toxins

Review
Polyvalent Snake Antivenoms: Production Strategy and Their
Therapeutic Benefits
Kavi Ratanabanangkoon

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand;

kavi.rtn@mahidol.ac.th; Tel.: +66-86-974-8874

Abstract: Snake envenomation remains an important yet neglected medical problem in many coun-
tries, with around five million people affected, and over a hundred thousand deaths annually.
Plasma-derived antivenoms are the main therapeutic agent available. Monovalent antivenoms are
produced via the immunization of large animals, e.g., horses, with one venom, after which the horse
serum can neutralize the homologous venom, with minimal or no cross neutralization against other
venoms. It is necessary, therefore, for the culprit snake to be identified, so that the appropriate specific
antivenom can be selected. Polyvalent antivenoms (pAVs) are produced via immunization with
a number of snake venoms, and the serum can neutralize all the venoms used in its production.
Thus, pAVs can be used to treat several venoms from a country/region, and the identification of the
culprit snake is not necessary. There are various parameters and processes involved in the production
of pAVs, depending on the requirements and resources available. Most commercial pAVs use a
mixture of both elapid and viperid venoms as immunogens, while some pAVs use either elapid or
viperid venoms. Some pAVs are produced through the mixing of more than one monovalent or
polyvalent antivenom. These various types of pAVs have their own characteristics, and have benefits
and drawbacks. The major benefits of pAVs are the wide coverage of many medically important
venoms, including many heterologous venoms. They also remove the need to identify the culprit
snake, and they can be produced at a lower cost than several monovalent antivenoms. Interesting
polyvalent antivenoms, termed ‘syndromic pAVs’ (s-pAVs), have recently gained attention. They
are produced for use according to the syndromes manifested in snakebite patients. The venoms that
produce these syndromes are used as immunogens in the production of ‘syndromic antivenoms’. For
example, ‘neurotoxic polyvalent antivenom’ and ‘hematotoxic polyvalent antivenom’ are produced
using the neurotoxic elapid and hematotoxic viperid venoms as immunogens, respectively. They
Citation: Ratanabanangkoon, K. were first marketed by the Thai Red Cross in 2012, and have since gained attention as a possible
Polyvalent Snake Antivenoms: therapeutic modality to help solve the problem of snakebite envenomation globally. The merits of
Production Strategy and Their these s-pAVs, including their efficacy and wide paraspecificities, are discussed.
Therapeutic Benefits. Toxins 2023, 15,
517. https://doi.org/10.3390/
Keywords: antivenom; snake; syndromic polyvalent; polyspecific; equine; neurotoxins; hematotoxins
toxins15090517

Received: 15 July 2023 Key Contribution: Various polyvalent antivenoms against snakes are presented with respect to
Revised: 16 August 2023 their production processes and characteristics. The potential of the recently described ‘syndromic
Accepted: 18 August 2023 polyvalent antivenoms’ in alleviating the snakebite envenoming problem in African and south Asian
Published: 24 August 2023 countries is discussed.

Copyright: © 2023 by the author.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Snakebite envenomation remains a seriously neglected tropical disease. It has been
distributed under the terms and
estimated that at least 1.8–2.7 million people are affected annually, with about
conditions of the Creative Commons 94,000–125,000 deaths [1,2]. Successful treatment requires the timely administration of an
Attribution (CC BY) license (https:// effective antivenom (AV). In the present article, animal-plasma-derived antivenoms will
creativecommons.org/licenses/by/ be discussed.
4.0/).

Toxins 2023, 15, 517. https://doi.org/10.3390/toxins15090517 https://www.mdpi.com/journal/toxins

