Jpn. J. Infect. Dig., 52, 234‑237, 1999

Original Article

Stability of Russell 's Viper Venom Toxoid (Lyophilized Fom) on Storage
Win‑Aung* , Khin‑Khin‑Hlaingl , Khin‑Pa‑Pa‑Kyawl , Mya‑Mya‑Winl and Aye‑Kyaw
Diagnostics and t4accine Research Centre, and lBiochemistTy Research Division,

Department of Medical Resewch, No. 5, Ziwaka Road, Dagon P.0., Yangon 11191, Myanmar(Received February 10, 1999. Accepted November 1 I , 1999)

StJMMARY: A previously developed Russell's vIPer Venom tOXOid in Myanmar is in a liquid form that shows
reversion in the form of a reduced number of formaldehyde linkages and toxicity during storage at 370C and atroom temperature. In order to have a safe, potent and stable toxoid, a lyophilized form was prepared in thepresent study from the liquid toxoid throughthe use of a freeze dryer. Both the liquidand lyophilized forms
were then stored at 4oCand at room temperature, and in addition to safetyand immunogenicity tests, biochemicalparameters such as the protein content, the activity of venom enzymes (proteinase, phospholipase A, phosphodi‑
esterase,and arginine esterase), and the released free formalin amounts were assessed at 3‑month intervals overaperiod of I year. The results indicate that under both conditions, the lyophilized toxoid shows minimum changes
in enzyme activity, a reduced tendency toward formaldehyde linkage, no toxocity, and more immunogenicity incompariSon with the respective liquid toxoids. It could therefore be hypothesized that Russell'S viper Venom
toxoid in a lyophilized form is more promlSlng in terms of efrlCaCy and stability for prophylactic use in humanimmunization than the conventional toxoid in a liquid fom.
Then, the toxoid in a liquid fom was treated by a freeze‑

INTRODtJC TION

drying process to obtain a freeze‑dried (lyophilized) form･

Development of a suitableand effective snake venom toxoid

Then, both forms of the toxoid were stored at 40C or at RT

for active immumization of people atrisk to snake bite is offor a 1‑yearperiod, during whichthe toxoids weFeperi∝hcallyutmost importance in tropical countries where snake bite is achecked for enzyme activity free formalin released, safety,

serious medical problem (1 ,2). In Myanmar, Russell's viper

and immunogenicity, With the ultimate aim of achieving a(Daboia russelii siamensis) venom (RVV) toxoid in a liquid
form has been successfully produced from RVV by a slow

more safe, potent, and stable toxoid to be used in human im‑
munization.

and stepIWise formalinization method in the Department ofMedicalResearch (DMR) for 2 decades (3). It has been found

MATERIALS AND METHODS

to be potent and immunogenic with minimum undesirable

side effects in immumized monkeys (4)and humanvolunteers

Desiccated crude RVV and lyophilized anti‑snake venom(5). However, there was a reduction in the number of fbmal‑(AS∨) were purchased from Myanmar Pharmaceuticaldehyde linkages with an appearance of toxicity and a reduc‑Factory (MPF), Yangon, Myanmar. All chemicals used in thisexperiment were ofanalyticalgradefrom Sigma Chemical Co･,
St. IJOuis, Mo. Adult mice of both sexes, Institute of Cancer

tion in the immunogenicity of the toxoid being stored

Especially at 3rCand at room temperature (RT) (6,7)･ TherelS an urgent need to develop a safe, stable form of the toxoid

Research (ICR) strain, weighing 20 ± l g were obtained fromthat maintains its potency during storage, as the morbidity

the Laboratory Animal SeⅣice Division of DMR, Yangon･

and mortality rates, i.e. more than10,000 snake bite cases

Detemination of intramuPctllar(i･n･) median lethal

per yearwitha mortalityrate of 10%, associated with Russell 'svlper remain relatively high in Myanmar. Many attempts havebeen made to improve the stability, safety,and immunogemiC‑
ityof the DMR toxoid by using only major purified fractions

dose (LDs.) of RVV inmice: I.m. LD5. Was determined byuslng Seven groups Of mice, each consisting of six animals

envenomed with different concentrations of RVV ranglng
from 181600 Ug/0.I ml. Deaths of mice within 24 h werenoted, and LD50 Was Calculated by the method of S.Karberof RVV (8), increasing the concentrations of fbmalin, andadding fomalin binding agent, i.e. sodium bisulfite to thetoxoids (9). No promising effects or applicable results have(13).

