Vaccine. Vol. 13, No. 13, pp.

1196-1206, 1995
Coovriaht 0 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0264-410X/95
$lO+O.OO

0264-410X(95)00059-3

The use of synthetic peptides can be a

misleading approach to generate
vaccines against scorpion toxins
Emma Soraida Calderon-Aranda,
Lourival Domingos Possani*

Timoteo Olamendi-Portugal

and

Seven peptides

corresponding to the amino acid sequence of toxin 2 from the scorpion

were chemicall_v synthesized, purtjied and assayed in mice for their
putative neutralizing properties against scorpion toxins. All the peptides were immunogenic and some produced neutralizing antibodies, as verified by injecting the antisera with
toxin into naive animals. However, direct challenge of pre-immunized
mice (with the
longest synthetic peptides of 27 and 57 amino acid residues) revealed an unexpected
sensitization phenomena: the animals did not resist injection of one LD,,, of purified toxin
2 (5% survival), but pre-immunization
of mice with native toxin protected 100% of
the animals. These findings suggest that vaccine preparations
with synthetic peptides
corresponding to the amino acid sequence of certain toxins should be unal,yzed cautiously.
Centruroides

Keywords:

Synthetic

noxius

peptide:

scorpion

toxin; vaccine:

Cetmwoides

no.xiu.r; sensitization

Scorpion venoms are rich sources of ion-channel

affecting toxins.
They disrupt
the normal
function
of
excitable tissues (muscles and nerves) of many animals,
including
man, by causing respiratory
and circulatory
problems that can eventually
lead to the death of the
individual
envenomed. . The occurrence
of scorpion
stings in humans is called scorpionism,
and in many
parts of the world it is a considerable
public health
problem.
For example, in Mexico alone, scorpionism
affects more than 200 000 peo le annually, with death of
approximately
700 people. Pj. For this reason, and
despite the fact that effective horse antisera exist and are
used with success,
it would be very important
to
develop a possible vaccine against these toxins. The
toxins are polypeptides
of 4-8 K which are very resistant
to degradation
by heat or enzymes,
and have been
classified according to the target they recognizes..
.
The sodium channel toxins are 60-70 amino acids long,
stabilized by four disulphide bonds.4.5.X, and are by
far the more important
ones in scorpionism.
If only this
family of peptides could be neutralized,
the public health
problem would have been solved-.
There are three
more classes of toxins in scorpion venoms that recognize:
K+-channels3..,
Ca+-channels
and
Cl _ channels.
However,
their general
contribution
to
human intoxication
is relatively minor, due to the fact

Department of Molecular Recognition and Structural Biology,

lnstituto de Biotecnologia,
Universidad National Autonoma
de Mexico, Avenida Universidad,
2001, Apartado Postal
51 O-3, Cuernavaca 62271, Mexico. *Corresponding
author.
(Received 24 November 1994; accepted 22 March 1995)

1198

Vaccine

1995 Volume

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that they are less abundant

components
of the venom,
or they are resent in species of scorpions not dangerous
to humans P
Since the primary structure of scorpion toxins is well
in this
fieldI.
21.24.25 have
known I,14.15.23, researchers
considered
that non-toxic
but immunogenic
synthetic
peptides corresponding
to the amino acid sequences of
these toxins could lead to the production
of synthetic
vaccines against scorpionism.
In this communication
we report the results in mice of
passive and direct immunization
protocols, using a series
of synthetic peptides prepared according to the amino
acid sequence of toxin 2, a major component
from the
venom
of Centruroides noxius, the most dangerous
scorpion of Mexico. This venom has an LD,, of 5 pug
20 g- mouse weightj. Toxin 2 has 66 amino acids and
its globular
structure
is stabilized
by four disul hide
bridges.
Since initial results from our group.- 7spsuggested that the N-terminal
amino acid region of the
J-scorpion
toxins was likely to be implicated
in the
active site of these peptides, emphasis on the synthesis
was oriented mainly towards this part of the molecule.

