History

It has long been established that animals can be actively immunized against snake venom
by repeated parental injections, with increasing doses of venom (Sewall, H., 1887.) Active
 immunity against snake venom has also been attempted and demonstrated in humans using
toxoid derived using the venom of the Australian Tiger snake (Notechis scutatus)
(Wiener, S., 1960.) A large-scale immunization trial was also implemented using a toxoid
of the venom from the Habu (Trimeresurus flavoviridis) (SAWAI et al. 1969).  The most
remarkable case of active immunity in humans is of one William E. Haast of the Miami
Serpentarium.   He originally obtained immunity using venom from the Cape Cobra,
 (Naja nivea) then later included venom obtained from the Indian Cobra, (Naja naja)
and King cobra (Ophiophagus hannah).   His case demonstrated cross-resistance between
 venoms of different species.  He subsequently was bitten by species other than was used in
the inoculations and suffered little or no systemic reactions.  The most remarkable followed
after being immunized with the Cape cobra (Naja nivea).  He was bitten by the Blue krait
(Bungarus caeruleus) and survived without any antiserum. This case unlike most other cases
of human immunization against snake venom involved using venom that was not detoxified 
 (Haast, W.E, Winer, M.L. 1955).  Later other venoms were included into his inoculations. 
Presently more than thirty venoms are used in his booster shots.  He has subsequently
survived more potentially lethal bites than any other human.  These bites included some of
 the most lethal species.  The Tiger snake  (Notechis scutatus) Russell's viper
(Daboia russelii) and Saw scale viper (Echis carinatus) are a few of the many notable
 species he has survived bites from.   He is presently ninety four years old and in good health.  
Mr. Haast pioneered human immunizations when started immunizations in 1948. 
(Haast, W.E., Nancy Miami Serpentarium 2005 personal communications)
                                                                                                                        1

It has long been established that animals can be actively immunized against snake venom
by repeated parental injections, with increasing doses of venom (Sewall, H., 1887.) Active
 immunity against snake venom has also been attempted and demonstrated in humans using
toxoid derived using the venom of the Australian Tiger snake (Notechis scutatus)
(Wiener, S., 1960.) A large-scale immunization trial was also implemented using a toxoid
of the venom from the Habu (Trimeresurus flavoviridis) (SAWAI et al. 1969).  The most
remarkable case of active immunity in humans is of one William E. Haast of the Miami
Serpentarium.   He originally obtained immunity using venom from the Cape Cobra,
 (Naja nivea) then later included venom obtained from the Indian Cobra, (Naja naja)
and King cobra (Ophiophagus hannah).   His case demonstrated cross-resistance between
 venoms of different species.  He subsequently was bitten by species other than was used in
the inoculations and suffered little or no systemic reactions.  The most remarkable followed
after being immunized with the Cape cobra (Naja nivea).  He was bitten by the Blue krait
(Bungarus caeruleus) and survived without any antiserum. This case unlike most other cases
of human immunization against snake venom involved using venom that was not detoxified 
 (Haast, W.E, Winer, M.L. 1955).  Later other venoms were included into his inoculations. 
Presently more than thirty venoms are used in his booster shots.  He has subsequently
survived more potentially lethal bites than any other human.  These bites included some of
 the most lethal species.  The Tiger snake  (Notechis scutatus) Russell's viper
(Daboia russelii) and Saw scale viper (Echis carinatus) are a few of the many notable
 species he has survived bites from.   He is presently ninety four years old and in good health.  
Mr. Haast pioneered human immunizations when started immunizations in 1948. 
(Haast, W.E., Nancy Miami Serpentarium 2005 personal communications)
                                                                                                                        2

