Snakebites get

renewed attention
Scientists look for alternative antivenoms that
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are easy to make and deploy to remote areas

DINSA SACHAN, special to C&EN In brief
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More than
n a sunny September morning in Nemmara, a small

O
100,000 people
township in Kerala, India, Ally Thomas headed out into die every year
from venomous
the woods. Her plan was to cut grass, feed it to her cattle, snakebites. In
and attend a prayer service in the evening—a regular day, 2017, the World
by all means. Then a sharp, stinging pain shot through her left foot. Health Organization
recognized
A snake had bitten her. The serpent slithered away through the grass snakebites as a
before she saw it. neglected tropical
When Thomas reached home, she col- had bitten her. But after the antivenom disease. Some
lapsed with pain. Her family didn’t know was administered, Thomas was still in in- researchers want to
what to do. They brought her to a tradi- tense pain. tackle the problem
tional healer, who told her to apply a paste So the next day, her family brought her by addressing the
of basil leaves and turmeric on her foot. It to the nonprofit Little Flower Hospital shortcomings of
didn’t help. and Research Centre, in Angamaly, Kerala. current antivenoms.
Two days later, she experienced excru- By this time, her limbs were numb and her These conventional
ciating abdominal pain, her urine turned vision was blurred. Noushad CK, a doctor therapies require
black, and she spat blood. at the hospital, says Thomas had gone into refrigeration for
Her family took her to a small private minor kidney failure, a complication that storage and trained
hospital nearby. “They told us she was often arises from the bite of a viper—likely personnel for
bitten by a very deadly snake and that either a Russell’s or pit viper—in this part delivery, both of
she might not survive,” recounts Rohan of the country. which rural areas
Thomas, Ally’s son. Springing to action quickly, Noushad’s in the developing
Doctors at the hospital gave Ally Thom- team provided Thomas with intravenous world often lack.
as antivenom, an antidote for snakebites fluids to improve her kidney function, and Also, producing the
that can save people’s lives if administered after two days, her condition improved. antivenoms involves
quickly enough. In India, doctors give pa- “It’s a miracle,” Noushad says. Thomas inoculating horses
tients a so-called polyvalent antivenom, didn’t receive antivenom until three days with snake venoms,
C R E D I T: EP HOTO CO R P/A L A MY STO C K P H OTO

which targets four of India’s most venom- after she was bitten. It should be given as which is expensive,
ous snake species—Russell’s viper, Indian soon as possible after a bite. Long delays is time consuming,
cobra, saw-scaled viper, and common before receiving antivenom normally and yields proteins
krait. The hospital hoped giving Thom- result in irreversible organ damage or, that could trigger
as this concoction would help her, even worse, death. allergic reactions
though doctors weren’t sure which snake As Thomas sat propped up on pillows in people. Several
research groups
are now developing
antivenom
alternatives to try
to get around these
issues.