According to the literature, formalimized TaiWancobra venom

Preparation of RVV toxoid (liquid form): The method
used forthe toxoidation of RVV (i.e. preparation of RVV
toxoid) (liquid form) is a modified method of Kondo et all

toxoid kept at 370C shows a reversion to toxicity, whereas the(14) described byAung Khin et al. (15), in which a slow andyet been obtained, however.

toxoid kept at 40C as well as freeze‑dried (1yophilized) toxoid

stepwise fbmalinization of RVV is pe血med by increaslng

kept at 370C remain non‑toxic (10,1 1). In Japan, Habu venom

the concentrations of fbmalin by 0.2% at 2‑day inteⅣals to

toxoid, which is prepared from the formalinization of two

obtain a final concentration of 0.8% formalin at day 10. The

hemorrhagic fractions of the venom, HR‑ l and HR‑2, followed

solution wasthen dialyzed against M/30 phosphate buffer

by lyophilization, has been found to be safeand highly immu‑

solution (PBS) to remove excess formalin. The adjuvant

nogenic f♭r various animals and human beings (12).

adsorption was then camied out by the addition of M/8･45
aluminum phosphate to the toxoid solution at a ratio of 1: 1

In the present study, toxoidation was ca汀ied out using thecrude RVV by a slow and step‑wise fomalinization method.

by volume.

Preparation of RVV toxoid (lyophilized form): Half thevenom of the whole toxoid (liquid form) was separated into*Co汀eSpOnding author: Tel: +95‑1‑25 1 508, Fax: +95‑ト25 1 514

234

Table 1. Determination of intramuscular medianlethaldose (LD5.)

1‑ml samplesthat were then added to the appropnate glass
bottles･ The samples were frozen for 48 h followed by afreeze‑

of RVV on micedrying process (i･e･ lyophilization) using a freeze‑dryer model

Concentrations or RVV

no･ 5, Labconco from Japan, to obtain a lyophilized form.

(Ug/0. 1 mg)

Therefore, atthis stage, twotypes of toxoid, the liquid form and/hU 3 1

Deathof mice within 24 h

0 0 知75371

6/6

lyophilized form, were obtained. Samples of each form werethen divided into two equalpartsand stored under two different

6/6
6/6

storage conditions, namely 40C and RT (i.e. approximately

5/6

2rC)･ Then, 1 ‑ml samples of the liquidand lyophilized forms

2/6of the toxoid stored at 40C and at RT were tested for stability

0/6

every 3 months for 1 year. These tests included the determina‑

NormalSaline

tion of pH and proteins, assays of enzyme activity, namely

0/6

Numerator indicates number of deaths and denominator indicatesproteinase, phospholipase A (Pu), phosphodiesterase (PDE) ,number of mice used.

and arginine esterase (AE), tests for chemical reversion, i.e.

CalCulated i.m.LD5｡=47 Ug/0･ l mL (i･e･, 2･35 Ug/gm body weight)

free formalin released, toxicity, and immunogenicity.

Just before every expenment, each sample of lyophilized

Table 2. Comparison of pH, protein contentand the activityof various
enzymes present in the liquid and lyophilized toxoids

toxoid was reconstituted with only 0.65 mJ of distilled water

to compensate for the loss of proteins duringthe process of

Li qui d Lyophi I ized

lyophilization during which 35% of proteinsin the liquid RVV

pH

were fわund to be lost.

Protein (mg/mL)

Protein determination and the assay of enzyme activity:

Ptoteinase (EU/mg protein)

The protein content of each form of stored toxoid was measured

by the method of Miller (16). Proteinase activity was deter‑
mined at pH 8.5 using casein as a substrate by the method of

Takahashi and Ohsaka (17). PLA activity was measured
according to the method described by Marinetti uslng Phos‑

76.̲ 5.1 0.打打

7
紀鴎̲ 6̲ 63

6.64

12.3

PLA (EU/mg protein)ll.34

PDE (EU/mg protein)

97.9

AE (EU/mg protein)

23.64

EU=Enzyme Unit

phatidylcholine (egg yolk lecithin) as a substrate (18). PDE
activity was assayed at pH 8.9 according to Kocholaty et al.