MATERIALS

AND

METHODS

Source of toxin
Venom from scorpions of the species C. noxius was
collected by electric stimulation
of animals maintained
alive in the laboratory,
as describedlh. The dried venom
was dissolved
in double-distilled
water, centrifuged
and the soluble part freeze-dried
and kept at - 20C
until use. Toxin 2 was purified by Sephadex G-50 gel

Vaccines against scorpion toxins: E.S. Calderon-Aranda

Table 1

Tox. 2
Chim.
pl-10
Pl-14M
Pl-14
Pl-27
P22-36
P30-40
Hexa P

Amino acid sequence

of toxin 2 and synthetic

et al.

peptidesa

10
20
40
60
AEGYLVDKNTG C-KYE-CLKLGDNDYCLREC?QQGYKGAGGYCYAF-ACWC?HLYEQAfWPLPNKRCS
KEGYLMKKSDGCGRGEGCNDYCLRECKQQGY
KGAGGYC-AGPAGCY
C-KLGDG-VAG
- TK- CKEGYLVDKNT
K- GYLVDKNTG C-KY
KEGYLVDKNTG C-KY
KEGYLVDKNTG C-KYE-CLKLGDNDYCLR
NDYCLRECKQQGY
KG
KQQGYKGAGGY
PNKSRC

Single letter code for amino acids was used. Gaps (-) were introduced in order to enhance similarities. Tox. stands for toxin P for peptide, and
Chim. for the chimaeric toxin. The first dioit of numbers on too of the amino acid sequence of toxin 2 indicates the actual position of that particular
amino acid in the sequence. Cysteines are in bold

filtration, and various cation-exchange

steps, as described.
Preparation

chromatographic

of synthetic peptides

Seven peptides with lo-27 amino acid residues, corresponding to different regions of the sequence of toxin 2,
plus one long peptide (chimaeric toxin of 57 amino acid
residues), shown in Table 1, were synthesized by the
solid-phase method of Merrifield, under the conditions
describedsO. using t-Boc-amino acid strategy (Peninsula
Laboratories Inc., Belmont, CA, USA). An additional
hexapeptide corresponding to the amino acid composition (not sequence) of the C-terminal region of the
toxin 2 (Pro-Asn-Lys-Ser-Arg-Cys)
was also prepared,
as an internal control. Peptides were named according to
the parent position of the amino acid residues in toxin 2.
For example, peptide l-10 means the peptide whose
sequence starts with amino acid at position 1 and
finishes at 10 of the corresponding native toxin. The
manual mode was used for the short peptides and an
automatic Applied Biosystems apparatus was used for
the chimaeric toxin (57 residues), with protocols given
by the company. After synthesis the peptides were
cleaved from the resin by hydrofluoric acid (HF), recovered in the presence of the reducing agent dithiotrietol
(DTT) and lyophilized. The eight peptides were purified
directly by high performance liquid chromatography
(HPLC) in a Waters 600E chromatograph, using a Cl8
reverse-phase column in the presence of 0.1% trifluoroacetic acid (TFA) in water and eluted with a gradient
from 0 to 60% acetonitrile as describedO. The chimaeric
toxin (long peptide) was previously separated on a
Sephadex G-50 column with the same conditions used
for the crude venom?, before HPLC. The correct synthesis was always verified by amino acid analysis, by the
method of Bidlingmeyer et al. Peptide l-14M has the
same sequence as that of 1-14, except that the glutamic
acid at position 2 is missing (M means modified peptide).
This situation was confirmed by sequencing peptide
l-14M in a ProSequencer, model 6600 from Milligen/
Biosearch (Division of Millipore).
Only peptides
containing the expected compositions were used for
immunization experiments.
Lethality tests and clinical symptoms of intoxication
scorpion toxin

by

Lethality tests were conducted in albino mice of the

strain CDl, weighing 20 g. Initial LD,, values for the
toxin were determined as described previously32, using

100 mice (10 groups of 10 animals each using different

concentrations of toxin). For the synthetic peptides,
various amounts of each (50-1000 ,ug mouse-), dissolved in 200 yl 0.015 M sodium phosphate buffer,
pH 7.2 containing 0.15 M NaCl, were injected intraperitoneally.
Mice injected with toxin have a peculiar reproducible
course of clinical manifestations: excitability, aggressiveness, sialorrhoea, profuse salivation, difficulty in respiration with a characteristic curvature of the upper
back, paralysis of limbs, mainly rear limbs, diarrhoea,
convulsions and death.
Three designations were used: lethal when the mouse
injected died within 24 h of injection; toxic if the animal
survived, but showed some of the symptoms listed; and
non-toxic if the mouse did not show any symptoms of
intoxication, like the control animals injected only with
the phosphate-saline buffer described above.

