Sea wasp toxoid: An immunizing agent against the venom of the box jellyfish, Chironex fleckeri

roxkar, 1975, Vol. 13, pp. 423-435. Pett;amon Prew Printed in Great Brltato.

SEA WASP TOXOID : AN IMMUNIZING AGENT AGAINST THE VENOM OF THE BOX JELLYFISH, CHIRONEX FLECKERI

E. H. BAxTaIt and A. G. M. MAxR ~ Commonwealth Serum Laboratories, Parkville, Victoria, 3052, Australia (Acceptedfor publication 10 June 1975) E. H. Bxxz~e and A. G. M. Mnax . Sea wasp toxoid : an immunizing agent against the venom of tho boxjellyfish, Chlronex fleckeri . Toxicon 13, 423-435, 1975 .-A method of preparing a toxoid from the venom of the sea wasp or box jellyfish (Chironex Jleckerl) is described. After adsorption on aluminium phosphate adjuvant the toxoid provides a vaccine which may be used for immunization of laboratory animals against the lethal action of the venom. The course of antibody dovelopment in the immunized animalsand the effect of booster doses after various intervals has been studied. It is concluded that the toxoid will provoke an adequate immunity level in animals, and the possibility of relating this to its future use in humans is discussed. INTRODUCTION

ExcovtvT~t in the tropical waters of northern Australia with a large specimen of the box jellyfish, commonly known as the sea wasp (Chironex,~feckeri), may have fatal consequences. Over sixty documented fatalities (CLBLAND and SOUTHCOTT, 1965) testify to this view, while sublethal envenomations cause disfiguring scarring. Knowledge of the very dramatic fatalities (death ensuing within a few min of stinging), deters much enjoyment and indeed use of Australian tropical beaches during the summer season. Availability of a specific antivenene, developed in these laboratories (BAXTER et al., 1973) has modified this situation, but mainly in respect to sublethal envenomations, as the extremely rapid action of the venom frequently causes death before antivenene can be injected. Active immunization is the obvious solution to the problem, and it would be expected that an immunization routine designed to maintain high circulating antibody levels would be the method of choice. Unlike the state of active immunity to bacterial infections, in which the immune system, primed by immunization, has time to produce antibody while the infection is in progress, an envenomated victim is presented with a lethal (often multilethal) challenge immediately, and adequate circulating antibody to neutralize the toxin appears essential. We report here the development of a sea wasp toxoid effective as an immunizing agent against multilethal challenge in laboratory animals. Its possible application to humans and, in adequately immunized animals, the non-correlation of circulating antibody level with resistance to lethal challenge are discussed. AN

MATERIALS AND METHODS herrons Venom of Chironex }leckeri was collected by the milking technique (BexIVFS, 1967) and supplied in the frozen state by Dr . J. H. Barnes of Cairns, Queensland. All venom was stored at -15°C until required and immediately prior to use was thawed and diluted in cold physiological saline containing 1 ~ Evens peptone. ~Deoeased . roxrcoN ~9~s vor.

r3

423

424

L. H. BAXTER and A. G.

M.

MARK

Venom units

The venom of Chirorrex }leckeri is heat-labile (B~xrex and Mien, 1969). Venom aliquots, although stored in the frozen state, often show a two-fold variation in lethality, and decline in lethality when stored for some montés . Additionally, as the work reported has extended over several years, a number of venom batches, of varying original potency, have been used . It is thus not possible to define lethal doses quantitatively in terms of ml, pg, etc. Experiment-to-experiment correlation is best achieved, therefore, by using the Lo s o dose for the species under test as the unit, and by confirming this r n, u dose on the aliquot of venom being used for the particular experiment . For the sake of simplicity, LDso control determinations have been omitted from the tables. /xtravenoris lethal doses (i.v . Ln6o)