Table 2. No apparent differences in pH, protein content, andAE assay was ca汀ied out by the method of Tu et al. uslng

the activityof enzymes were observed between the liquidand
lyophilized forms of the toxoids. Therefore,the comparison of

Na‑p‑Tosy1‑I‑Arginine Methyl Ester CTAME) as a substrate

the potency of liquid and lyophilized toxoids in our study(20).

was found to be valid.

Using his‑p一mitrophenyl phosphate as a substrate (19). The

Test for chemical I℃Vel苫ion: Determinations of free formal‑

Figure 1 shows the changes in the protein content and thedehyde released from four different samples of stored RVV
toxoid were carried out by the method of Greenfield et al. atactivity of various enzymes present in liquidand lyophilized
forms of the RVV toxoid stored at 4oCand at RT measured at
3‑month intervals for 1 year. There was no apparent change

3‑month inteⅣals (21).

Toxicity test (safety test): One milliliter of each toxoid

in the protein content in either fbm of血e toxoid upon storage,(containing 5 mg venom protein, i.e. more than60 LD5.) stored

especially in toxoids stored at 4oC. However, the enzyme

at different temperatures was injected intramuscularly in an

activitywas found generally to gmdually increase withincreases

undiluted fbm to each test group. The groups consisted offive mice, and deaths within 24 h were noted. Toxoidsthat

in the time stored. The changes were more apparent in theliquid toxoid than in the lyophilized toxoid, and also in those

did not causeany death within 24 h were regarded as safe

samples stored at RT compared to those stored at 4oC･ Marked

and used fわr immunization (14).

changes in the enzyme activity were usually fわund at 6 months

Immunogemicity test: The immunogenicity of the toxoids
was tested according to Sawaiand Kawamura (22). Dilutedof storage and onwards.

(1 :9 withM/30 PBS) 0.I‑ml samples of the toxoids (contaimingfrom liquid and lyophilized toxoids stored at 4oCand at RT for

Fi gure 2 demonstrates the amount offree formalin released

O･05 mg of venom protein) Were injected subcutaneously to

l year. There was little change in the amount of free formalineach test group (five mice per group), followed by threereleased from both toxoids stored at 4oC. Although a sigmiflCantamount offree formalin was released from liquid toxoid stored

booster injections of the same dosage at the 3rd, 4th,and 5th

weeks. One week afterthe last booster dose of toxoid, all mice
were sacrificed, serum samples were collected, and circulating

at RT at 6 months of storageand onwards, only a small amountvenom antibodies were detected by the radioimmunoassay

at the same temperature.

of free formalin was released from lyophilized toxoid stored

Data from the safety test of the various stored toxoids are(RIA) method (23).

glVen in Table 3. Only some of those mice injected with liquid
toxoid stored at RT for 3 months and onwards died within 24

RE S tJIJTS

h after injection. These findings indicate that the liquid toxoid

Before prepanng the RVV toxid, the i.m. LD5. 0f RVV

stored at f汀becomes toxic at 3 months of storageand onwards,

used in the expenment was determined. It was found that

whereas liquid toxoid stored at 4oCand the lyophilized toxoids

LD50 and the minimum lethal dose (LDl00) of RVV in thestored at both 40C and RT did not show any toxicity fわr up to

mice were 2.35 and 4.7 Ug/gm body weight (i.e. 47 and 94
Ug/20 g mice), respectively, as calculated by the S. Karber

1 year of storage.

method (Table 1).

mice immunized with liquid and lyophilized toxoids stored

Figure 3 illustrates the serum venom antibody levels in

A comparison of the bi∝hemiCalcharacteriStics of the liquid

at 4oCand at RT. Mice immunized with lyophilized toxoids

and lyophilized toxoids (主ust after reconstitution) is shown instored at both 4oC and RT, respectively, showed a slgnificant