Coupling of peptides to carrier proteins

Peptides with less than 27 residues were coupled either

to bovine serum albumin (BSA) for ELISA assays, or to
thyroglobulin (T) for immunization protocols, following
the technique described by Goodfriend et aL3 Peptide
l-27 was used in two ways: peptide alone and peptide
bound to thyroglobulin. The chimaeric toxin and native
toxin 2 were also used without coupling to carrier
proteins.

Mice immunization

protocols

Albino mice of the strain CDl, 48 weeks old, were

used for immunization with synthetic peptides and
native toxin. For synthetic peptides, the first injection
consisted of 100 ,ug of each, covalently attached to
thyroglobulin and emulsified with complete Freunds
adjuvant. Three additional immunizations with the same
peptides were conducted at intervals of two weeks, in
presence of incomplete Freunds adjuvant. Groups of 18
animals were used for immunization protocols. For the
chimaeric toxin, 50 pg mouse were used also in the
presence of complete Freunds adjuvant for the initial
application and incomplete Freunds for the subsequent
immunizations. Toxin 2, due to its high toxicity, was
used at a dose of 2.5 yg toxin mouse-, adsorbed onto
nitrocellulose membranes (capacity 80-100 pg cm-,
from BioRad Lab., Richmond, CA, USA), which decreases significantly the lethality of this toxin, according
to protocols previously described by our groug4.

Vaccine

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Vaccines against scorpion toxins: E.S. Calderon-Aranda

et al.

All mice immunized

were bled 9 days after the third
immunization
for sera collection.
Sera were kept at
- 20C until used for titration or for passive neutralization experiments.
Only animals that had completed
the fourth
immunization
treatment
were used for
challenging
with native toxin.

Immunization of rabbits
Rabbits (female strain New Zealand, 2 kg weight at
the start of the immunization
protocol) were also immunized four times at intervals
of 2 weeks, to obtain
antisera against the chimaeric toxin and native toxin 2.
The chimeric toxin was used at 150 pg dose- with
Freunds complete adjuvant
the first time, and incomplete adjuvant
for the subsequent
applications.
Native
toxin 2 was used at 7.5 ,ug rabbit-
adsorbed
onto
nitrocellulose
membranes,
as described above.

ELISA titration
Polyvinyl plates (Costar, cat. No. 2596, Cambridge,
MA, USA) were coated independently
with four classes
of peptide: 0.15 pg well of each synthetic peptide alone;
0.15 pg well of each BSA-coupled
synthetic peptide;
0.15 ,ug well- of either the chimaeric
peptide or the
native toxin 2. The coating for all these peptides was
performed in the presence of 20 mM sodium bicarbonate
buffer, pH 9.4. The wells were them blocked with 1%
BSA in 15 mM sodium phosphate
buffer, pH 7.8, plus
0.15 M sodium chloride (PBS solution) and incubated
for 1 h at 37C. Wells were washed with PBS and all sera
obtained
(mice or rabbits) were assayed on the coated
plates using doubling serial dilutions, starting with 1:50
(serum:PBS-BSA
1%). After 2 h incubation
at 37C. the
wells were washed with PBS plus 0.05% Tween-20, and
the attached antibody measured with a second antibody
coupled to horseradish
peroxidase.
Two preparations
were used, either goat anti-mouse-IgG,
or goat antirabbit-IgG,
both from BioRad Lab. (Richmond,
CA,
USA). Colour was developed with o-phenylenediamine
and HzO, as substrates.
The reaction was stopped by
addition
of 4 N sulphuric
acid and the absorbance
read at 490 nm in an ELISA reader model 1550 EIA
(BioRad. Lab., Richmond,
CA, USA).

Protection assays
All mice immunized
with synthetic peptides or native
toxin 2 were challenged 9 days after the fourth immunization, by intraperitoneal
injection of one LD,,, of native
toxin 2 (0.025 pug g- mouse weight of strain CDl).
Groups of 10 non-immunized
mice were used in parallel,
as controls,
for both strains. The survival rate was
recorded 24 h after challenge.