White mice each weighing 25 g were used . The i.n6o was established by i.v . injection into the caudal vein of groups of mice using an injection volume of 0~2 ml of each of a graded series of dilutions of venom. For range-finding assays, two mice were used with each dilution . For final assays, four mice were used with each dilution . The animals were observed for 24 hr. The tn, o end point was determined by the method of Reen and MUE2VCH (1938). Twice the i,n, o dose was observed to kill all animals tested . Rabbits of a coloured strain or a black and white Dutch strain each weighing about 2~5 kg were used . The Lobo was established by i.v. injection into the marginal ear vein of groups of rabbits using an injection volume of 0~5 ml of each of a graded series of dilutions of venom . Groups of two to five animals were used with each dilution . A preliminary result was obtained after about 1 hr observation, but for the final result the animals were observed for 24 hr. The i n, o end point was determined by the method of REen and MtJSNC~~ (1938) . Twice the i.n, o dose killed all animals tested . Monkeys (Maeaca jascieularis) each weighing about 2 kg were used . The c.n~ o was established by i.v . injection into the saphenous vein of groups of monkeys using an injection volume of 0~5 ml of each of a graded series of dilutions of venom. Groups of two to five animals were used with each dilution . A preliminary result was obtained after about 4 hr observation, but for the final result the animals were observed for 24 hr. The Ln so end point was determined by the method of RsEn and MuaxcH (1938). Twice the Ln, e dose killed all injected monkeys. Mouse, monkey and rabbit challenge doses

Guided by the preliminary in, o determination for the species to be challenged, which was carried out on each occasion of challenge (i .e . the control animals), dilutions of venom were prepared to contain 2, 4, 8, etc. assumed tnbo doses in the injection volume appropriate to the series. Two immunized animals were challenged intravenously at a chosen level, and, guided by the trend of the result, further groups of animals were challenged at higher or lower levels . The true challenge levels were determined in terms of the Lobo dose found in the control animals at the end of the observation period . All challenges of immunized animals were made 'blind', that is, antibody titrations of the blood taken immediately prior to challenge were not carried out until after the challenge. There was thus no bias in selecting high responding animals for high challenges in a group (see Table 8 for a single exception) . Necrotic dose (N.D .)

The necrotic dose of the venom was established by injecting i.d. into the shaved skin of albino guinea pigs (about 350 g each) 0'1 ml volumes of a graded series of dilutions of the venom. The necrotic dose is defined as the minimum amount causing ulceration of the skin greater than 5 mm in diameter, when observed at 72 hr. Preparation ojformol toxofd by the dialysis method

Thawed venom (20,000 mouse tnso/ml) was diluted to 4000 r~, o/ml in saline containing 0~1 ~ casamino acids (acid hydrolysed casein, Difco Laboratories, Detroit, Michigan, U.S.A .) and centrifuged for 60 min. at 4°C to remove turbidity (Spinco ultracentrifuge rotor type 30, average r.c.f. 13,620 g). The supernatant was sterilized by filtration through a 4~7 cm Millipore ® filter, pore size 022 pm (Millipore Corp ., Bedford, Mass ., U.S .A .) . Suitable lengths of dialysis tubing (Visking Dept ., Union Carbide Co., Chicago, Ill., U.S.A .) were made into bags attached to stoppered glass tube fittings . For sttrilization, the bags were filled with 1 formalin and placed in Basks containing 1 ~ formalin . After standing overnight the bags and flasks were rinsed out aseptically with sterile physiological saline, and the sterilized venom solution placed in the dialysing bags . The flasks were filled with sterile dialysing solution (volume equal to 10 times the diluted venom volume) containing 085% (w/v) sodium chloride, 0~1 ~ (w/v) casamino acids (Difco), 0~05~ (v/v) formalin [Merck Formaldehyde Solution (Formol) 35 ~ w/v, guaranteed reagent] and 001 ~ (w/v) thiomersal (Lilly Merthiolate, N.F.) . In batches prior to FT9, peptono was used in lieu of casamino acids as the stabilizing agent. Dialysis was allowed to proceed at room temperature (18-23~ in the dark, and samples were taken at intervals commencing at 28 days to determine residual toxicity (mouse i.v . bubo and necrotic dose) until the residual toxicity was .fudged to be at a satisfactory level. The fittings and bags containing TOXICON 1973 Yol. J3