JANUARY 28, 2019 | CEN.ACS.ORG | C&EN 31

Snakebite hot spots
Most venomous snakebites in the world happen in these three regions.
Laos
South and Southeast Asia
Estimated venomous snakebites per year:
231,866–961,508 89,009–417,343
Estimated snakebite deaths per year:
14,902–52,760
Notable venomous snakes: Russell’s viper
(Daboia russelii), Indian cobra (Naja naja),
and Malayan krait (Bungarus candidus)
Russell’s viper
Source: PLOS Med. 2008, DOI: 10.1371/journal.pmed.0050218.
in the intensive care unit of Little Flower,
she expressed how fortunate she felt. “I
feel I’ve had a rebirth.”
Although Thomas lived to tell her tale,
many in India don’t. Snakebites kill nearly
50,000 people there every year. But snake-
bites aren’t India’s problem alone. Africa’s
snakebite-related mortality rate is estimat-
ed at 30,000 per year, with sub-Saharan
Africa being most affected. In total, more
than 100,000 people die from venomous
snakebites around the world each year.
In June 2017, the World Health Organi-
zation took notice, recognizing snakebites
as a neglected tropical disease. Since then,
the world body has established a working
group of researchers, physicians, and pub-
lic health experts to create a plan for im-
proving access to treatment and improv-
ing the quality of antivenoms, especially
in the developing world. It also approved
32 C&EN | CEN.ACS.ORG | JANUARY 28, 2019
Sub-Saharan Africa
Estimated venomous snakebites per year:
Estimated snakebite deaths per year:
3,243–31,751
Notable venomous snakes: Black mamba
(Dendroaspis polylepis), black-necked
spitting cobra (Naja nigricollis), and puff
adder (Bitis arietans)
Black mamba
a resolution at the World Health Assembly
in May 2018 to create a framework for
helping countries tackle snakebites.
Many countries struggle with treating
snakebites because they lack the infra-
structure or logistics planning needed to
get antivenoms to people. For example,
many clinics in the developing world don’t
have adequate facilities for refrigerating
antivenom serum. Also, many rural hos-
pitals are often not staffed during the
evenings or are remotely located, leaving
victims with no choice but to rush to a tra-
ditional healer for immediate treatment,
just like Thomas and her family did.
Another challenge is that some coun-
tries lack the expertise to distinguish good
antivenoms from bad ones, says Jean-
Philippe Chippaux, a French physician
who has worked in sub-Saharan Africa.
The trouble is, bad ones are often cheap-
Mexico, Central America, and
Tropical South America
Estimated venomous snakebites per year:
71,723–99,268
Estimated snakebite deaths per year:
293–1,760
Notable venomous snakes: Coral
snakes (Micrurus), hog-nosed pit vipers
(Porthidium), and South American
rattlesnake (Crotalus durissus)
Eastern coral snake
CR E D I T: SA LE E M H A M EE D/ WI KI MED I A ( RU S S E LL’S V I P E R ) ; SA FA R I T RAVE LP LU S/
er—and more dangerous. They contain
high concentrations of preservatives or
protein-degradation products, indicating
poor storage conditions, Chippaux says.
Government officials generally pick the
WI KI M ED I A ( BL AC K MA M BA ) ; S H UT TE RSTO CK ( CO RA L S N A KE )
more inexpensive antivenom when de-
ciding which one to stock hospitals with,
Chippaux adds. “If the criteria is just price,
it is not a good thing.” Moreover, not all
antivenoms have gone through clinical tri-
als and have therefore not been regulated.
Regulatory concerns aside, some snake-
bite researchers think antivenoms them-
selves need an update. The method for
making antivenom was standardized in the
early 20th century. It involves milking a
venomous snake and then immunizing an-
imals, mostly horses, with diluted venom.
The horses’ immune systems generate an-
tibodies against the venom molecules over
a few months. Technicians then take blood
 plasma from the horses and extract the
antibodies to make the antivenom serum.
It’s a long and expensive process, and
some companies cut corners, especially in
purification. “In Africa, the purification of
antibodies is not sufficiently achieved, and
we have antivenom with high risks of side
effects,” Chippaux says.
Venoms also contain many compo-
nents, including peptides, proteins, and
enzymes. So antivenoms need to counter
combinations of these rather than a single
component to be effective. And the anti-
bodies in antivenoms come from horses
rather than humans. So injecting them
into a person brings with it a risk of an
allergic reaction to foreign substances,
especially if the purification process hasn’t
been carried out properly.
Because of these reasons and more, sci-
entists have started to look for alternatives
to current antivenoms, keeping in mind the
challenges posed by the poor public health
infrastructure in countries such as India.
Skipping the fridge
Some attempts to make alternative
antivenoms focus on providing a therapy
that is easier to use and easier to store.
Current antivenoms are not stable unless
refrigerated.
“The major gap in antivenom’s utility
is that it can only be administered in the
hospital, but the vast majority of snake-
bites—upward of 75%—occur before
victims can get to the hospital,” says Mat-
thew Lewin, an emergency physician and
the director of the Center for Exploration
and Travel Health at the California Acade-
my of Sciences.
Lewin’s company, Ophirex, is investigat-
ing a therapy that wouldn’t have such stor-
age needs. It is an off-patent compound
named varespladib, first developed by the
pharmaceutical firm Eli Lilly and Compa-
ny, for its venom-fighting properties.
The small molecule counters phos-
pholipase A2, an enzyme that is found in
the venoms of most snakes around the
world. The enzyme degrades the lipids