Toxins 2023, 15, 517 2 of 11

Antivenoms are produced via the immunization of large animals, such as horses,
donkeys, sheep, or camels, using the crude venom(s) or toxin(s) from the relevant snake.
The plasma from these animals can be prepared, with the red blood cells being returned to
the source animals. The plasma is then fractionated to obtain the immunoglobulins, which
may be further treated with proteolytic enzymes to give Fab or F(ab’)2 antibodies.
Originally, antivenom was produced via the immunization of an animal with a single
venom, and the antiserum obtained was used to treat patients envenomed by the cog-
nate snake venom used in its production. This type of AV is called a monovalent AV, is
usually effective only against that particular venom, and often shows little or no cross-
neutralization with other heterologous venoms [3]. Therefore, it is necessary for the species
of the culprit snake to be correctly identified, in order to select the specific antivenom for
effective treatment.
However, correct identification of the culprit snake is frequently difficult, as snakebites
usually take place in the dark, or in bushy areas. In Thailand, culprit snakes are identified
in about 44% of envenoming cases [4]. In addition, the development and use of snake diag-
nostic test kits are expensive. Such tests may require about half an hour to provide a result,
and this will delay the appropriate antivenom administration. Further, the half-life of these
diagnostic tests is usually rather short, and this makes the test quite expensive. For these
reasons, it is advantageous to produce polyvalent antivenoms (pAVs) that can effectively
neutralize a spectrum of snake venoms that are medically important in a country/region,
without the need to identify the culprit snakes.
Polyvalent or polyspecific antivenoms are produced with the purpose of neutralizing
multiple snake venoms and, thus, can provide effective therapy against a selection of
the medically most important snakes within a geographical area. It is expected that the
widespread adoption of pAVs will negate the necessity of identifying culprit snakes. A
single pAV, instead of a battery of different monovalent antivenoms, would, thus, suffice for
snakebite treatment in each country or region. It should be mentioned here, however, that,
after the administration of the appropriate antivenom, it is useful to know, or to deduce,
the likely causative species, so as to predict the clinical course, and to provide the optimal
supportive treatment and clinical management.

2. Various Parameters Involved in the Production of Polyvalent AVs and the

pAV Characteristics
The first pAV was produced by Vital Brazil in 1898, at the Instituto Butantan, São Paulo,
Brazil [5]. Since then, a variety of procedures have been employed in pAV production,
resulting in differing therapeutic characteristics [6–9]. The following section details various
procedures used in pAV production, and the properties of the resulting pAVs.

2.1. The Number of Venoms to Be Included in the Production of a pAV

Conventional pAVs are prepared via the immunization of horses with a mixture of
various crude venoms. Naturally, producers want to include as many immunizing venoms
as possible, so that all the target venomous snakes are covered. As each snake venom can
contain more than 100 different proteins [10], immunization with a mixture of more than
5–6 venoms may result in a total protein antigen load that can overwhelm the immunized
animal, and result in a low titer of the produced antibodies. Even after fractionation
and concentration, the final antivenom products might be ineffective against some of the
homologous venoms. This could possibly be due to the effect of ‘antigenic competition’ [11]
resulting in lower potencies in the AV produced. Therefore, there are certain upper limits
to the number of snake venoms that can be used in the immunization of horses. Usually
about 5 or 6 are used [9].

2.2. Use of Crude Venoms or Toxin Fractions

One way to circumvent the perceived ‘antigenic competition’ problem is to first
fractionate the venoms, to remove highly immunogenic but non-toxic proteins from the
Toxins 2023, 15, 517 3 of 11

venoms, and then use the separated lethal toxin fractions for immunization [12]. In this
way, the amounts of immunizing toxin proteins are greatly reduced, allowing an increase
in the number of venoms used in the pAV production, up to a total of 12 different venoms.
As the horse B cell antibody repertoire is almost infinite, antibody paratopes against all the
toxin epitopes could be generated [13]. This should significantly increase the paraspecificity
of the pAVs produced. It has been shown that this approach could lead to pAVs with
an improved potency [14] and a wider paraspecificity [12,15]. However, this approach
is applicable only in cases where all the toxins lethal to humans are known, and can be
fractionated without too many hurdles.

2.3. Antagonism and Synergism between the Constituent Venoms Used in pAV Production
It has been demonstrated that the Crotalus simus and Crotalus durissus ruruima venoms
exert a deleterious effect on the antibody response towards Bothrops asper venom when
used as co-immunogens in pAV production [8]. This study is interesting and important,
and it raised awareness regarding the potential for positive or negative interactions among
venoms in pAV production. Interestingly, it has also been shown that a mixture of specific
venoms can stimulate the antibody response to a greater extent than would immunization
with only a single venom in the production of bothropic–crotalic pAVs [8]. In this case,
it seems that various venoms may act as ‘adjuvants’, to enhance the immunogenicity of
another venom. This study could have significant implications in pAV production, and
must be considered in any pAV production plan. More research is also needed, to shed
light on the mechanisms underlying these interactions.