Neutralization assays
The sera from immunized
rabbits and mice were used
for neutralization
assay using groups of 8-10 nonimmune CD1 mice. Total serum and its dilutions were
pre-incubated
for 1 h with one LD,, of native toxin 2, at
37C with agitation, and injected intraperitoneally
into
mice (total volume of mixture was adjusted to 200 ~1
animal-). A control group of 10 mice was also injected

1200

Vaccine

1995 Volume

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with a non-immune
serum, pre-incubated
with one LD,,
of toxin 2, using the same conditions.
Survival rate was
read after 24 h of assay.

RESULTS
In Figure 1 are the profiles of HPLC purification
of the
synthetic
peptides,
listed in Table 1, including
the
chimaeric
toxin, which was initially separated
by gel
filtration on Sephadex G-50, prior to HPLC. Stars on
top of each component
of Figure 1 indicate the peptides
which had the expected composition,
as determined
by
amino acid analysis (data not shown). The recoveries of
the synthetic peptides were usually from 50 to 60% of
total synthesized.
Peptide 22-36 was better than 80%,
while the recovery of theJ-chimaeric
toxin was no more
than 12% of the total prepared.
Injection
of the synthetic peptides into mice, using
amounts
as high as 1 mg mouse- did not caused
symptoms of intoxication.
However, the chimaeric toxin
(57 residues long) was lethal to mouse at levels of 400 lug
animal- (strain CDl). Furthermore,
this chimaeric toxin
was shown to affect Na+ currents of N 18 neuroblastoma
cellsA5, in a very similar way to a typical J-scorpion
toxin, thus affecting the site 4 of the Na-channels
of
these cells.,
as shown by our group in a previous
publication.
Scorpion toxins have been classified into
a-toxins, those that affect the inactivation
mechanism of
the Na-channels
(site 3 of the channel) and J-scorpion
toxins, those that modify the activation mechanismj6.
The synthetic peptides were all immunogenic.
These
results are shown in Figure 2, where a titration curve was
obtained
for each peptide, using sera of immunized
mice. The results of Figure 2 were obtained by coating
the ELISA trays with free peptides. The only negative
result was obtained
with the C-terminal
hexapeptide
(internal control). The level of antibodies titrated, when
peptides were covalently attached to a carrier (BSA) and
then used to cover the ELISA plates, have similar
behaviour,
but with a better titre (data not shown). In
the latter case, the hexapeptide also gave a slight positive
titre, assayed with the homologous
anti-serum.
For
immunization,
the peptides were coupled to thyroglobulin as carrier. In the case of the long peptides 1-27 and
thefl-chimaeric
peptide, the titre obtained was independent of the use of a carrier protein for immunization
(Figure 2). The best titre was obtained
with the
J-chimaeric
peptide.
Similar titration
curves were obtained,
using native
toxin to cover the ELISA plates (Figure 3). However, it
is clear that only some of the antisera generated against
synthetic peptides were capable of recognizing
native
toxin 2. These were the homologous
serum (toxin 2
against anti-serum
toxin 2), and the sera against the
J-chimaeric
toxin, peptide l-27, peptide l-27T and to a
much lesser extent, sera against peptide 1-14, I-14M
and l-10. Sera against peptides 22-36 and 3040 had the
lowest reactivity, and the hexapeptide
was negative.
Since some of the antisera against synthetic peptides
were capable
of recognizing
the native toxin 2, we
decided to test their neutralizing
capacity
in naive
animals. Table 2 shows the results obtained. Two types
of sera were used: mouse anti-sera
for the synthetic
peptides, and rabbit sera for native toxin 2, and the

Vaccines against scorpion toxins: E.S. Calderon-Aranda

0.
0.

l-14

l-10

,E
8
(Y 0.

E
c
g

et al

0.