235

200 proTein

10

Table 3. Safetytest of the various stored toxoids on immunized

40C

expenmentalmice

Deathofmice within 24 h

碑二二二Monthsofstorage

Liquid*4oC

Lyophilized*RT

0 0 0 0 0/ / / / / 5 5 5 5 5

0 3 ′0 9 2

0 2 1 1 1 / / / / /5 5 5 5 54oα α α α α5 5 5 5 5CRT

0 0 0 0 0/ / / / / 5 5 5 5 5A)T 3t!auJ(ZaputZ一ua)O3u!9701dtZ!)tJ!Oagqu931d

1

Numerator indicates number of deathsand denominator indicates

number ofmiccused.
*Dose of toxoid administered=l mL (containing morethan60 LD50
of RVV)advantage over liquid products in that they can be stored atordinary RT for a long period without deterioration (24). In

this study, lyophilized RVV toxoid was developed from crude
RVV by detoxification and polymerization of venom proteinsinduced by formaldehyde. As a result, the RVV formed
methylene bridges with amino acid side chains to fb‑ large
molecular weight protein complexes. These polymerized large

molecules are non‑toxic but immunogenic and canbe used asa snake venom toxoid (25). Therefore, the stabilityof a toxoid
totally depends on the stability of the formaldehyde linkages
fbmed in the toxoid molecule. If there is a dissociation ofthese formaldehyde linkages free formaldehyde will be
released from the polymer and can be detected in the toxoids.

In the present study, we examined the effects of different

0

3

6

9

12

storage temperatures (4oCand RT)and different storage forms(liquid and lyophilized) on the stabilityof the RVV toxoid.
Regarding the storage temperature, both liquidand lyophilized
0

3

6

9

12

toxoids stored at 40C showed little changes in protein content,Months of storagea reduced increase in enzyme activity, minimal chemical

Fig･ 1･ Changes in the protein contentand the activity of various
enzymes in liquid and lyophilized toxoids during storage at 4oCand
RTfor I year.
●●‑.Liquid

0‑‑〇一→ Lyophilized

OuJ的TJ)SP^9tApOq!tLn!^ qunlaS

rise in serumantibody levels compared to those induced byimmunization with liquid toxoids at co汀eSpOnding inteⅣals

throughout the 1‑year storage period. In addition, a more
slgmificantrise inantibody levels was found in mice immu一山zed with the lyophilized toxoid stored at 40C than with thesame toxoid stored at RT.

DIS CUSS ION
It is well known that freeze‑dried biologlCals have anTt! )!u!JOa qu931aJ
SuO!)t!17u93t103
0

3

6

9

J2

Months of storage

Fig. 3･ Serum RVVantibody levels inmiCe imJnumized with RVV
toxoids; 1iquidand lyophilized forms stored at 4oCand Rr for 1
year･
0

3

6

9

12

0

3

6

9

12

Each value represents mean± SEm (n=5)

Statistical evalutions wereperformed between the antibody levels
induced bythe lyophilized toxoids and those of the respective liquidMonths of storage
Fig･ 2･ Changes intheamounts of free formalin released from liquid

toxoids, using the Student'Sunpaired "I" test.

and lyophilized toxoids during storage at 4oCand RT for 1 year･

NS means not significant at the 5 % probabilitylevel･

一･一一Liquid

一一一HLiquid

8‑14‑‑ Lyophilized236‑.一一一Lyophilizedreversion, no toxicity, and greater immunogenic properties(1992): Reversion of formaldehyde linkage in Russell's

compared to the respective toxoids stored at RT throughout

vIPer Venom tOXOid on storage. Snake, 24, 147‑150･

the studyperid of 1 year. Wlth storage at higher temperatures,
there was an apparent chemical reversion, with a release of

7. Kyaw, K. P. P., Maung, K. M., Myint,A･ A･, Kyaw, A･and Pe, H. (1994): Effect of storage temperature on thefree formalin detected at 3 months of storage and onwards,

stability of Russell's vlper Venom tOXOid, p･ 59･ Abstr･

indicating the release of more enzyme molecules that had

Myan. Health Res. Congr., 12 to 19 December, DMR,

been previously linked in the molecular mass of the toxoid,

Yan 甚on.

resulting ln increased enzyme activity. This increase may also

8. Che汀y (1996): Study on stability of purified Russell's

have been due to the autolysIS Of proteins stored at highviper venom toxoid on storage. M.Sc･ (Zoology) Thesis･

temperatures for a long period. At 4oC, there was lower

Univ. Yangon.

incidence of formaldehyde‑linkage reversion in both forms

9. Aye, S., Kyaw, A. and Su, K. W. (1997): Effectivenessof the toxoid, resulting inthe stable polymerized molecule tx:ingof sodium bisulfite om the stability of Russell's vlper

retained with persistent antigenic properties and no toxicity.

