USARMY MEDICAl, RESEARCH LABORATORYFORT KNOX, KENTUCKY 40121

REPORT NO. 744

IMMUNIZATION STUDIES WITH NAJA NAJAVENOM DETOXIFIED BY PHOTOOXIDATION

(Final Report)by

Walter F. Kocholaty, Ph.D.Edith B. Ledford, A.B.

Thoma A. Billings, B.S.Joyce C. Goetz, B.A.

00 and

BillyD. Ashley, M. S.

30 June 1967

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UNITED STATES ARMYMEDICAL RESEARCH AND DEVELOPMENT COMMAUD

OCT 2 1967:1 UUJ ,L, ,u U L ,

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i q 1 erDeatroy this report when no longer needed, Do not return it to

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In conducting the research described in this report, the investi-gatore adhered to the "Guide for Labcratory Animal Facilities and Care, 'as promulgated by tht Committee on the Guide for Laboratory Animal,Resources, National Academy of Sciences. National Research Council,

t'

AD

REPORT NO, 744

IMMUNIZATION STUDIES WITH NAJA NAJAVENOM DETOXIFIED BY PHOTOAOXMXION

* (Final Report)

byWalter F, Kocholaty, Ph.D.

Edith B. Lzjdford, A.B,Thomas A. Billings, BS.

Joyce C. Goetz, B.A,and

Billy D. Ashley, M.S,

Biochemistrv DivisionUS ARMY MEDICAL RE3EARCH LABORATORY

Fort Knox, Kentucky 40121

30 June 1967

Production of Polyvalent AntiveninsWork Unit No. 149

In-House Laboratory Independent ResearchTask No. 00In-House Laboratory Independent Research

DA Project No. 3A01 3001A91C

Distribution of this document is unlimited,

USAMRL Report No, 744DA Project No, 3A013001A91C

ABSTRACT

IMMUNIZATION STUDIES WITH NAJA NAJAVENOM DETOXIFIED BY PHOTOOXIDATION

OBJECTIVE

The object was to detoxify cobra venom by photooxidatlon in sucha way that the immunogenic properties of this venom were preserved.

RESULTS

Photooxidation of cobra venom by visible light in the presence ofmethylene blue resulted in a relatively slow detoxification of the ven-om. The detoxified venom when injected in rabbits caused a i ary weakimmunogenic response.

CONCLUSION

The phenomenon of photooxidation in the detoxification of venomsappears to be generally applicable; however, the conditions for the im-m'unogenic response may vary from venom to venom.

IMMUNIZATION STUDIES WITH NAJA NAJAVENOM DETOXIFIED BY PHOTOOXIDATION

INTRODUCTION

Detoxification of snake venonti by photooxidation with visible lightin the presence of methylene blue was demonstrated first on Crotalusatrox venom (I) and extended to other venoms (2). Three of the photo-

oxidized venoms tested for their immunogenic properties gave rise inrabbits to antibodies which protected mice from the toxic effects of thesevenoms. In an attempt to apply similar techniques to Naja naja venomsome differences and difficulties were encountered which necessitatedmore detailed investigations reported herewith,

EXPERIMENTAL PROCEDURE

The venom was collected at the snake colony of this laboratoryunder ice, quick-frozen and lyophilized, The pooled collections wereground and sieved as described previously (1,2) and stored at 4'C ina desiccator in the dark, The protein content of the centrifuged and fil-tered (Millipore Filter of 0.45 gm pore size) venom solution was 82.5%(biuret). Phompholipase A activity was determined as previously de-scribe~d (3). No proteolytic activity against casein could be demonstratedand e~terase activity against TAME (p-toluene sulfonyl-L-arginine meth-yl ester) was absent. Photooxidation was carried out at pH 4.0 and 8.5.Toxicity and qualitative precipitin tests were conducted a& described

previously (2). The LD 5 0 for this particular batch of venom was 7.5 yI.V. and 8 y I. P. (20-25 g mouse).

RESULTS

Effect of Photooxidation on Loss of Toxicity and Phospholipase AActivity. Table I summarizes the rapid loss of phospholipase A activ-ity and concomitant loss in venom toxicity with increasing 02 uptake and

time of photooxidation.