Sea Wasp Toxoid

42S

toxoid were then removedaseptically from the Basks to another set of sterile Basks containing sterile dialysing solution without formalin . After dialysis in the cold overnight to reduce the residual formalin concentration ten-fold, the toxoid was removed aseptically from the dialysis bags. Formulation of the vaccine

The toxoid was centrifuged for 60 min at 4°C to remove turbidity (Spinco ultracentrifuge rotor type 30, average r.c .f. 13,620 g) and the supernatant sterilized by filtration through a 4~7 cm Millipore® filter, pore size 022 pin. The supernatant toxoid was followed through the filter by ores-f9fth of its volume of an isotonic buffer solution pH 6~7 made by mixing 11 volumes of 001 M Na,HPO, in 014 M NaCI with 9 volumes of 001 M KH,PO, in 014 M NaCI . To prepare an adsorbed preparation of the toxoid the filter was disconnected and sterile freshly prepared A1P0, suspension (10 mg per ml in 0~9 ~ NaCI containing 001 ~ w/v thiomersal and 1 ~ w/v CH,000Na. 3H,0) was slowly added aseptically with gentle mixing. The volume of A1P0, suspension used was equal to two-thirds of the diluted toxoid volume . After standing for 2 days for adsorption, the toxoid was dispensed aseptically in 0'SS ml volumes in glass ampoules. The final preparation contained the equivalent of 2000 mouse rn,° original venom and 4 mg of ALPO, in each ml . To prepare a fluid preparation of the toxoid, wo asepticallyadded to the diluted filtered toxoid two-thirds of its volume of a 1 in 6 dilution of the isotonic phosphate buffer in saline. The toxoid was dispensed aseptically in O~SS ml volumes in glass ampoules . The final preparation contained the equivalent of 2000 mouse LD,° original venom in each ml . Immunization and bleeding of the animals

Animal numbers are identification numbers allotted on issue from the breeding unit among animals to be used for many purposes, and their sequence (or lack of it) has no significance to this report . Arrangement within a group in any table was made to facilitate observation of challenge results. Animals were immunized by subcutaneous injection into the mid-flank. Alternate sides were used for sequential doses. Mice were sacrificed and rabbits and monkeys were test bled . Following severing of axillary vessels of anaesthetized mice, blood was collected quickly with a Pasteur pipette and transferred to a centrifuge tube . Rabbits were bled from an incision in the marginal ear vein which was enclosed in a receiver under gentle vacuum . Ten ml of blood was quickly taken as the test bleed. Seven ml of blood was taken by syringe from the monkey femoral vein. After clotting, the separated sera were centrifuged to remove residual red cells, and inactivated by placing in a water bath at S6°C for ~ hr. The sera, to which thiomersal was added to a final concentration of 001 ~ (w/v), were stored at 4°C. Antibody unit and assay of sera

The antivenene prepared by hyperimmunizing rabbits (Bnxrett et al., 1968) and later established as the reference standard, was assigned the value of 2000 antilethal units per ml. The antilethal unit was defined as that amount of serum capable of neutralizing one mouse i.v . 50 ~ lethal dose (c n, °) of the venom in use at that time. All later preparations of antisera are refereed to this standard . Antilethal assays were carried out employing mixtures of serum and venom at various levels, in comparison with similar mixtures of a working standard antivenene aad venom. Mixtures were incubated for 45 min at room temperature (18-23°C) in the dark . The antilethal titration was performed at a 10 antilethal units per 0~1 ml level, but for sera of lower titre a preparation containing 10 antilethal units per 0~4 ml was used . The test dose of venom contained about 10 mouse r n, ° in 0'1 ml. The injection volumes of the serumvenom mixtures were, respectively, 0~2 ml and O~S ml. Testing was by intravenous injection into white mice of 25 g, using 2 mice pea dosage level. RESULTS

Early immunization experiments in mice, rabbits and monkeys established the fact that circulating antibody levels and some resistance to challenge could be achieved with two doses of 0~5 ml of toxoid each containing the equivalent of 1000 mouse Lobo of original venom, given at a 1 month interval, and challenging 14 days later. Some results for rabbits and monkeys appear in Table 1. A tentative correlation was established for each species between the circulating antibody levels found present by pre~hallenge blood sampling and the number of lethal doses resisted under challenge, using data derived from several preliminary experiments (unpublished) giving two immunizing doses at a 1 month interval . This was to provide guidelines when monitoring antibody responses and avoid the necessity of providing groups of TOSICON 1975 Vol. 13