2.4. Production of ‘Mixed pAVs’

Another approach to overcoming the problem of too many immunizing venoms in
pAV production is to produce 2 or more pAVs, each using fewer immunizing venoms, and
then mix the IgG or F(ab’)2 of these pAVs, to obtain a ‘mixed pAV’. This approach has
been successfully employed via the mixing of the F(ab’)2 of one pAV covering five Bitis
and Echis venoms with the F(ab’)2 of another pAV covering six elapid venoms, to give a
‘mixed pAV’, which could neutralize 11 snake venoms. This ‘mixed pAV’ was shown to
have a wide paraspecificity against the venoms of numerous snakes in sub-Saharan African
countries [9]. The mixing of monovalent antivenoms to give a pAV has also been tried [16].
However, the drawback to this approach is the increased expense, due to the requirement
for more horses, the additional cost of fractionation, etc. Another concern linked to this
approach is the possible mutual dilution of the constituent antibody pools. The approach
can provide a wider paraspecificity, but may reduce the potency per unit volume of the
resulting ‘mixed pAV’.
Victims of snakebites most likely suffer from either elapid or viperid envenoming.
When they are treated with a ‘mixed pAV’ that contains both anti-elapid and anti-viperid
antibodies, one of the two antibody populations is not used, and is, therefore, wasted. In
addition, the unused antibody immunoglobulins may themselves contribute to adverse
reactions. Further, a ‘mixed pAV’, such as that described by Ramos-Cerrillo et al. [9], that
showed a good potency and paraspecificity would, without the mixing of the two groups
of F(ab’)2 antibodies, serve as two independent syndromic pAVs, one for use against elapid
envenoming, and the other against viperid envenoming (see below). Finally, in the same
total quantity, these two syndromic pAVs could be used to treat twice as many snakebite
victims as the ‘mixed pAV’.

2.5. pAVs with Combined Anti-Elapid and Anti-Viperid Activities

Many pAVs are produced using a mixture of elapid and viperid venoms. These
‘combined pAVs’ (c-pAVs), which offer both anti-elapid and anti-viperid activities, have
been produced, and are widely used in many countries [14,17–19], where they have saved
countless lives. However, there are some possible drawbacks associated with this type
of pAV.
 Many pAVs are produced using a mixture of elapid and viperid venoms. These
‘combined pAVs’ (c-pAVs), which offer both anti-elapid and anti-viperid activities, have
been produced, and are widely used in many countries [14,17–19], where they have saved
countless lives. However, there are some possible drawbacks associated with this type of
pAV.
Toxins 2023, 15, 517 4 of 11
The major commercial pAVs produced in India are against the four most medically
important snakes, “The Big 4”, consisting of two elapid and two viperid venoms (Naja
naja, Bungarus caeruleus, Daboia russellii, and Echis carinatus). It was apparently assumed
The major commercial pAVs produced in India are against the four most medically
that all the venom
important snakes,toxins were
“The Big 4”,immunologically equivalent.
consisting of two elapid and two However, this is not
viperid venoms the case.
(Naja
The lethal
naja, toxinscaeruleus,
Bungarus of theseDaboia
two types of snake
russellii, are carinatus).
and Echis vastly different, not only chemically,
It was apparently assumed but
also immunochemically.
that all the venom toxins Almost all elapid venoms
were immunologically contain
equivalent. neurotoxins
However, this is and cytotoxins
not the case. of
The lethal toxins of these two types of snake are vastly different, not only chemically,
the 3-finger toxin (3FTx) family, with molecular weights of around 7 kilodaltons [20,21], but
andalso
areimmunochemically.
poorly immunogenic Almost all elapid
[22,23]. In venoms
contrast,contain neurotoxins
the viper venomsand cytotoxins
mostly of high
contain
MW thetoxins
3-fingeroftoxin (3FTx)
around family,
20–30 with molecular
kilodaltons, withweights
enzymaticof around 7 kilodaltons
activities, [20,21],
e.g., phospholipases
and are poorly immunogenic [22,23]. In contrast, the viper venoms mostly contain high
A2, zinc-dependent metalloproteinases, and serine proteinases; these hematotoxins are
MW toxins of around 20–30 kilodaltons, with enzymatic activities, e.g., phospholipases
quite immunogenic.metalloproteinases,
A2 , zinc-dependent Therefore, it is and not serine
surprising that these
proteinases; thesehematotoxins
pAVs contain are more
antibodies
quite immunogenic. Therefore, it is not surprising that these pAVs contain more antibodies1) [23].
against the viperid protein toxins than against the elapid toxins (Figure
against the viperid protein toxins than against the elapid toxins (Figure 1) [23].