Case

This case, before us now, is another using venom that is not detoxified.  This case involved
a male subject, the author, who is of good health and condition.  The immunization was carried
out as a prophylaxis in case of envenomation, while engaging in venom extraction.  Liquid,
non-lyophilized venom was used for  convenience.   All doses are in the liquid natural form.  
Detoxification was not attempted in order to eliminate any epitope or antigenic change.  
No adjuvant was added.   Prior to doses of .01 ml of pure undiluted venom, dilutions were
made using bacteriostatc water with 0.9 % benzyl.   Inoculations were administered parenterally
by intradermal injections.  The course of immunization was carried out from September 2001
 to March 2005. Inoculations were given one week to one month apart.  On two accounts
there where delays between inoculations of a period of two months.  Immunization commenced
 using venom from Egyptian cobra (N. haja) and Forest cobra (N. melanoleuca) at a dosage
 of .001 ml.  These venoms were rotated between inoculations.  Later Cape cobra (N. nivea)
venom was used as a substitute to replace other naja venoms.  This variation from the
original immunization schedule was done because of the known toxicity of N. nivea venom. 
It being the most toxic of all cobra venoms (U.S.  Department of the Navy, Bureau of Medicine
 and Surgery, POISONOUS SNAKES OF THE WORLD, 1991).   It is the hypothesis of the
author, that because of its toxicity, N. nivea would provide the best immune protection against
most other similar naja venoms.  The use of N. nivea venom commenced after the inoculation
 doses of the other naja venoms were .01 ml.  At this stage of the immunization when the use
of different, but similar venom was implemented, a lower dose of .005 was used initially. 
In this case, immunity obtained using the other naja venoms proved affective in that no
substantial reactions occurred  following initial and subsequent injections with N. nivea venom.
   Due to the absence of systemic reactions and only minor local reactions, these doses were
doubled with each inoculation until the previously used dose as with the other venom was
obtained.  This stage of immunizations continued with these venoms until a dose of .02 ml of
 undiluted liquid venom was used.  During this course no serious systemic effects were
experienced.  General malaise was experienced during earlier inoculations.   On a few
occasions pyrexia was also experienced.    

                                                                                                                                 3

Systemic effects decreased with repeated injections of the same amount.  Local reactions
always consisted of an immediate wheal, erythema, ecchymosis, and subsequent swelling
distal to injection site.   Edema sometimes encompassing the entire limb and usually subsided
within 2 days. Initial and higher doses always resulted with local necrotic ulcerations, acute
cellulitis and subsequent holes when the necrotic tissue was debrided and/or sloughed from
injection site.  The necrotic ulcerations and cellulitis would also progressively show signs of
healing within 2 days.   During the course of these inoculations, it was also noted that after
lapses of two months, the only noticed reduction in immunity or immune responses were

noticed in these local effects.   Shorter intervals between inoculations produced less of a
local reaction.  Systemic involvement remained more consistent, with no changes in reactions
after these 2-month lapses. The course of immunization using venom of the Eastern Green
Mamba (Dendroaspis angusticeps) was started when the dose of naja venoms were .02 ml. 
The same initial dose, as used with the others, of .001 ml was used.  The effects of this of
D. angusticeps venom proved substantially different, than the naja sp, used with the previous
inoculations.   It proved different with its extremely rapid onset of symptoms.  This was possibly
due to the facilitating ability of the dendrotoxins and / or to its extremely light viscosity.   Snakes
belonging to the Dendroaspis genus have a particular dreaded reputation.  This is partly due to
this rapid onset of systemic symptoms.   Prior to the development of mamba antivenoms, there
where difficulties experienced in their development.  The animals often died, as a result of the
venom acting so rapidly (Krush, Haast Cobras in His Gardens 1963).   Initially, in each
case of these earlier inoculations, using D.A. venom, numbness and tingling sensations of the
lips, mouth and brow was experienced immediately

upon injection.   Sore throat and muscular aches later followed these symptoms of the
earlier inoculations.  Some of these earlier doses which resulted with notable reactions, would
persist into the next day with symptoms similar to those following other intoxications or
flu-like symptoms, i.e. headaches, sore muscles and joints.                        

                                                                                                                                4

 The symptoms lessoned with subsequent inoculations of the same amount.  However symptoms
 would return with increased doses.  Inoculation doses, up to this point, where; .001, .002,
.003, .004, .005, .006, .007ml.  When inoculation doses of this venom were .008 ml,
symptoms came on quickly, like with a “rush”, then would begin to subside within 2 hours.  
During these occasions it was required to lie down in bed.   In two accounts, recorded blood
pressures were noted with a decrease in systolic and diastolic pressure.   On one of the
inoculation using the dose of .008, the BP of 78/40 was recorded initially.  BP then fluctuated,
but gradually increasing and returned to within normal parameters within one hour.   Pulse
rates increased considerable after injections initially along with the feeling of a “flush” hot
sensation.  Pulse rates as high as 105 per minute were recorded.  The pulse rate would
then drop to as low as 40 per minute.  The advent of these symptoms were within two
(2) minutes following injections.  Vital signs would return to normal parameters within
2 hours. The reversal of these rapidly developed symptoms were as notably felt as
with the onset.  This is interpreted as a positive immune response.  The subsequent dosage
following the above inoculation and adverse
symptoms was reduced to the last previous dosage without these symptoms, so that the
previous dose, in this case .007 ml. was repeated.   Tonic muscular spasms associated with
flexion and extensions while walking, resulting with extra movements while taking steps, were
also experienced.  These spasms persisted after all vital signs returned to normal limits, then
would gradually return to normal.   However sensations and reflex actions attributed to this
persisted into the next morning.  These spasms were possibly indications of the fasciculins or
dendrotoxins, that facilitate the release of acetylcholine, without endogenous regulation and
control.  Initially this venom did prove difficult to use as an antigen for inoculations because
of this rapid onset of symptoms and effects.   However, once the dose of .01 ml of undiluted
was reached, all subsequent increases of dosage were uneventful.  No remarkable deviations
of vital signs were experienced following subsequent injections of higher doses.  It was after
a dose of .04 ml. undiluted D. angusticeps venom that dose increases of venoms; N. nivea
and D. angusticeps in increments of .01 until a dosage of .15 ml undiluted venom with no
systemic effects.  At this dose of .15 ml, D.A. venom caused only erythemas and edema of
limb distal to inoculation                                                                                                               