(Daboia russelli siamensis) venom toxoid under various

Therefore, there was a greater immunogenic response withconditions. Myan. Health S°i. Res. ∫., 9, 70‑73.

no lethalityin mice injected with either toxoid stored at low

10. Fukuyama, T. and Sawai, Y (1975): Study on reversion

temperature.

of toxicity of cobra venom toxoid. Snake, 7, 91‑94.

Regardingthe storage forms of the toxoid, 1yophilized toxoid

ll. Sawai, Y.and Fukuyama, T. (1978): Study on Taiwan
cobra venom toxoid, p. 91‑101. In Rosenberg, P･ (ed･),Tbxins: Animal, plant and microbial. Pergamon Press,

stored at both 40C and RT was also found to have a lower

incidence of fbmaldehyde‑linkage reversion. As a result, the

New York.

protein content was more stable, as was the enzyme activity,
and the toxoid therefore remained safe and elicited a stronger

12. Sadahiro, S., Rondo, S., Ohsaka,A., Fukushima, H. and
Murata, R. ( 1 978) : Standardization of Habu (TrimeTleSuruS

immune response, Compared to the respective liquid toxoids,

stored at both conditions, over a period of I year. Therefore,jlavoviridis) snake‑venom toxoid. Toxicon, 1 6, 2751282･

it is apparent that the lyophilized toxoid is more potent and13. Karber, S. (1981): Calculation of LD5.. Progress in the

more stable than the corresponding liquid toxoids at any

characterization of venoms and standardization ofstorage temperature･ Althoughthe lyophilized toxoids stored

antivenoms. WHO offset publication, 58, 23･

at 4oCand at RT showed little difference in potency, safety,

14. Rondo, S., Sadahiro, S., Yamauchi, K., Rondo, H. andand immunogenicity, the stability of the lyophilized toxoid

Murata, 良. (1971): Preparation of standardization of

stored at 40C was slightly superior to that stored at RT, at

toxoid from the venom of Trimeresurus jlavoviridis

which temperature there was slight chemical reversion with

(Habu). Jpn. ∫. Med. S°i. Bio1., 24, 281‑194･

15. Khin, M.A. (1980): The problem of snake bites in Burma.

an appearance of free formaldehyde at 6 months of storageand onwards. Hence, the most potent formof the stored toxoid

Snake, 12, 125‑127.

16. Miller, G. L. (1959): Protein determination for large

possesslng nO toxicity and high immunogenicity was thelyophilized toxoid stored at 4oC. This was followed bynumber of sample. Anal. Chem., 31, 964.
17. Takahashi, T. and Ohsaka, A. (1970): Purification andlyophilized toxoid stored at RTand liquid toxoid stored at
4oC･ The least potentand the most dangeroustoxoid was liquidcharacterization of a proteinase in the venom of r.

toxoid stored at RT.

jiavoviridis. Complete separation of the enzyme from

In conclusion, it is evident that lyophilized toxoids storedhemo汀hagic activity. Biochem. Biophys･ Acta, 198, 293‑

at 4.C and at RT are totally safe (i.e. non‑toxic) and are also

307.

18. Marinetti, G. V. (1965): The action of phospholipase A

potent enough to induce an immunoprophylactic antibody

response in expenmentalmice. In addition, the potency of theon lipoproteins. Biochem. Biophys. Acta, 98, 554‑565･

lyophilized toxoid is apparently stable for 1 year, even when

19. Kocholaty, W. F.,Ledford, E. B., Daly, J. G.and Billings,

stored at RT･ Therefore, the prophylactic use of lyophilized

T. A. (1971): Toxicity and some enzymatic properties

toxoid for humanimmunization in theruralareas of Myanmar,
where people are atrisk of snake bite and proper cold storage

and activities in the venoms of Crotalidae, Elapidae andViperidae. Toxicon, 9, 131‑138.
20. Tu,A. T., Cheu,A.and James, G. P. (1966): Proteolytic

facilities are lacking, could be considered in the near future.

enzyme activities in a variety of snake venom. Toxicol.

Appl. Phamacol･, 8, 215･

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237