Figure 1. The horse antibody response to immunization with viperid (Bitis arietans arietans, Echis
Figure 1. The horse antibody response to immunization with viperid (Bitis arietans arietans, Echis
ocellatus) and elapid (Dendroaspis polylepis, Naja nigricollis) venoms, using different immunological
ocellatus) and elapid (Dendroaspis polylepis, Naja nigricollis) venoms, using different immunological
adjuvants. The antibody response is expressed as mg venom neutralized per ml of antiserum. This
adjuvants. The antibody response is expressed as mg venom neutralized per ml of antiserum. This
Figure was published in Arguedas et al. ‘Comparison of adjuvant emulsions for their safety and
Figure was published in Arguedas et al. ‘Comparison of adjuvant emulsions for their safety and
ability to enhance the antibody response in horses immunized with African snake venoms’. Vaccine X
ability to enhance the antibody response in horses immunized with African snake venoms’. Vaccine
2022, 12, 100233 [23].
X 2022, 12, 100233 [23].
Various immunochemical studies have shown that more antibodies in the c-pAVs
Variousand
recognize immunochemical studiesproteins,
neutralize viper venom have shown
while thethat moretoxins
elapid antibodies in the
are not well rec-c-pAVs
ognized [24].
recognize Thus, these viper
and neutralize pAVs often
venom show higher while
proteins, neutralizing potencies
the elapid against
toxins are the
not well
homologous viper venoms, but usually a poor or even absent neutralization
recognized [24]. Thus, these pAVs often show higher neutralizing potencies against the against the
elapid venoms [25]. Put another way, the neutralizing potencies of the anti-viper activity in
homologous viper venoms, but usually a poor or even absent neutralization against the
the c-pAVs are often higher than those of the anti-elapid activity [26]. Thus, the producers’
elapid venoms [25]. Put another way, the neutralizing potencies of the anti-viper activity
recommended initial doses of c-pAV for the treatment of elapid and viperid envenoming
in may
the be c-pAVs areBut,
different. often
as thehigher than is
culprit snake those
usuallyofnottheidentified,
anti-elapid activity
choosing [26]. Thus, the
the appropriate
producers’ recommended initial doses of c-pAV
initial dose of c-pAV for treatment may be challenging. for the treatment of elapid and viperid

2.6. Paraspecificity of pAVs

It has been widely observed that various pAVs may provide cross-neutralization
against homologous venoms with significant geographical variations in venom profiles [27],
Toxins 2023, 15, 517 5 of 11

or against other heterologous venoms, even from different genera [19,28–32]. This assumed
paraspecificity may be useful in providing a wide coverage against other venoms in places
where no specific AV is available. The paraspecificity of pAVs is more pronounced than
that observed in monovalent AVs. A possible basis for this is that the horse, when exposed
to the ‘diverse toxin repertoire’ present in a mixture of many immunizing venoms [12],
could respond by producing the corresponding diverse antibodies capable of reacting
with similar epitopes in the toxin isoforms of other related venoms [15,27]. An example
is the neutralization of Hypnale hypnale venom from Sri Lanka by Hemato Polyvalent
AV, produced in Thailand [33,34]. In Central American and European countries, and
elsewhere, pAVs against various vipers have been produced, and they have shown a wide
paraspecificity against numerous other vipers in the region [19,29,35–38].

2.7. The Benefits and Advantages of pAVs

pAVs are produced against the venoms of many snakes that are medically important,
and they could save lives, especially in areas where no specific antivenoms are available.
pAVs could be used to treat envenoming by unidentified snakes in specified coun-
tries/regions. As most snakebites occur in the dark, or in bushy areas, the culprit snakes
are rarely identified. The use of specific monovalent AVs may be ineffective, cost lives, and
lead to the wastage of expensive antivenoms. In contrast, with regard to the selection of the
appropriate antivenom for treatment, pAVs remove the need to identify the culprit snake,
and the use of snake-identifying test kits, which are expensive and time consuming.
pAVs can be administered immediately, as there is no need to identify the culprit snake.
The rapid administration of an effective pAV could reduce the severity of envenoming, and
save lives.
Another advantage of pAVs is the lower cost of production, in that fewer groups
of horses are needed, compared to the number required to produce several monovalent
antivenoms. Moreover, the cost of the fractionation processes for the plasma samples, and
the quality control of the products, packaging, inventory, etc., can be reduced, and this can
make the final products less expensive and more affordable.
pAVs can replace some monovalent AVs against snakes that cause serious, but low,
incidences of envenoming. For example, in Thailand, the O. hannah (king cobra) is a WHO
Category 2 snake and, although it causes very few incidents of envenomation, at seven cases
or 0.22% out of 3091 cases of venomous snakebites [4], a monovalent AV is produced. The
inclusion of O hannah in the Thai pAV could eliminate the need to produce the monospecific
anti-O hannah AV.