                                                                                                                                 5
 

site, with no significant signs of systemic changes.   No cellulitis or necrotic effects were
noted following any inoculations using venom of D. angusticeps, only an immediate wheal
followed by erythema and subsequent swelling was observed in the immediate injection
site.  A challenge dose of .15ml N. nivea venom caused no systemic effects, only the
previously described local necrotic effects.  The effects on the tissues in the immediate areas
did appear dose dependant, but only to a point.  A dose of .15 ml caused only slight increase
in the local reaction to that of. 05 ml.  This is consistent with the observations of Wiener 
(Wiener,S.1960).  The venom of the Western Green Mamba (D. viridis) is 3 to 4
times more lethal than D.A venom.  (Harvey et al, 1984 J. Toxicol) However, inoculations
of D.V with venom doses of .01, .02, .04, .08, 10, .15ml., given one week a part,
subsequent to D.A. immunization, were uneventful.  It remains unknown what effects
an initial higher dose of D.V. venom and what degree of immune protection would have
been provided following D.A. immunization.  However, it is evident with the accelerated
 immunization using D.V. venom without ill effects are an indication of substantial protection.  
This is possibly because the toxic constituents of D.V. venom are similar to the combination
of D.A. and N. nivea venom.  D. A. venom does not contain potent post synoptic neurotoxins
as found in both D.V. and N. N. venom. (Harvey et. al, 1984 J. Toxicol) The absence of notable
systemic symptoms following a dose of .15ml undiluted venom from these species, as well as the
 rapid rate at which symptoms subsided, following earlier inoculations is evident of some substantial
immunity.  The unsuspected adverse reactions, as noted following D.A. dose .008 is an example of
difficulties that can be experienced during immunizations of snake venoms that have not been attenuated. 
On the 21st of April 2005, a bite was sustained from a four foot Eastern
diamondback rattlesnake, Crotalus adamanteus.  The bite was to the right middle finger
with both fangs and a scratch to the index              

                                                                                                                                     6

 finger.  The snake remained attached for a couple seconds.  Upon it's release one of the
snake's fangs remained in the wound.  This bite was followed by definite signs of
envenomation, i.e. blood oozing from both fangs puncture holes, swelling and severe burning
sensation.  No major systemic symptoms were experienced.  Swelling was limited to the
hand and wrist, which was less than double the normal size.  Only the wound area was
discolored, and with small blebs.   No antivenin was administered.   Laboratory test; CBC,
Fibrinogen level and prothrombin time revealed no gross abnormalities.  Discharge from the
hospital  was the same night.  It appears evident that despite the known marked differences
between crotalid and elapid venoms, there was substantial immunity demonstrated in this case. 
*This bite sustained from the Crotalus adamanteus is inconclusive as an indication
of an immune response. Even so it should not be over looked entirely.  It should stimulate
curiosity.  Booster inoculations are continued on a monthly basis.  The author is confident and
predicts that the unique properties of Dendroapsis venom will hold some significance regarding
the further understanding of the generation, transmission and reception of acetylcholine.  There
also appears to be evidence, here, which suggest an immunopotentiation between different
snake venoms as antigens.  Further investigations could reveal more information regarding
the immune responses between antigenic toxins and antibodies produced heterologously by
inoculation of different venoms.  This phenomenon may be due to the affinity at which different
antibodies bind to antigens exogenous to the production of that specific antibody or further
research may reveal new information regarding antibody production and differentiation of
undifferentiated B cells.  This needs further investigation. It should be made known that the
foregoing paper is not intended as an account of any study, but rather of the author's own
immunization schedule used and of the observations of the effects experienced during its course.
The author wishes that this information be made available to any and all researchers that may
find interest. 
                                                                                                                                          7