in cell membranes, causing cells to break
down and a range of symptoms to appear,
including paralysis, bleeding, and muscle
destruction. Varespladib binds to the hy-
drophobic active site of the phospholipase
enzyme and destabilizes it so it can’t go to
work on lipids. The molecule also appears
C R E D I T: D I N SA SACH A N
to fight inflammation in the victim’s body.
In test tubes, varespladib neutralizes the
membrane-munching activity of venoms
from 28 snake species, including the black
mamba and death adder. Intravenous and
subcutaneous injections of the molecule
led to a 100% survival rate for rats and
mice injected with venom from European
adders or Eastern coral snakes (Toxins
2016, DOI: 10.3390/toxins8090248). Pigs
that have been given coral snake venom
also respond to varespladib. All the animals
treated with the compound, either orally or
intravenously, survived for the period stud-
ied, 120 h. But untreated pigs did not (Tox-
ins 2018, DOI: 10.3390/toxins10110479).
The small molecule offers many ad-
vantages over conventional antivenoms,
Lewin says. It is inexpensive to make and
thus would be affordable in resource-poor
parts of the world. It would also be shelf
stable in tropical climates. Lewin points
out that varespladib is thousands of times
as potent as a conventional antivenom at
inhibiting phospholipase. “Further, it can
penetrate tissues that antivenom cannot,
such as muscle and neurons.”
But varespladib’s biggest advantage is
Ally Thomas nearly lost her life to the bite of a snake in her hometown of Nemmara in
Kerala, India.
that anyone can give it to a victim right
on the spot after a snakebite, Lewin says.
Only trained health-care practitioners can
administer traditional antivenom therapy
to patients because the agents typically
have to be given intravenously, and their
side effects need to be monitored.
Still, Lewin emphasizes that the mole-
cule probably would be used in combina-
tion with standard antivenoms. “Antiven-
oms have a longer half-life, circulating
longer in the body, and a broader effect
on nonphospholipase components of ven-
om,” Lewin explains.
Lewin hopes to begin a Phase I or Piv-
otal Phase II clinical trial in early 2020,
in time for the taipan snakebite season in
Papua New Guinea. “We hope to partner
with manufacturing firms that have glob-
al capabilities so that we can keep costs
down and get the drugs to the people that
need them most,” Lewin says.
While Lewin is exploring a small-mol-
ecule alternative to standard antivenoms,
a team led by Kenneth Shea, a chemist
at the University of California, Irvine, is
investigating a hydrogel consisting of poly-
mer nanoparticles.
The gel works by exploiting a phenome-
non that happens when polymer nanopar-
ticles are mixed with a solution containing
proteins. The proteins glom on to the
particles, creating what is called a corona.
Shea and colleagues previously deter-
mined that they could get specific proteins
to associate with the particles by tuning
the composition of their polymers.
So the researchers developed nanopar-
ticles that would form coronas of phos-
pholipase A2, selectively sequestering the
enzyme from snake venoms (J. Am. Chem.
Soc. 2016, DOI: 10.1021/jacs.6b10950). Shea
likes to describe the polymer nanopar-
ticles as plastic antibodies because the
particles are plastic and they tie up the en-
zyme, preventing it from acting on cells.
To find their antivenom nanoparti-
cles, the researchers experimented with
different combinations of monomers,
such as acrylic acid, N-phenylacryl-
amide, N-isopropylacrylamide, and
N,N′-methylenebis(acrylamide), to pro-
duce particles with different polymer
compositions. They tested this library of
nanoparticles against different types of
venoms, and they chose the particles with
the highest affinity for phospholipase A2
for further study.
JANUARY 28, 2019 | CEN.ACS.ORG | C&EN 33
What’s in a snakebite?
A closer look at five toxins found in snake venoms.
Snake venom
metalloproteinases
Snakes with the toxins: Most members
of the Viperidae family, including South
America’s Bothrops jararaca pit viper
Effects of the toxins: The enzymes
produce a wide range of damage. For
example, they help cause bleeding by
breaking down components of capillaries,
and they can disrupt blood clotting.
Phospholipase A2
Snakes with the toxins: Most snakes in the
Viperidae and Elapidae families
Effects of the toxins: The enzymes break
down the phospholipids in cell membranes,
leading to muscle damage and paralysis.
Shea and his team collaborated with
José María Gutiérrez of the University
of Costa Rica to test the best-performing
nanoparticle in mice. They injected ven-
om from an African cobra into the skin of
the mice at a dose that would cause skin
necrosis. Mice given several doses of the
nanoparticle-based gel at the site of venom
injection showed no signs of necrosis after
72 h, while untreated mice did (PLOS Ne-
glected Trop. Dis. 2018, DOI: 10.1371/journal.
pntd.0006736). According to further
studies, the nanoparticles end up in the
liver, where they are quickly captured and
cleared from the body.
Because the synthetic polymers are sta-
bler than antibodies, this gel could survive
in harsh conditions in tropical regions,
Shea says. “You don’t have to worry about
refrigeration.”
But Shea says this so-called nanodote
is not intended to replace traditional
antivenom either. “It could be injected di-
rectly into the site of the bite by the victim
or a companion,” he says. The gel would
help keep the victim in stable condition
until they get medical help, he adds.
Before moving to clinical trials, Shea and
his team want to test the nanodote against
other relevant venomous snake species.