3. Syndromic Polyvalent Antivenoms (s-pAVs)

An interesting version of pAVs is called syndromic polyvalent antivenoms (s-pAVs).
As the name implies, they are produced for the treatment of snake envenoming based on
the signs and symptoms or syndromes manifested in the victims. Mixtures of venoms that
can cause these syndromes are used as immunogens in s-pAV production.
In brief, the medically important venomous snakes causing most snakebite envenom-
ing can be classified into two families, i.e., Elapidae and Viperidae [39,40]. The elapids
produce mainly neurotoxins and cytotoxins of the 3FTx family, while the vipers produce
hematotoxins. Thus, the syndromes that the snakebite victims present are used to de-
termine which s-pAV treatment is appropriate. For example, victims showing signs of
neurotoxic poisoning would be treated against neurotoxins, while those showing signs of
hematologic disorder would be treated against hematotoxins. Specifically, patients with
bleeding and coagulopathy (signs of hematologic disorder, Figure 2A) would be treated
with a ‘syndromic hemato pAV’ produced using various viper venoms as immunogens.
On the other hand, patients with muscle weakness, respiratory paralysis, or bilateral ptosis
(signs of neurotoxin poisoning, Figure 2B) would be treated with a ‘syndromic neuro
pAV’, produced using local elapid venoms as immunogens. The syndromic approach and
syndromic antivenoms were first described by Williams et al. in 2011 [41].
 On the other hand, patients with muscle weakness, respiratory paralysis, or
ptosis (signs of neurotoxin poisoning, Figure 2B) would be treated with a ‘sy
neuro pAV’, produced using local elapid venoms as immunogens. The sy
Toxins 2023, 15, 517 approach and syndromic antivenoms were first described by Williams6 ofet11al. in 20