Immunization of Rabbits and Mice with Photooxidized Venom ofNa naja. Venom ofN, na, was photooxidized to .n uptake of 20 jd02/mg protein usitig the reaction mixture and conditions described inTable I and stored in ice and in the dark. Prior to injections the photo-oxidized venom was diluted with physiological saline and mixed with anequal amuunt of Algivant (Colab Laboratories, Inc.). The weekly

immunization echedule for rabbits and mice s identical. Each animalreceived three injections a week (Monday, Wednesday, Friday), mice for3 and rabbits for 4 weeks consecutively. The first two doses for mice(mature white Swiss, 30 to 40 g) were 32y of photooxidized venom andthe rest 64 y adirlnistered subcutaneously in a volume of 0. 1 ml for atot of 512Y.

Rabbits (white New Zealand) weighing 2-3 kg, received I mg ven.0M pxotein in 1. 0 ml fox the first two injections and 2 nig in 1. 6 ml forall subsequent injections for a total of 22 mg per anitzoa. Injectionswere administered intramuscularly and alternated between the two hindlegs. After a rest period of I week, mice were challenged with unal-tered venom and rabbits were bled to death by heart puncture.

Neutralization and Protection Studies. When the mice which hadbeen immunized with the photooxidized N. naja venom were challengedintravenously with increasing amounts o unaltered venom, all micedied within 24 hours ever with the lowest dose (2 LD 5 0 ) employed.

Of the 10 rabbits (23-31, 33) immunized with photooxidized venom(Table 2), four did not show a positive (qualitative) percipitin test withthe highest concentration of venom used, and one was positive only tothe fourth dilution. The rest werc weakly positive up to the sixth dilu-tion.

The sera of the rabbits showing the highest (#23, 24, 25, 31, 33= "A") and the lowest (#26-30 = "B") titer were combined and their y-globulins isolated separately (4).

The neutralization and protection studies were carried out on miceusing the Y-globulin obtained from rabbits of group A. As shown inTable 3, the globulins were practically inert in the neutralization aswell as in the protection studies.

Since the results of the neutralization and protection studies weiedisappointing, five more rabbits were immunized with twice the amountof photooxidized venom for a total of 44 mg using the same adjuvant andan Adentical immunization and bleeding schedule as before. No lesionsof significance were recognized in the tissues of all animals injectedwith N. naia venom. This information was furnished by Colonel M. A.Ross, VC, formerly Director, Pathology Division, USAMRL. One rab-bit died during this period from causes not related to the immunizationprocedure.

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All the rabbits (#3Z. 3A. 37. 3 - "C") immunined with the hlghtramounts (44 rng total) of photooxidized venom responded with a positivethough somewhat erratic precipitin test (Table Z). The v-globulinswere lolated as described before. The results shown in Table 3 arealmost identical with the ones obtained in the prev,01ir experiment, whereonly one half as much photooxidized venom had beeii used in the immr-u-

aauon schedule.

Photooxidatiun of Naja raja Voenom at Acidic pH and at Low Temp.erature. The comparatively low protection obtained with the rabbitimmune globulins prompted a more detatled investigation into the va-louis paramctcrs of the photooxidation mechanism of the particular van-ont. Photooxidation may be carried out in a more selective m.annerutilizing an acidic reaction thus excluding histidine and tyrosine fromphotooxidative destruction (5, 6). Another specific variationmay beintroduced by working at temperatures below 37 (7).

Examinztion of the effect of pH on detoxification by photooxidationof this venom revealed the usual pattern with a pH optimum at about8. 5* and a steep drop toward the acid side, similar to that demonstratedwith Crotalus atrox (1).

Studies of other parameters revealed that apparently the onlyfactor related to the decrease in toxicity is the amount of 02 uptake permg venom protein irrespective of the concentration of the venom, thelength of time of photooxidation, the pH of the reaction mixture (8. 5 or4. 0), and the temperature (37' or 10" C). No significant volumes ofCO 2 were developed during any of the variations in the photooxidationprocedure. A sumrm.ary of these experiments is illustrated in Figure 1.