Biotech lends a hand

Snake venom serine proteinases
Snakes with the toxins: Some snakes in both
Viperidae and Elapidae families, including the sharp-
nosed viper found in southern China
Effects of the toxins: These enzymes disrupt
multiple aspects of the blood-clotting process.
Three-finger toxins
Snakes with the toxins: Most snakes in the
Elapidae family, including the Malayan krait
(Bungarus candidus)
Effects of the toxins: These molecules are potent
neurotoxins that can cause paralysis.
While Lewin’s and Shea’s teams are
looking for alternatives to antivenoms,
some researchers haven’t given up on an-
tivenoms’ main component: antibodies.
These scientists are looking to biotechnol-
ogy to establish less-cumbersome methods
of producing venom-deactivating antibod-
ies. The idea is to avoid milking dangerous
snakes or injecting venom into horses.
One such approach involves developing
human monoclonal antibodies. Monoclo-
nal antibodies are genetically engineered
proteins that are synthesized by cloned
immune cells and are identical to each
other in amino acid sequence. Some can-
cer therapies use such antibodies to target
specific proteins on cancer cells. A human
monoclonal antibody is capable of target-
ing a specific venom toxin. An antidote
that is a mixture of monoclonal antibodies
could target multiple venom toxins to save
the lives of snakebite victims.
C R E D I T: P ROT EI N DATA BA N K ( A LL)

If researchers could find monoclonal

Dendrotoxins
Snakes with the toxins: Many snakes in the Elapidae family,
including the eastern green mamba
Effects of the toxins: These neurotoxins block potassium channels
on nerve cells.
Source: Nat. Rev. Dis. Primers 2017, DOI: 10.1038/nrdp.2017.63.
antibodies that target specific venom ingre-
dients, they could produce the antibodies
on a large scale in mammalian cells. “Ev-
erything can be produced in big fermenta-
tion tanks in the laboratory,” says Cecilie
Knudsen, a researcher at the Technical Uni-
versity of Denmark. “This is faster than im-
munizing animals.” The process takes days,