REFERENCES

Haast, W.E., Winer, M.L 1955.: Complete and Spontaneous recovery from the bite of
 a blue krait (Bungarus caeruleus) Ameri. J. trop. Med. Hyg. 4 1135-1137  

Haast, W.E, Nancy Miami Serpentarium 2005: Personal communications

Harvey, A.L., Anderson, A.J. Mdugka, P.M., and Karlson, E. (1984): Toxins from
Mamba venoms that facilitates neuromuscular transmission. J. Toxicol. Toxin Rev.3 91-137

 Harvey, A.L. editor, 1991: SNAKE TOXINS International Encyclopedia of
Pharmacology and Therapeutics   ISBN 0-08-040294-1   

Kursh, H. 1965 Harvey House, Inc. Pub.  Cobras In his Gardens, biography W.E. Haast

 Menez, Andre., Immunology of Snake Toxins (1991):  Harvey, A.L editor, SNAKE TOXINS
 pp 5-90   ISBN 0-08-040294-1

U.S Dept. of  Navy (1991) ( Bureau of Medicine and Surgery) POISONOUS SNAKES
OF THE WORLD,   ISBN 0-486-26629-X    

SAWAI., Kawamura, Y., T., Okonogi, T., Ebisawa, I  (1969a): Studies on improvement
of treatment of habu  (Trimeresurus flavoviridis) bites  Jap. J. exp. Med. 39, 109-117 

SAWAI, Y., Kawamura, Y., T., Okonogi, T. Ebisawa I  (1969b):  Field trial of prophylatic
 inoculation of  the habu (Trimeresurus flavoviridis) venom toxoid.  Jap. J. exp. Med. 39, 197-203

Sewall, H. (1887):  Experiments on preventive inoculation of rattlesnake venom., J. Physiol
(Lond) 8, 203-210 

Wiener, S., (1960)Active immunization of man against venom of Australian tiger snake
(Notechis Scutatus),  Amer. J. trop. Med Hyg. 9, 28                                                                                                           

LEE MOORE          
Serpentoxin Laboratory   
P.O Box  565
Fort McCoy, Florida 32134- 0565
USA
email   LeeMoore@Serpentoxin.com

 

  1         
Professor Alan Harvey
Department of Physiology and Pharmacology
University of Strathclyde
27 Taylor Street Glasgow G4 0NR, UK      

RE: AH/2005/000156

          HUMAN IMMUNIZATION TO ACQUIRE ACTIVE IMMUNITY
                                         AGAINST SNAKE VENOM   

Dear Dr. Harvey,  

I apologize for the delay with responding on this issue.  I feel the subject of this
letter is important.  I pray it is given proper consideration. 

I wish to make it clear that I commend Toxicon's and The International Society
 on Toxinology for its work and dedication to the ends of the standardization
and organization of information concerning snake toxins. These efforts have
provided a forum, unparalleled by any other.  It has enabled researchers from
 across the globe a means to participate as a whole, towards the advancements
 in toxinology. 

I do wish, however, to express some concerns directly related to the journal's
written evaluations relating to the above manuscript.  It is to be expected that
any “risky” protocol such as the one presented would be under harsh scrutiny. 
However, the reviewers of this paper where not objective and did not represent
the papers most obvious objectives. The objective was merely to make available
information concerning this particular case of immunization.  It is not intended as
 a proposed immunization schedule for others to use, but merely as an account
of the reactions experienced.  Allow me first response to “Reviewer No. 33 title
“Mere Masochism or certifiable madness?” Some of the greatest advances came
 with risk.  Medicine has always been latent with risk.  One of the first thoughts
that came to mind was that of Edward Jenner's vaccine and how many had not the
convenience of not having to weigh the risks between exposing themselves and
their own “young” to the vaccine (cowpox virus) to protect from the risk of smallpox.
This was a new concept that was far from being fully understood and with many
unknowns.  I would like to add a discovery actually made by a young uneducated
 milkmaid. 

My life's work has directed me in a direction involving risks related to snake venom
extraction. The point being is that these risks can be logically decided upon. The risk
of snake bite is very real regardless of the caution exercised.  Reviewer No. 33 also
seemed alarmed at the mention of swelling and necrosis.  It is not only “risk” but
usually at some physical consequence taken by either animal or human.  A review of
immunization schedules and processes of the immunization of horses, one would see
similar reactions to a lesser degree, of course, due to the Dose-Response relationship
because of the mass/dose ratio. The horse obviously has much more of an advantage. 
To address reviewer No. 33 statement “There is no demonstration of the induction
of specific or paraspecific immunity, merely his apparent tolerance of increasing doses
of venoms.” This statement is a contradiction in terms. “......... tolerance of increasing
 doses of venoms.”? Tolerance of increasing doses is definitive of immunity.  I will
address this further later in this letter.               