Figure 2. (A) Patients with coagulopathy and bleeding (signs of hematologic disorder) caused by a
Daboia
Figure 2.russelli bite. (B) A with
(A) Patients patientcoagulopathy
showing bilateraland
ptosisbleeding
(signs of neurotoxic
(signs ofpoisoning caused disorder)
hematologic ca by an
elapid (Bungarus caeruleus) bite. Photographs courtesy of Professor David A Warrell.
Daboia russelli bite. (B) A patient showing bilateral ptosis (signs of neurotoxic poisoning
an elapid
The(Bungarus caeruleus)
elapid and viperid bite.used
venoms Photographs courtesy
in production of Professor
are usually David
those of WHO A Warrell.
Category
1 snakes, which are medically important snakes of the country or region. For example,
in Thailand,
The elapid two types
andofviperid
syndromic pAVs have
venoms been studied
used and produced,
in production are one against those
usually
neurotoxic venoms [6], and the other against hematotoxic venoms [42,43]. These s-pAVs
Category 1 snakes, which are medically important snakes of the country or reg
are called ‘Neuro Polyvalent Snake Antivenin’ and ‘Hemato Polyvalent Snake Antivenin’,
example,
and they in Thailand,
have been producedtwo types of syndromic
and marketed by Queen pAVs haveMemorial
Saovabha been studiedInstituteand
of produ
against
The Thai neurotoxic venoms
Red Cross Society [6],
since and
2012 the other
(Figure 3). Theagainst hematotoxic
‘neuro s-pAV’ is prepared venoms
using [42,4
immunogens from three medically important elapid neurotoxic
s-pAVs are called ‘Neuro Polyvalent Snake Antivenin’ and ‘Hemato Polyvale venoms of the country
(Naja kaouthia, Ophiophagus hanna, and Bungarus fasiatus) [6] and, later, another elapid
Antivenin’, and they have been produced and marketed by Queen Saovabha M
(Bungarus candidus) was added. This s-pAV is used in cases where snakebite victims show
Institute of The Thaiwith
signs of neurotoxicity Redmuscle
Cross Societywith
weakness, since 2012palpebral
bilateral (Figure ptosis
3). The ‘neuro
(Figure s-pAV’ is p
2B) [40].
using immunogens
The ‘hemato s-pAV’ is from
produced three
using medically
three medicallyimportant
important elapid neurotoxic
hematotoxic venoms venom
(Daboia russellii, Calloselasma rhodostoma, and Trimeresulus albolabris) as immunogens [42,43];
country (Naja kaouthia, Ophiophagus hanna, and Bungarus fasiatus) [6] and, later
it is used when the patient shows signs of persistence of incoagulable blood and bleeding
elapid
in the(Bungarus
whole bloodcandidus)
clotting testwas added.for
(20WBCT) This
more s-pAV
than 20ismin
used in cases
(Figure where
2A) [44]. snakebit
Thus,
show signs of neurotoxicity
the syndromic approach allows with muscletoweakness,
the physician observe the with
signs andbilateral
symptomspalpebral
of the ptosi
patient to select the appropriate s-pAVs, and use the criteria
2B) [40]. The ‘hemato s-pAV’ is produced using three medically important hem (bilateral ptosis or 20WBCT)
as indicators of systemic poisoning and the appropriate time to administer the antivenom.
venoms (Daboia russellii, Calloselasma rhodostoma, and Trimeresulus albola
In these cases, the identification of the culprit snake is not needed.
immunogens
It should be[42,43]; it is used
mentioned, at this when thethere
point, that patient shows
are some signsimportant
medically of persistence
elapids, of inco
blood
such asandNajableeding in Naja
nigricollis and theashei,
wholewhose blood
venoms clotting testto (20WBCT)
do not lead signs and symptomsfor moreof than
neurotoxicity. They do, however, give rise to a dominant syndrome
(Figure 2A) [44]. Thus, the syndromic approach allows the physician to observe of cytotoxicity. This
type of venom could be considered as an additional immunizing venom in the production
and symptoms of
of ‘non-neurotoxic’ the for
s-pAVs patient to select
the treatment theenvenoming
of a snake appropriate s-pAVs,a clinical
that produces and use the
(bilateral
syndromeptosis or 20WBCT)
dominated as indicators
by procoagulant, hemorrhagic, of systemic
or cytotoxicpoisoning
effects. and the appropr
to administer the antivenom. In these cases, the identification of the culprit sna
needed.
 It should be mentioned, at this point, that there are some medically important
elapids, such as Naja nigricollis and Naja ashei, whose venoms do not lead to signs and
symptoms of neurotoxicity. They do, however, give rise to a dominant syndrome of
cytotoxicity. This type of venom could be considered as an additional immunizing venom
in the production of ‘non-neurotoxic’ s-pAVs for the treatment of a snake envenoming that
Toxins 2023, 15, 517 7 of 11
produces a clinical syndrome dominated by procoagulant, hemorrhagic, or cytotoxic
effects.

Figure3.3.The
Figure Thetwo
twosyndromic
syndromicpolyvalent
polyvalentantivenoms:
antivenoms: “Neuro
“NeuroPolyvalent
PolyvalentAntivenin”
Antivenin” (right)
(right)and
and
“Hemato Polyvalent Antivenin” (left), produced by Queen Saovabha Memorial Institute, The Thai
“Hemato Polyvalent Antivenin” (left), produced by Queen Saovabha Memorial Institute, The Thai
Red Cross Society.
Red Cross Society.