.
+

/
mln

0.3

l-

l-27

l-14M

mln
0.5

0.1

30-40
22-36

Figure 1 Fractionation of synthetic peptides. Synthetic peptides (loo-250 pg), indicated by their corresponding numbers in each individual
chromatogram, were applied to a Cl 8 reverse-phase column and separated by HPLC, using a linear gradient from 0.12% TFA in water to 0.10%
TFA in 60% acetonitrile, over a period of 1 h. The bottom graphs represent, respectively: Sephadex G-50 gel filtration of the chimaeric toxin into
a 0.9 x 200 cm column (left panel) and separation of fraction II from Sephadex G50 into the HPLC system described (right panel). Stars on top
of HPLC peaks indicate the peptide that gave the expected amino acid composition. In the case of the Sephadex column, fraction II corresponds
to the nearest composition expected for the chimaeric (57 amino acids) peptide, and is also the place where native Na+ channel toxins co-elute
toxin.
Ten non-immune
mice were used for
each case. One LD,, of native toxin 2 was incubated in
the presence of the various antisera
as described
in
Materials and Methods, and injected into the animals.

Bchimaeric

Survival rates were recorded after 24 h and showed an

interesting
results: the control group of mice gave the
expected
LDjO, while both toxin 2 and J-chimaeric
antisera protected 100% of the mice. Peptides l-27 and

Vaccine 1995 Volume 13 Number 13 1201

Vaccines against scorpion toxins: ES. Calcieron-Aranda et al.

-Log

dilution

Figure 2 ELISA titration of sera anti-synthetic

peptides. ELBA plates were pre-coated with free synthetic peptides (no carrier attached)
antibodies were titrated by using the sera obtained from the immunized mice, according to the scheme described in Materials and Methods.
values given are the means of triplicates and the absorbance (A 490 nm) of pre-immune sera (controls) was subtracted. Similar results
obtained when the plates were coated with peptides attached to EGA (data not shown), including the hexapeptide, which in this figure
negative results

I-27T showed a slight protection,

with 70 and 80%
survival, respectively.
All the other cases were in the
range of 30-60%, except for peptide 3040, which gave
an unexpected low value of 10% survival. Since these are
final survival rates, it is noteworthy
to describe the
clinical observations
of the experimental
assay. Mice
injected with neutralizing
antibodies,
i.e. anti-toxin
2
and anti-chimaeric
peptide, showed none or very few
symptoms
of intoxication,
and these are very much
delayed in time. The larger peptide l-27, and l--27T had
the onset of clinical symptoms of envenomation
delayed.
when compared with the control (50% survival) or with
the shorter peptides 3040 and l-10. Encouraged
by the
possibility of using a synthetic chimaeric toxin (200-fold
less toxic than native peptides) or the larger peptide l-27
for immunization
of experimental
animals,
we have
proceeded to assay the effect of directly challenging
the
pre-immunized
mice with one LD,, of native toxin 2
(Table 3). The most important
data are the lOO/o
survival rate for the mice immunized with native toxin 7,
and the 95% death
obtained
with the mice preimmunized
with either the J-chimeric
toxin and larger
peptides l-27 and l-27T. These results were both unexpected and disappointing.
in terms of obtaining
a
possible synthetic vaccine against scorpion stings. It is
worth mentioning
the 50/0 survival of the controls, and
also the low survival of the mice pre-immunized
with
peptide l-14M. Other shorter peptides make little difference. Here also, the clinical observations
are important. Mice pre-immunized
with &chimaeric
toxin, l-27
and l-27T developed symptoms exactly similar to naive

1202

Vaccine 1995 Volume 13 Number

13

and
The
were
gave

animals injected with native toxin, and died in approximately the same time, or a little before the others. The
pre-immunized
mice with native toxin 2 showed very few
symptoms of envenomation,
and the onset of these was
very much delayed compared to controls (50% survival).

DISCUSSION
In the last three years, more than 100 papers have
been published describing the synthesis of peptides corresponding
to the amino acid sequence of fragments of
proteins
from viruses,
bacteria
and other sources,
prepared with the purpose of possible aylications
towards the development
of new vaccinesT6 . The model
using toxic peptides
from venoms
has also been
described-..
In the present report, the rationale for choosing the
peptides we have designed (Table 1) emphasizes
the
N-terminal
region of toxin 2, because initial results
obtained by our group
with a synthetic decapeptide
corresponding
to amino acids l-10, from C. rroxius toxin
10, showed toxicity in mice. Work performed
with
synthetic peptides of another different class of toxin
(u-scorpion
toxin) from Androctonus australis also indicated that this segment was important
for toxicityZO.
Furthermore,
as shown by othersis, a minimum
of six
amino acid residues were needed for antigenic purposes.
Thus, most of our peptides
contained
10 or more
residues. The amino acids of toxin 2 among positions
43-60 are hydrophobic
and peptides in this region are