2

I agree entirely that the bite sustained from the Crotalus adamanteus is inconclusive
as an indication of immunity.  Even so it should not be over looked entirely.  It should
stimulate curiosity.   It was apparent that it was not entirely a “dry bite”, due to the
subsequent surgical debridment of some subcutaneous necrotic tissue.

I will have to disagree entirely concerning Reviewer No. 33 concerning the spurious
opinion that Bill Haast has not demonstrated a high degree of immunity.  His case
demonstrated cross immunity as was indicated with the well documented case of the
bite of the blue krait in the New England Journal of Medicine. This citation can be
found in the letter to the editor.  His blood’s serum has been used as an antiserum
and has saved lives.  This is well documented.   

The fact that serious envenomation will always be a risk regardless of immunization
 was never disputed in the submitted manuscript nor is it here.  Immunity of snake
toxins is relevant to the amount of toxin vs. antibody titer.  It is always possible to
overwhelm the antibodies and a very real risk! There is no “silver bullet” as
prophylaxis for envenomation.  However a remarkable IgG, antibody titer is of
obvious advantage.  Along with this consideration the term resistance may be more
suitably used in its stead. 

Reviewer No. 33 also stressed that the author has “proved nothing”.  It was not the
paper's intention to prove anything. There was no attempt to present this data as a
study or experiment.  It was just intended to relay what had been done to immunize
a human, myself.  “......... merely his apparent tolerance of increasing doses of venoms.”
Maybe, but an injection of .15 ml of N. nivea or D. angusticeps venom, presently
without any effects is an indication that there would be an advantage in the case
envenomation and a subsequent higher dose.  The undisputable fact remains that
this dose would ordinarily be critical.  

Reviewer No. 13 was concerned and stated that “Dosages such as .001ml is not
reliable data to describe or estimate venom activity”.  An argument that measured
doses in liquid form are not exact could well be made”.  However to state “…is not
reliable data to describe or estimate venom activity”, is unreasonable.  It is merely,
as stated by this reviewer, an estimate and does present consistency.  Lyophilized
venom of these liquid doses would reveal consistency in dry milligram doses, would it
not? 

I admit there where some potentially serious consequences.  These are noted and
described in the manuscript.  These facts where not omitted.  They could have easily
been omitted and just presented the procedure as without any difficulty, but that
would not have been objective or ethical.  In response to the reviewer No. 33 concern
of not addressing the “very important theoretical objections ....” the facts that would
clearly raise these important objections are clearly presented.  Despite the comment
by reviewer No. 33 that this paper, “would be of interest only in the psychiatric
literature”.   The author did not present the manuscript to such and therefore felt it
not appropriate to convince anyone on ethical issues. 

The risks are very real when one injects foreign substances, especially those that
are termed and classified as dangerous venoms into the body.  However, the risks
of these venoms are also very real when one engages in venom extraction.  I argue
that these risks can logically be “weighed” and decided upon, but only by the
individual involved.  Perhaps the paper the reviewers would prefer would be titled
Snake bite Vs. Immunization? The theory behind this immunization is fundamental
and has been accomplished numerously with animals. The risks and ethical issues,
as stated earlier, must be evaluated by the individual.  I do strongly advise against
this procedure, except for cases involving an extreme risk of envenomation.  In
which cases, the risks and potential benefits should be fully investigated and decided
upon by the individual.  I have continually been adamant that I would never encourage
nor assist any individual with their own immunization. The risks of miscalculations,
adverse effects directly related to the different toxins and anaphylactoid reactions
are all very possible and real risks.  There are numerous risky variables. This,
however, was not the subject of the presented manuscript.
                                                                                                                3   

I have found no other more comprehensive work on the subject of toxinology than
Snake Toxins edited by yourself and that of Venomous and Poisonous Animals by
Dietrich Mebs. The accomplishments of the International Society On Toxinology are
unparalleled.  I do understand the Journals concerns, whereas it is the journal's
responsibility to express such concerns, it should equally be responsible to relay
documented information.  If the information is challenged, investigate it further.   

To my knowledge, there have not been many immunoassays done in humans. The
long term effects of snake venoms on humans have not yet been investigated.  This
 information would be of obvious benefit to the investigation of using snake toxins in
human medicine.