Scorpions comprise around 2400 species, and belong to an ancient and ecologically successful group of animals that has been exemplified in the fossil record for some 400 million years [1
]. They pose a public health threat in many countries, but particularly in North Africa [3
]. Scorpions most dangerous for humans belong to the Buthidae
family, which, with 82 genera and 756 species, is the largest scorpion family, found on every continent except Antarctica [5
]. About twenty Buthidae
species are known to be lethal to humans. Some of these lethal species belong to the Androctonus
genus, as A. australis
in Algeria (Hector
morph) and in Tunisia (garzonii
sub-species) and A. mauretanicus
in Morocco. These sizeable animals can inoculate up to 500 µg of a venom that is particularly rich in toxins. In the Maghreb, these two species are responsible for about 100,000 stings per year and, 1 to 7% lead to death [7
]. Their median lethal dose (LD50
) by subcutaneous (s.c
.) injection is between 1 and 5 µg per mouse (20 g), and they are therefore considered to be the most lethal scorpion species in the world for mammals and humans [8
]. Other venomous Androctonus
species are Androctonus amoreuxi
, Androctonus aeneas aenaes
, Androctonus crassicauda
, and Androctonus bicolor
. However, the chemical and pharmacological properties of their venoms are still poorly studied, compared to what is currently known about the Androctonus australis
and Androctonus mauretanicus
venoms. The three Pasteur Institutes in Maghreb have largely added to our knowledge of the Androctonus mauretanicus
and Androctonus australis
venoms, their main objective being the production of specific and efficient antivenoms for serotherapy purposes [10
Victims of scorpion stings suffer various pathologies, involving both sympathetic and parasympathetic stimulation as well as central manifestations such as irritability, hyperthermia, vomiting, profuse salivation, tremor, and convulsions. The clinical signs and symptoms observed in humans and experimental animals are related to an excessive systemic host inflammatory response to stings. In addition to cardiac dysfunction, pulmonary edema, and respiratory failure, systemic inflammatory response seems to be strongly implicated in the pathogenesis of scorpion envenomation. The complexity of scorpion pathogenesis and its severity reduces the efficacy of treatment. Thus, improving serotherapy is a key challenge for scientists and antiserum producers.
Scorpion venoms are complex mixtures of peptides and proteins, for which many have yet to be assigned a function. The polypeptide toxins from scorpion venom have very specific actions, and mainly interact with different ion channels and receptors in excitable membranes. Four different families of scorpion neurotoxins have been described, which specifically recognize voltage-gated sodium, voltage-gated potassium, voltage-gated calcium, and chloride channels [13
]. These neurotoxins are present in the venom as a few percent of the dried venom weight. In Androctonus
venoms, s.c. venom toxicity in mammals has mainly been attributed to the activity of long polypeptide chain toxins, which bind with high affinity to voltage-gated sodium (Nav
) channels [14
]. Indeed, Nav
channels are very critical for generating the rising phase of an action potential by promoting a rapid flux of ions across the membrane [16
], an action that is disrupted by scorpion toxins. With their high number of disulfide bonds (four), which hold together their rather small molecular size (60–70 residues), these toxins can persist in a hostile environment because they are highly stable and resistant to denaturation. They display a high degree of relatedness at the level of three-dimensional (3D) structure, despite having more limited sequence homology.
Neutralization of scorpion venoms by heterologous antivenoms has been extensively investigated. However, the effectiveness of each commercial available antivenom, produced in a different geographical area, in neutralizing homologous and heterologous scorpion venoms has been a matter of debate [17
]. Nowadays, antivenom specificity can be explained by the large amount of chemical and immunological data accumulated so far.
In this review, we will tackle recent research progress that led to our understanding of (1) the mechanisms contributing to the pathophysiology and inflammatory response after envenomation, (2) the chemistry of Androctonus venom α-toxins and their immunochemical interrelations, and (3) the set-up of an appropriate serotherapy with the most recent developments, and possible future directions.
2. Immediate Envenomation Symptoms
Commonly, the symptoms of scorpion stings are mainly observed in the peripheral nervous system. Stings in children, the elderly, and immunocompromised people are much more dangerous than in healthy adults. Following a sting, symptom progression is rapid. However, serotherapy is very effective when a specific antiserum is rapidly injected; victims typically recover within one hour after administration.
Three stages of severity are described [18
]. First, an immediate intense and persistent pain (up to two hours) is the dominant clinical sign. During this unthreatening stage I, other discrete general symptoms can be observed such as agitation, febricula, sweats, nausea, feeling of general faintness, and alternating blood pressure (hypertension or hypotension). During stage II, which is considered a severe envenomation, the body temperature increases and sweats, epigastric pain, vomiting, colic, diarrhea, priapism, hypotension, bradycardia, pulmonary obstruction, and dyspnea can appear. Vomiting indicates huge severity and necessitates specific monitoring. Stage III is only seen in 5–10% of stage II cases, and is potentially fatal. At this late stage, cardiac arrhythmia and myocardial ischemia explain the risk of cardiovascular collapse, associated with severe respiratory complications such as pulmonary edema, bronchospasm, and cyanosis.