Vaccines against scorpion toxins: E.S. Calderon-Aranda

-Log

et al

dilution

Figure 3 ELISA titration of anti-synthetic peptide sera on toxin 2-coated plates. ELISA plates were pre-coated with toxin 2, and the antisera
obtained from mice immunized with thyroglobulin-attached
synthetic peptides (except chimaeric toxin) were assayed. The results obtained with
serum against native toxin 2, synthetic chimaeric toxin and peptide l-27 with no carrier, are also reported. Values are means of triplicates and
the absorbance (A 490 nm) of preimmune sera (controls) were subtracted

Table 2 Neutralization of one LD,, of toxin 2 from C. noxius with

anti-synthetic peptide sera
Survival
Anti-sera

A/-Y

Dilution
factor

Control
Toxin 2
l-10
1-14
30-40
22-36
l-14M
l-27
1-27T

5/l 0
10110
3/l 0
3/l 0
1110
6/l 0
7110
700
8110
lo/lo

50
100
30
30
10
60
70
70
80
100

1.05
8.00
1.05
1.05
1.05
1.05
1.05
1.05
1.05
2.60

Table 3 In vivo protection

C. noxius in immunized mice

against

one

LD,,

of toxin

2 from

Survival
Group of mice

AI-T

Control
Toxin 2
l-10
1-14
l-14M
22-36
30-40
l -27T
l-27
Chimaeric

9118
18118
7117
8117
4118
7118
10118
l/l8
1118
1118

50
100
40
45
20
40
55
5
5
5

A means number of mice alive; T, total number of animals assayed

aA means number of mice alive; T, total number of animals assayed

insoluble
in physiological buffers, complicating
their use. Finally, we designed and synthesized a fulllength peptide (chimaeric toxin) with 57 residues by
applying the following rationale: (i) the relative positions
of the principal cysteine residues were respected; (ii) the
positions of the secondary structure forming stretches,
for a-helix and J-pleated sheets were also respected,
based on the X-ray diffraction pattern obtained from a
similar toxin, variant 3 of C. sculpturatu.s44, with minor
conservative substitutions; (iii) the N-terminal sequence
of toxin 2 was conserved, because if that was the case,
the new chimaeric toxin should behave as a&scorpion

toxin; (iv) the C-terminus was shortened and corresponded to the amino acid sequence of the a-scorpion
toxin class, to test the possibility of the C-terminal part
being involved in the active site of a-toxins; (v) an
important number of glycines (14 of 57) was introduced
in highly variable regions of the primary structure5, to
permit the toxin to have free movement in those regions
which are not very important for the three-dimensional
folding, (vi) the loop-forming regions44 were considerably changed, to test the hypothesis of these variable
length segments in differentiating between an a- and a
J-scorpion toxin.

usually

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Vaccines against scorpion toxins: E.S. Calderon-Aranda