34 C&EN | CEN.ACS.ORG | JANUARY 28, 2019

 compared with the
months that it takes to
work with horses.
And because the
antibodies are human,
they are less likely
to cause the allergic
reactions seen with
antivenoms made us-
ing horses. “Moreover, Polymer nanoparticles (left) capture phospholipase A2 (center) and render it
monoclonal antibodies inactive.
can persist in the hu-
man body for a longer time—they don’t get is the venom of the coral snake Micrurus
cleared out by the immune system the way
foreign antibodies would,” Knudsen says.
Scientists generally have two approach-
es to find a human monoclonal antibody
that targets a specific venom protein.
Hybridoma technology involves injecting
the target protein into mice, isolating the
genes for the mouse antibody that forms
in response to the protein, then tweaking
the gene in specific ways to humanize it.
“You’ve got to keep
in mind that venom
is very complex.”
—Paulo Lee Ho, biochemist, Instituto synthesize and churn out
Butantan proteins containing the epi-
The other route is to use phage display,
which won the 2018 Nobel Prize in Chem-
istry, to screen a library of human anti-
body genes to find the antibody that binds
a target protein.
Knudsen and her colleagues from
Denmark and Costa Rica used the latter
method to find human monoclonal an-
tibodies against the venom of the black
mamba, a highly venomous snake from
sub-Saharan Africa. They looked for
antibodies that could bind to so-called
dendrotoxins inside the black mamba’s
venom. The team made two prototype
antidotes and tested them in mice, find-
ing that treated mice survived injections
of the mamba venom (Nat. Comm. 2018,
DOI: 10.1038/s41467-018-06086-4).
Black mamba venom contains another
group of deadly components called α-neu-
rotoxins. Knudsen’s team next wants to
develop antibodies against those mole-
cules. The researchers’ goal is to find an-
tibodies for all the key mamba toxins and
C R E D I T: KE N N E TH S H EA
then mix them together into one antidote.
Meanwhile, Paulo Lee Ho, a biochemist
at Instituto Butantan, in São Paulo, and
his team are also harnessing biotechnology apply its technique to horses and harvest
techniques to derive an antivenom against
a particularly tricky snake. Their target
corallinus, one of the deadliest serpents in
Brazil.
“The problem with making antivenom
against coral snake is obtaining venom to
immunize the horse. It doesn’t have enough them would be a marathon task.
venom in the venom gland,” Ho says.
Instead of milking these snakes for their
paltry supply of venom, the team wanted to says in the near term, these alternatives
find a way to make the proteins themselves. could work in conjunction with current
They examined the amino acid sequences
of the venom’s five most important toxins
and then mapped their epitopes—the parts
of the toxins to which an antibody would
actually bind. They then constructed genes
that encoded just those epitopes.
The researchers injected pieces of DNA
containing these genes into mice so that
the animals’ cellular machinery would
topes. The rodents’ immune
systems then started to
produce antibodies against
these epitopes. Basically, the
scientists used the pieces of
DNA to immunize the mice O O
against the venom—like HO O trials will be needed, and
injecting horses with the ac-
tual snake toxins.
Varespladib
The team found that
immunizing the mice first with these DNA
pieces and then with a booster shot of
proteins containing just the epitopes was
fairly successful. The animals’ survival rate height, body weight, and amount of venom
when injected with the snake venom was
60%. All the untreated mice died.
Ho wants to improve the survival rate.
He thinks that the researchers haven’t yet
isolated epitopes for all the significant
toxins in the coral snake’s venom. “You’ve
got to keep in mind that venom is very
complex.”
Ho thinks this epitope immunization
method could work well for snakes that
have limited amounts of venom or highly
venomous serpents that are difficult to
handle. To produce antivenoms for people
rather than mice, the team would have to
the antibodies produced after immunizing
them.
A long wait
ahead
Some antiven-
om researchers are
excited about these
alternatives but think
it’s still early days in
their development. In
particular, Chippaux,
the French physician,
thinks it will take a
while for monoclonal antibodies to make it
to the clinic for testing against snakebites.
The venom of a single species of snake, he
says, contains an eclectic cocktail of toxins,
and making antibodies against every one of