3.1.The
3.1. TheParaspecificity
Paraspecificityofofs-pAVs
s-pAVs
Polyvalentantivenoms
Polyvalent antivenomshave havebeenbeen shown
shown to cross-react
to cross-react withwith
otherother heterologous
heterologous ven-
venoms,
oms, andcross-reaction
and this this cross-reaction is usually
is usually more pronounced
more pronounced than thatthan that observed
observed with monova- with
monovalent antivenoms. This paraspecificity is beneficial in
lent antivenoms. This paraspecificity is beneficial in the treatment of snake venoming, as the treatment of snake
itvenoming,
can provide as ait wider
can provide
coverage a wider coverage
of a larger of a larger
number of snakes.numberThisof is snakes.
likely toThis is likely
be the case
to bes-pAVs.
with the case with s-pAVs.
Eachs-pAV
Each s-pAV is produced
is produced against
against venom venom
toxins toxins
that give that
risegive rise signs,
to similar to similar signs,
symptoms,
symptoms,
and syndromes; and i.e.,
syndromes; i.e., they
they are raised are raised
against against pharmacologically
pharmacologically similar toxins.similar toxins.
For example,
inFortheexample,
case of the in neurotoxic
the case ofs-pAV,the neurotoxic
the venoms-pAV,immunogens the venom are fromimmunogens
neurotoxicare from
elapids
neurotoxic elapids whose lethal components are the post-synaptic 3FTx family and,the
whose lethal components are the post-synaptic 3FTx family and, for some elapids, for
phospholipase
some elapids, Athe 2 -containing
phospholipasepresynaptic neurotoxin
A2-containing family. These
presynaptic toxins are
neurotoxin chemically
family. These
similar,
toxins areand chemically
are structural homologs.
similar, and areThus, the paratopes
structural of the Thus,
homologs. antibodies raised are likely
the paratopes of the
to cross-react
antibodies with are
raised the likely
epitopes of the toxinwith
to cross-react isoforms, or the proteoforms
the epitopes of other elapid
of the toxin isoforms, or the
venoms
proteoforms[13]. This couldelapid
of other lead tovenoms
the wide[13].
paraspecificity
This could observed
lead to the in thewide neurotoxic s-pAV,
paraspecificity
and numerous
observed in thepublished resultss-pAV,
neurotoxic attest to thisnumerous
and conclusion published
[15,31,45–47]. results attest to this
Similar situations
conclusion [15,31,45–47]. may prevail, or be present, for the viperid venoms. They contain
various enzyme
Similar toxins, may
situations each prevail,
with almostor beidentical
present, functions,
for the viperid e.g., serine
venoms. protease, phos-
They contain
pholipase A2 , zinc-dependent
various enzyme toxins, each metalloproteinases,
with almost identical hyaluronidase,
functions, etc.e.g.,
Theseserine
enzymes from
protease,
different vipers are
phospholipase likely to have some
A2, zinc-dependent structural similarity,
metalloproteinases, at least around
hyaluronidase, the active
etc. These sites
enzymes
of the enzymes. Antibodies raised against these enzymes have
from different vipers are likely to have some structural similarity, at least around the been shown to cross-react
with
activethe corresponding
sites of the enzymes. enzymes from other
Antibodies raisedvipers
against [48,49]. The pAVshave
these enzymes produced against
been shown to
several viper venoms have shown a very wide paraspecificity [30,50].
cross-react with the corresponding enzymes from other vipers [48,49]. The pAVs produced
against several viper venoms have shown a very wide paraspecificity [30,50].
3.2. Possibility of Wider Paraspecificity in the Syndromic pAVs
The wide paraspecificity of the s-pAVs described above is inherent to their production
process. However, it should be possible to further increase their paraspecificities, to widen
the coverage of snakes in neighboring geographical areas. The number of immunizing
venoms for each s-pAV is quite low, compared to those used in the ‘combined pAVs’, in
which both elapid and viper venoms are used as immunogens. This will allow for an
increase in the number of venom–immunogen cocktails in s-pAV production. Thus, it
should be possible to produce an effective s-pAV covering a wide geographic area, such as
various countries in Asia and in Africa.
Toxins 2023, 15, 517 8 of 11

For South Asia, the elapid venoms required to produce a ‘neuro s-pAV’ for use in
seven countries in mainland South Asia may include a total of about seven WHO Category
1 elapid venoms: Naja oxiana, Bungarus caeruleus, Bungarus niger, Bungarus walli; Naja
kaouthia, Naja naja, Bungarus sindanus, and possibly some other Bungarus species [51].
Similarly, a ‘hemato s-pAV’ may involve seven WHO Category 1 viper venoms: Echis
carinatums, Macrovipera lebetina, Cryptelytrops erythrurus, Daboia russelii, Protobothrops jerdonii,
Echis carinatus, Hypnale hypnale [51]. Each of these venoms must be obtained from male,
female, young, and adult specimens, and from various locations/countries, to cover the
venom’s geographical variation. Furthermore, in the case of elapid venoms, toxin fractions
containing only the lethal neurotoxins, and devoid of high-MW immunogenic non-toxic
proteins, can be used [12]; this could further increase the number of immunizing elapid
venoms, e.g., WHO Category 2 snakes and sea snakes, to widen the paraspecificity of the
syndromic neurotoxic polyvalent AV [15].
It should be mentioned that, as well as the wide paraspecificity of the s-pAVs, and the
removal of the need to identify culprit snakes, the potency of the s-pAVs, at least with regard
to the ‘neuro s-pAV’, should be higher than that of the corresponding ‘combined pAV’
discussed in the form of the anti-elapid-plus-anti-viperid AV (Section 2.5). Furthermore,
the s-pAVs have been shown to exhibit immunochemical and biochemical properties
comparable to those of the corresponding monovalent antivenoms prepared under similar
conditions [52].