DISCUSSION

The photooxidation of proteins by visible light in the presence ofmethylene blue permits gradual and subtle alterations of certain aminoacid side-chains, which can Le further modified by carrying out thereaction varying the pH and the temperature. The changes resultingin the molecule affected have provided an insight into functional groupsof enzymes and a method for the detoxification of toxins and venoms(8).The latter has proven useful in providing modified venoms capable of

At pH 9. 0 and a concentration of 10 mg venom protein per ml a pre-cipitate forms very rapidly in the initial stages of photooxidation.

3

slciting antibodies in experimental animals with simultaneous abolish-ment of the necrogeilc and h imolytic properties.

Why Immunisation with the photooxidized venom failed to producean irnmunoglobulln capable of protecting mice is not understood at thepresent time. Attempts to vary the conditions for photooxidation (Fig. 1)revealed nothing of relevance to conduct photooxidation hi -A more prom-iing way, or to indicate abnormnalitici in the chemical nature of thisvenrsn.

One may, however, speculate on two facets which stand ouL bycontrast and, although not explainable at thi time, maiy haveL a bearingon the failure to produce an immunoglobulin.

One, i the observation that the photooxidative detoxification ofNaja maja venom proceeds at a considerably slower rate and with a highO consumption compared with the other venoms investigated. Fromtha& one might suspect a unique arrangement of the fo'ir amino acidssusceptible to photooxidation. The isolation of cobrotoxin, one of #' etoxic proteins from Formosan cobra venom, was reported recently byYang (9). The amino acid composition of this compound has not yetbeen reported. Its molecular weight is 11, 000. In this connection it isof interest to compare the rapid loss of phospholipase A activity with

the very slow decrease in toxdcity, a fact that would tend to indicatethat this enzyme is, at least in its native form, a minor factor in thetoxicity of this venom.

Two, Is the observation of De Vries and his group (10) that theVipers palestinae neurotoxin by itself is weakly antigenic, but when com-bined with carboxymethyl cellulose (CMC) the immunogenic propertiesare enhanced to such a degree that minute amounts of carboxymethylcellulose-bound neurotoxin elicit a high antibody response. The Viperapalestinae neurotoxin recenLly isolated by these workers (11) has a lowmolecular weight (1I, 6 ')0) similar to that of the cobrotoxin of Yang.

Do Vries and co-workers conclude that if a low molecular weightcharacterizes venom neurotoxins the increase in immunogenicity ob-tained by binding to carboxymethyl cellulose "may be a reflection of theincrease in molecular weight of the antigen."

SUMMARY

Photooxidation of cobra venom by visible light in the presence ofmethylene blue resulted in a relatively slow detoxification of the venom.

4

The y-globulin Isolated trnrr rabbit. i mmunizad .-,tih ihe detoxified 'en-

orn gave poor protection in the animal experiments. Possible reasons

for the weak response of the immune aurum are discussed.

LITERATURE CITED

1. Ru ohulaty, W. Detoxification of Crotalus atrox venom by

photooxidation in the present of methylene blue. USAMRL

Report No. 628, 1965 (DDC AD No, 4(6948); Taxicon, 3: 175,1966,

1 I'toehuaty. W. and 13. D. Ashley. l)etoxification of the ven-

oms of the Russell's viper (Vipers ruoselli) and the water

moccasin (Agkistrodon piscivorus) by photooxidation, USA-MR1, Report No, 629, 1965 (DDC AD No, 466949); Toxicon,

3: 187, 1966.

3, Kocholaty, W, A rapid and simple phospholipase A assay.

USAMRL Report No, 65Z. 1965 (DDC AD No, 485846): Toxi-con, 4: 1, 1966.

4. Campbell. D, H. , J, S. Garvey, N. E. Crerner, and D. H.

Sussdorf, Methods in Immunology New York: W, A,

Benjamin, Inc,, 196j.

5. Sluyterman, L. A. AE. Photooxidation sensitized by pro-

flavine of a number of protein constituents. Biochini. Bio-

phys. Acta, 60: 557, 1962.

6. DasGupta, B. R. and D, A. Boroff. Incr.ased selectivity

of photooxidation for amino acid residues. Biochim. Biophys.

