more effectively (better absorption) like cobra venom toxins [
18]. Additionally, in sea snake bites, there is very minimal interaction between the local tissue and toxins (virtually no local tissue inflammation or necrosis) to retain the venom
in situ [
4]. This is in contrast to some Viperidae venoms which can show exceptionally low systemic bioavailability from a non-vascular injection site [
19].
Since the neurotoxins (NTX) in
H. schistosus venom are typically the short-chain type, cross-neutralization was first investigated using the Taiwan bivalent antivenom (TBAV), as
Naja atra venom, one of the venoms used to raise the antivenom, consists mainly of short-NTX [
20]. Using the antivenom-venom preincubation method, TBAV was found to be only weakly effective, with potency values of 0.016 mg/mL and 0.019 mg/mL for cross-neutralizing the venoms of
H. schistosus and
H. curtus, respectively, in mice (
Table 2). The Thai
Naja kaouthia monovalent antivenom (NKMAV) appeared to be more effective than TBAV in this regard (
Table 2), with potency values approximately twice that of TBAV, even though Thai cobra venom is known to consist mainly of long α-neurotoxins. These findings suggest that NKMAV may cross-react more substantially with the sea snake venoms. This supports an earlier report that Thai cobra long NTX-specific F(ab')
2 cross-reacted with
H. curtus venom [
11], although the major NTX in sea snake venom is of the short NTX subtype [
21]. The new polyvalent antivenom (neuro polyvalent antivenom, NPAV) produced by QSMI, which targets neurotoxic envenomation, showed a distinctively higher neutralizing potency, approximately twice that of NKMAV and four times that of TBAV, at doses standardized according to the respective guidelines for clinical use. Cross-neutralization by NPAV was also observed for sea krait (
Laticauda sp.) venoms, which mainly comprise NTX of the short-chain type called erabutoxins [
22]. In comparison with NKMAV, the apparently higher potency observed in NPAV in cross-neutralizing
H. schistosus and
H. curtus venoms could be due to synergistic cross-reacting effects from the polyvalent F(ab')
2 against toxin components originating from other elapids, in particular possible neutralization of sea snake basic PLA
2 in addition to the neurotoxins. A previous study [
23] demonstrated that prior administration (10 min) of CSL tiger snake antivenom into the chick biventer cervicis nerve-muscle preparation was able to attenuate the neuromuscular blockade effect of the venoms of several sea snakes, except that produced by
E. schistosa (synonymized as
H. schistosus). The current
in vivo study indicated that the antigenic properties of
H. schistosus venom may be closer to that of the
Naja cobra than the tiger snake. The cross-neutralization phenomenon is also supported by the immunological cross-reactivity result, where ELISA cross-reactivities between NPAV with
H. schistosus and
H. curtus venoms were shown to be 58.4% and 70.4% against blank, respectively (
Figure 1). Comparatively, the ELISA reactivity of NPAV with the homologous
N. kaouthia venom was high (103.3% against blank). The data indicate that the sea snake toxins have some degree of binding avidity toward NPAV, although the ELISA cross-reactivity values are not necessarily congruent or in proportion with the neutralizing potency against the different venoms. In contrast, the absorbance value for
C. rhodostoma venom (14.4% against blank) is remarkably low, representing non-specific binding between venom proteins and NPAV without effective neutralizing activity.
The cross-neutralizing effectiveness of NPAV was further tested in an experimental envenomation model, where different doses of NPAV were administered intravenously into mice that were subcutaneously pre-envenomed with 2.5 LD
50 of
H. schistosus or
H. curtus venom. NPAV was proven effective
in vivo for neutralizing both Malaysian sea snake venoms in the experimental envenomation model (
Table 2). From the experience of venom milking (using the method of induced biting through a film), we estimate the average
H. schistosus venom yield per bite to be 6.1 ± 3.7 mg (
n = 22, range 2–12 mg, adult snake > 1 m) and average
H. curtus venom amount per bite to be approximately 1 mg (
n = 3, range 0.7–1.2 mg, adult snakes of approximately 1 m). Based on the potencies (
Table 2) translated into 0.4 mg/vial and 0.8 mg/vial of NPAV for
H. schistosus and
H. curtus venoms respectively, a total of 10–20 vials of NPAV may be required for neutralization of the venom injected per bite. In cobra envenoming, this is considered an acceptable amount for antivenom dosage; the initial dose is typically 10 vials, followed by additional doses as required clinically [
24].
Figure 1.
Absorbance values from indirect ELISA indicating cross-reactivities between neuro polyvalent antivenom (NPAV) with venoms from Calloselasma rhodostoma, Hydrophis schistosus, Hydrophis curtus and Naja kaouthia. Values were expressed as mean of triplicates with standard deviation. Asterisks indicate significant difference between means (p < 0.05).