7. Advanced Scorpion Envenomation Therapies
Despite progress in understanding the pathophysiology of scorpion envenomation and its treatment, challenges still remain [122
]. As such, the development of effective, cheap and safe treatments is greatly needed. Understanding the cellular and molecular events involved offers promising future therapeutic and diagnostic opportunities. Such possible therapeutic targets include adhesion molecules, matrix metalloproteases (MMPs), and inflammatory cytokines and their receptors. Given the potential involvement of oxidative stress in the pathogenesis of envenoming, there are reasons to believe that antioxidant compounds might also be beneficial.
Nowadays, antivenom obtained from hyper-immune horses is the only treatment used for envenomed humans. However, current limitations of serotherapy require an efficient alternative with a high safety margin, target affinity, and more promising venom neutralizing capability [123
Neurotoxins are major lethal constituents of scorpion venoms, and are found in relatively low abundance in the venom (i.e., about 3–10 % in weight for an electrically obtained crude venom). Moreover, because of their small size, they are frequently not able to elicit a strong immunogenic reaction. As such, they may not always induce the production of high-quality and sufficient quantity of antibody molecules. Consequently, a balance between the injected doses, the toxicity towards the animal and high-quality antibody production has to be obtained, often empirically.
In spite of the cost and time needed, isolation of the main toxic venom components to develop antisera has advantages, compared to using the crude venom as an antigen to raise therapeutic antibodies. Purified neurotoxins constitute a remarkable tool for the preparation of highly protective antivenom. In this case, isolated antibodies are specific to the scorpion toxin family injected. Such antibodies are useful, for example, in the detection and isolation of scorpion toxin polypeptides present in biological samples.
A complicating factor in antiserum design is reports of controversial results stemming from the use of different antidote preparations in trials and clinical practice against local scorpion species. As a result, there is an absence of consensus among researchers. African and Brazilian authors note that the availability of potent and reliable antivenom sera in these regions is limited [102
The improvement of the serotherapy efficiency could be based on the immunization protocol, including type of antigen (venom vs. toxoid), adjuvant type, immunization dose, frequency and intervals of immunization, and animal choice (horse vs. camelid or chicken). The choice of animal producer is an important factor in the generation of effective antibodies. The use of mammals seems to be ineffective due to the secondary reactions, including anaphylactic shock. Other strategies using animals phylogenetically distant from mammals could avoid these reactions [124
]. A camel immunized with the toxic major fraction from Aah venom led to a high titer in conventional IgG and HCab (heavy-chain-antibody), lacking light chain and CH1 domain. Obtained serum was able to successfully neutralize venom toxicity [113
]. Fragments of recombinant VHH antibodies obtained from HCab antibodies expressed in recombinant bacteria were characterized by very high stability, low immunogenicity, and high production [120
]. However, some properties of VHH needed improving in order to be fully implemented. Affinity, specificity, and size properties were functional limitations in pharmacogenetics of VHH. Additionally, the therapeutic efficiency of VHH was also limited by the short serum half-life [126
Another alternative that has proved its usefulness in experimental therapy is the use of IgY obtained from egg yolks [127
]. Compared to mammals, chickens are economically attractive in terms of polyclonal antibody production, since antibody levels are higher (5–6 times more than a in a rabbit over a 2-week period) and production lasts a long time, since a laying hen can be productive throughout the laying period and beyond [130
]. This economic benefit is reinforced by: (i) the lower cost of feeding and housing chickens, compared to other animals such as horses or sheep, and (ii) the ethical production of IgY from the egg yolk [129
]. Recently, it has been reported that IgY antibodies effectively neutralize the Aah venom lethality, and could prevent its pathological effects [132
It is worth noting that a prophylactic treatment could be used to prevent the induced pathophysiological effects and the lethality of scorpion envenomation [133
]. The development of a vaccine-like product could enhance the immune responses to a specific antigen without deleterious side effects caused by adjuvants [136
]. It was reported that the Aah venom and its toxic fraction FtoxG50 vaccine nano-formulations present a high immunogenicity, an important immune-protective effect, and a low reactogenicity [137
Finally, all these academic studies showed that, from a practical point of view, laboratories producing antivenoms should use at least partially purified venom fractions, or a mixture of pure antigens, to obtain antivenoms able to neutralize neurotoxins belonging to the different structural and immunological groups. Serotherapy as a specific treatment is usually recommended after scorpion stings in most of the regions at risk. However, to be of better effectiveness, this therapy needs to be improved, optimized, and standardized considering the limitations (delay, antibody format, soluble or freeze-dried, dose, route of injection, etc.), and also the scientific and technological advances.