The immunogenicity of all synthetic peptides, covalently attached to a carrier molecule (thyroglobulin)
was evident by the titration curves of Figure 2, including
the hexapeptide used as internal control. Interesting
results were described in Figure 3, where, as expected,
not all antibodies generated against short segments of a
given protein (toxin) are able to recognize the native
form of the toxin, confirming similar results found by
others in this respect43. The peptides capable of eliciting
antibodies that recognize native toxin 2 are larger molecules (from 14 to 57 amino acid residues) and all have
the N-terminal segment of the toxin intact. The chimaeric synthetic toxin was lethal to mice and was shown
to belong to the&scorpion toxin class, thus supporting
the idea that the N-terminal amino acid sequence of
toxin 2 is possibly part of the active site of this toxin.
Synthetic segments of the internal part of the molecule
slightly recognize the parent toxin 2 in the ELISA assay
(Figure 3). The crystal structure of variant 3, from the
scorpion C. sculptur~tus~~ gives two types of information
relevant to the latter findings: first, the N-terminal part
of the toxin is very much exposed on the surface of the
molecule; secondly, the internally situated peptides are
compromised in a very structured region of the toxin,
tightly joined together by three of the four disulphide
bridges and by the u-helix and the three antiparallel
J-sheet forming structures. Thus, the amino acid sequences of highly organized regions of the toxin are
bound to generate different types of recognizing antibodies, when their sequential segments are synthetically
prepared, disrupting putative conformational epitopes.
This draws attention to the important finding that
antibodies generated by the native toxin are capable of
eliciting a protective response, in either protocols: i.e.
neutralization (Table 2) and direct challenge (Table 3).
However, the presence of neutralizing antibodies (Table
2), generated by synthetic peptides, with arbitrarily
chosen segments of the parent protein (toxin 2) is not a
guarantee that the respective antibodies will protect
experimental animals in a similar fashion to that of
native toxin (Table 3). This conclusion is supported by
the conflicting results of Table 2 and Table 3, in the
case of large synthetic peptides @-chimaeric peptide
and peptide l-27). In our opinion this means that the
manner by which the immunological system of the
mouse processes a given peptide is quite different, depending on whether the peptide is a native one, or is a
synthetic segment, that do not correspond to the size
and exact structure of the native toxin.
We have no clear explanation for the sensitization
phenomenon described here. Maybe other immunological events associated to the liberation of substances from
endogenous cells of pre-immunized animals can enhance
the performance of the native toxin, when the system has
been previously primed with synthetic peptides similar
to that of the native peptide. It could also be that a
protective effect is conferred by the antibodies to the
toxin molecules, against endogenous proteolytic digestion or against other mechanisms of clearing foreign
proteins from the body, or that the antibodies increase
the concentration of toxin near the site of action (ionchannel). These are some of the possible explanations
for the sensitization phenomenon described here.
Another aspect to be observed concerning our results
is that the clinical manifestations
of intoxication

1204

Vaccine 1995 Volume 13 Number 13

et al.

occurring in our experimental conditions are not distinguishable from normal mice injected with toxin. In our
opinion this phenomenon is not an artefact. Additionally, there are several lines of evidence that support our
conclusions: (1) the ubiquitous presence of circulating
antibodies in the blood of mice immunized with synthetic peptides, as demonstrated by ELISA titration in
vitro (Figure 2); (2) the restricted recognition of native
toxins only by antisera against synthetic peptides,
J-chimaeric peptide and native toxin, also as determined
by ELISA assays (Figure 3); (3) although neutralization (Table 2) and active immunization (Table 3) suggest that peptides in the range of 6-14 amino acids are
not effective as neutralizing or sensitizing peptides, as
judged by the number of surviving animals, those with
longer peptide chains (27, 57 amino acids) protect naive
animals (at a level of 70%100%) and exacerbate the
response in the direct challenging protocol (5% survival).
These results, in our opinion, cannot be attributable
to experimental error due to the number of animals
used.
Another important point is that identical results were
obtained with another strain of mouse (BALB/c) immunized, following the same experimental protocols, with
native toxin 2 and synthetic peptides: J-chimaeric, l-27,
l-27T, and 1-14 (data not shown).
Additionally, the clinical symptomatology observed
during the course of experimentation is very important
in confirming the arguments presented above. The
course of evolution of the intoxication process in mice,
described in Materials and Methods, is clearly evident
whether passively or actively challenged with toxin 2.
The onset of the clinical symptoms of envenomation
were much delayed, or practically abolished, in animals
that had been injected with antibodies from the longchain immunized mice (Table 2), while in the direct
challenge experiments (Table 3) only the animals preimmunized with native toxin did not show any symptoms of envenomation when injected with one LD,,
value of toxin. Again, the animals that died showed
symptoms indistinguishable from control mice injected
with toxin.
The conclusion of this work is that the approach of
synthetic peptides for generation of a possible vaccine
against scorpion toxin must be studied with much care.
It is likely that the determination of the exact neutralizing epitope of the toxin is needed.
ACKNOWLEDGEMENTS
We thank Georgina Gurrola-Briones for helpful discussions during the synthesis of peptides, and Dr Fernando
Esquivel for critical reading of the mansucript. The
technical assistance of Fernando Zamudio is greatly
appreciated. This work was supported in part by grants:
Howard Hughes Medical Institute (75191-527104).
DGAPA-National
University of Mexico (IN 205893),
CONACyT-Mexico
(001%N9105), and US-Mexico
Foundation (56-CH- 166) to L.D.P.
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