3.3. Future Prospects for Producing ‘Universal s-pAVs’

It has been hypothesized that, when a horse is exposed to a ‘diverse toxin repertoire’,
the animal should respond by producing a widely paraspecific antiserum against the
toxins. Thus, it has recently been shown that, through the use of the toxin fractions of
12 Asian elapids as immunogens, a ‘universal’ antivenom against at least 36 elapid snakes of
28 species in 10 genera, inhabiting over 20 countries on four continents, was prepared [15].
It is expected that, with the ‘diverse toxin repertoire’ immunization strategy [13], a similar
achievement could be demonstrated for ‘universal’ s-pAVs against neurotoxic venoms.
With regard to the ‘universal’ s-pAV against hematotoxic viper venoms, a similar
strategy may be applicable. A possible approach is to use affinity chromatography to purify
the major toxic enzymes of various medically important viperid venoms, and use them as
immunizing antigens. For example, affinity matrixes can be prepared for the purification
of snake venom serine proteases [53,54], for phospholipase A2 [55,56], and for hemorrhagic
metalloproteases [57,58]. After a dozen or so medically important viper venoms have been
sequentially passed through such affinity columns, the purified enzymes from each of these
venoms can be eluted from the columns, and collected. A mixture of 2–3 µg of each of these
enzymes from each venom can then be used as the primary immunogen, to generate a
widely paraspecific ‘hemato s-pAV’. With the previously observed wide cross-reactivity of
the antisera generated against these enzymes [48,49], it is likely that the antisera generated
from the above ‘diverse toxin repertoire’ immunization [13] could result in a ‘universal
hemato s-pAV’.
If this experiment were to be successfully carried out, then the two ‘universal’ syn-
dromic antivenoms should be able to cover the few dozen countries of sub-Saharan Africa,
and possibly South Asia. With the economy of scale, it should be possible to produce these
‘universal’ s-pAVs in large volumes at a reduced cost, and allow them to be affordable in
various poverty-stricken areas of the world. If realized, these ‘universal’ s-pAVs would
greatly reduce the fatalities caused by snakebite envenoming.

4. Conclusions
Snakebite envenoming causes a high rate of morbidity and mortality in developing
countries. Although specific plasma-derived antivenoms have been used for about a cen-
tury, they are not all effective, and are expensive and in short supply. Polyvalent antivenoms
have the advantage of neutralizing many different snake venoms from a wide region. In
Toxins 2023, 15, 517 9 of 11

addition, they remove the need to identify the culprit snake (a requirement in selecting a
specific monovalent antivenom), and they are cheaper to produce. Different procedures
have been used to produce polyvalent antivenoms, with ensuing advantages and draw-
backs. Syndromic polyvalent antivenoms constitute a relatively new therapeutic modality,
with the potential to alleviate snake envenoming problems. The syndromic polyvalent
antivenoms described here are in line with the first WHO public-benefit Target Product
Profiles for snakebite antivenoms for sub-Saharan Africa [59]. While novel therapeutic
alternatives based on recombinant antibody technologies are being rigorously pursued,
plasma-derived polyvalent antivenoms, especially syndromic polyvalent antivenoms, are
already available as lifesaving therapeutics.

Funding: This research received no external funding.

Acknowledgments: The author is deeply grateful to David A. Warrell and Jose Maria Gutierrez,
Timothy W. Flegel and James M. Dubbs for valuable advice and suggestions. The author thanks
Pavinee Simsiriwong of the Chulabhorn Research Institute, Bangkok, for assistant in preparing the
manuscript.
Conflicts of Interest: The author declared no conflict of interest.

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