Acta, 97: 157, 1965.

7, Weil, L. On the mechanism of the photooxidation of aminoacids sensitized by methylene blue. Arch. Biochem. Biophys,

110: 57, 1965.

8. Spikes, J. D. and B, W. Glad. Photodynamic action, Photo-

chem. & Photobiol, 3: 471, 1964.

9. Yang, C, C. Crystallization and properties of Cohrotoxin

from Formosan cobra venom. I. Biol. Chem. 240: 1616,1965.

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IC. Morox. C., N, Goldblurn, and A, De Vries. Preparation ofVipers paestine antineurotoxin using carboxymethyl-cellu-lose-bound neurotoxin as antigen. Nature, 200: 697, 1963,

It. Moroz, C. , A. De Vries, and M. Sela. Isolation and charac-terization of a neurotoxin from Viper palestinae venom, 13io-

k ch"i. Biophys. Act&, 14_4: 136, 1966.

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TABLE 1

EFFECT OF PHOTOOXIDATION ON LOSS OF TOXICITYAND PHOSPHOLIPASE A ACTIVITY

% loas inphospholipase A 41 O uptake/ Time of LD 5 0

activity mg venom protein photooxidation tolerated(min) (20 g mouse)

69 5 10 2

93 o 29 4

100 20 l11 8

- 27 258 16

Reaction mixture: 5 mg venom protein and 0. mg methylene blue in0. 1 M TRIS buffer, pH 8. 5: total volume 2 ml; t = 37"C. No measur-able CO Z production w. , observed during the time of photooxidation.

7

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TABLE 2

RESULTS OF THE QUALITATIVE PRECIPITIN TESTSOF SERA OF RABBITS IMMUNIZED WITHPHOTOOXIDIZED VENOM OF NAJA NAJA

Total amount Tube No.' "

of photooxidizedvenon. injected Rabbit No. 1 2 3 4 5 6 7 8 9

23, 24, 25 + + + + + + - -2 mg 26 + + + +-

27, 28, 29, 3031, 33 + + + + + + - - .

32 - - + + + - -

44 mg 34 + - + + - - - -

37 + - + - +

38 + : * + + +- *-

First tube contained 10 rmg venom protein in 0.5 ml "dilute saline-borate buffer," pH 8.4 (4). All subsequent dilutions were 1:5. Toeach tube, two drops of serum were added. After 60 minutes at 37"C, the tubes were placed in the cold and readings were taken 24 hourslater. Controls contained either saline and serum or saline and ven-om (10 mg) in amounts stated above. Both controls were negative.

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OCCUMIWY CONTROI DATA - N 0&0060w460405 of No. "Ewelm! 1101 WAh sp 01tDn mut bo sipRf N a* #P"& ft*&H It es *M*ldj

IMMNIZTIO STDIE WIH NJANAJA VE:NOM DETOXIFIrD BY PHOTO-

Walter F. Kocholaty. Edith B, Ledford. Thomas A. Billings. Joyce~ C, ("setv-and Billy D. Ashley

a Maros?Ot 04#. 1& Y AL NO. or PooI co. O parUs

30 June 1967 10 I Ic r4 S *Z. My I. a. so. 04101HAYOCS1 1119001 oNUlasftiso

& 048ac Io. 3A0 13001 A91 C74

4*Tauk No.00 a. OT NISNPORY 000181 (APWJ ows moo Not Ry 64.@*1~

'Work Unit No. 14910. 0O4TROUVION 5VAYRUaft?

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18. AISYNAST

Photooxidation of cobea venom by visible light in the presence of rnethyleneblue resulted in a relatively slow detoxification of the venom. The y-globutinisolated from rabbits immunized with the detoxified venomn gave poor protectionin the animal experimenta. Possible reasons for the weak response of the im-mune serum are discussed. (U)

DDI Rev .14 473 :2:P : [JNC1?*g DN 54 W1EU

UNCLASS1FIED

"IV*It#ON A &ifWa 0 W C. . . ... . . .. . ..... .-. - - . - -,

VenomnsImmune Serumns

ImmunizationToxoidToxicology

AG 1869-1-Army-Knox-Oct 67-90 UNCLASSIFIED

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