Julien Potet, a policy adviser for the
nonprofit group Doctors Without Borders,
antivenom therapies. For example, he says,
monoclonal antibodies “could be added to
the conventional animal antibody prepa-
rations in order to fortify them against
selected snake venoms that are hard to
neutralize with existing products.”
In general, these novel antidotes will
have to prove a lot before they’d be ad-
opted in the field, experts say. First, Potet
says, other researchers will
need to confirm in the lab
that the antidotes neutralize
their targeted venoms. After
that, scientists will need to
N
investigate any possible tox-
icities of the novel agents.
And then large-scale clinical
O NH2 those are expensive.
“You need detailed stud-
ies that involve thousands
of people for these novel antidotes be-
cause venom works differently on differ-
ent people’s bodies, based on individual
injected,” says Priyanka Kadam, founder
of the India-based nonprofit group Snake-
bite Healing and Education Society and a
member of the WHO panel on venomous
snakebites.
Unfortunately, most of the antivenoms
that are supplied to sub-Saharan Africa
and India aren’t clinically tested. “Like for
other neglected tropical diseases, there is
very limited funding for the development
of antivenoms,” Potet says. “For many
producers, the money is just not there to
finance the clinical trials. Only a couple of
the dozen existing antivenoms intended
for use in sub-Saharan Africa have been
evaluated in robust randomized clinical
trials.” And that doesn’t stop countries
JANUARY 28, 2019 | CEN.ACS.ORG | C&EN 35
with lackadaisical regulatory bodies from
registering antivenoms anyway. “It’s a
shame,” Potet says.
Meanwhile, Chippaux, who has treated
snakebites in sub-Saharan Africa for de-
cades, believes there is actually no need
for novel antidotes. Just improving the
quality and quantity of conventional an-
tivenoms could significantly help Africans.
“There is a need for a greater quantity of
good-quality antivenom. While fewer than
100,000 doses are sold annually in sub-Sa-
haran Africa, actual needs are estimated to
be higher than 1 million doses,” he says.
Potet also urges antivenom manu-
facturers to keep improving the current
inventory. “This is not rocket science, and
the investments that need to be made are
probably not very high, but they need to
happen,” Potet says.
He points to incremental improvements
at all stages of antivenom production,
including how the venom immunization
mixture is prepared and how the final
product gets purified. “Not many produc-
ers have focused on these incremental
improvements,” he says.
In 2016, the WHO commissioned an
assessment of the antivenoms sold in Afri-
ca and the facilities, many of which are in
India, where they are produced. Manufac-
36 C&EN | CEN.ACS.ORG | JANUARY 28, 2019
turers were asked to submit information
about their products to the organization.
WHO officers also inspected the facilities
to check if they followed best practices
of production—for example, they deter-
mined whether the horses were kept in
“Like for other neglected tropical
diseases, there is very limited funding
for the development of antivenoms.”
—Julien Potet, policy adviser, Doctors Without Borders
healthy conditions. The WHO hasn’t yet
disclosed its findings but plans to release
a list of trusted suppliers after this assess-
ment. “This will clarify which antivenoms
are the most capable of neutralizing the
venoms of the different African snake spe-
cies,” Potet says.
Moreover, Doctors Without Borders is
gearing up to host and implement clinical
trials of existing antivenoms in sub-Sa-
haran Africa because it wants to find the
most effective antivenoms for African
snake species. The organization will start
after the WHO completes its assessment
of African antivenoms. “Once this assess-
ment is finalized, we will be able to select
the antivenoms that we’d like to test in
our clinical trial,” Potet says.
No matter which of these efforts—im-
proving current antivenom production,
developing antivenom alternatives, or
creating human-antibody-based antiven-
oms—is most successful, it’s clear that
changes are needed. “Ninety-five percent
of people affected by snakebites come
from impoverished backgrounds, and they
aren’t aware of their fundamental rights,
including the right to basic health care,”
Kadam says. “These are voiceless victims,
and not a priority for policy makers.” She
says the developing world badly needs a
novel antidote that could reduce treat-
ment costs. “It would be a game changer.”
Dinsa Sachan is a freelancer writer based in
New Delhi.