426

E. H.

BAXTER and

A. G. M.

MARR

animals for frequent challenge episodes throughout the course of immunizations. It was difficult to find a correlation in mice as there seemed to be an upper level of resistance of 2~ lethal doses despite achieving very high circulating antibody levels . The levels found, and used for rabbits and monkeys are shown in Table 2. Monitoring of antibody levels revealed that these fell rapidly, and after 3 months were below what would be expected to be protective levels in terms of Table 2. Vaccine containing double the amount of antigen provided no advantage. As booster doses appeared essential, a regime of two basic doses followed after an interval of 3 months by two booster doses at 1 month's interval was investigated . Six animals were immunized in a similar manner with an adsorbed vaccine prepared from antigen which had been fully converted to toxoid (Toxoid FI'S~. At the same time, four animals (rabbits 185, 186, 190, 191) were given a single booster dose 6 weeks after basic immunization (Table 3). There appeared to be no advantage in using two closely spaced booster doses rather than a single one, and after 2 months titres declined to below what would appear to be protective levels in terms of Table 2. TABLE

1.

ANTIBODY TITRES AND LETHAL CHAS!F_NGE RESi3rANCE AFTER BASIC IMMUNIZATION OF RAHHrIS AND MONKEYS WITH SEA WASP TOXOID MSmal ~o .

~

I

14 Aay tit=n

~

IInite/u1

-

Hebbit

i

Cha;lenge . .- Level

-~

j

179 180 181

420 500 233G

182 183 1ßa

233G a2D 717n

j I j

4LD50 4LD 4II`5~

I ~

~50 5D ~50

ReNlt

0 _ ..

D

Monkey 715 AG 11B ü3

5b0 233G 117U SBU ~asn 2520

ue

ü9

a~50 a~50 0~50 ß~50

I. L D D

16LD5 0

Two doses of toxoid, each of 0~5 ml, were given subcutaneously 1 month apart . Adsorbed toxoid (Batch No . FTS) containing in the toxoid used for its formulation a residual lethal titre equal to 2 ~ of the original titre was used in these studies. Challenge carried out at time of bleeding. LD bo represents the LDaD dose for the species challenged, determined in controls concurrently (see text). L = lived. D = died . TABLE

2.

CORRELATION LEVELS OF ANTIBODY TITRES AND LETHAL CHALLENGE RESL4rED IN MONKEYS AND RABBITS IMMUNIZED WITH SEA WASP TOXOII) rarr :ecns tmrrs,"7W nsçulxrv rrr

I

R

I

200

~

a00

I

7r .n

11n0

775n

7aJn

'Lethal doses are intravenous lethal doses for species-1 monkey LDsD 10 mouse LDy D . Animals of both species approximately 2 kg weight . TOXICON 1975 Vo( . 13

7~0

= 100 mouse

LD, o .

1

rabbit LD =

42 7

Sea Wasp Toxoid

TASi .e 3. ANTiHODY 1tE4PONSE IN RAHHTIS AND MONK&YS AFl'ER 1 AND 2 sooeren DosFS or seA wASe ~roxoro 'toxoid öntch 1:0 .

An1an1 No .

14 dey titre

OreI booeter titr" Unit"/ml Unit"/ml

_aaSbit Pf5

195 186 1:0 191

1170 1630 1168 233G

I

j

I

.: .~ . N.D . re .D . a.D . N.D " N.D . N.D . N.D . N.D . I : .D . ::.D .

I 1 1 I

1G33 816 1152 all"

I

3.5 Sb-the

I

u

A3 A5 A6 AB A10 A13 A20

I

ln7 638 580 2336 ]170 1824 580 3600

_Aabbitl FfSP

1"t 2ad Unit"/nl

1

2

3

43 44 45 46 47 4B,

228 114 456 63B <57 , 319

~

I I

408 32G4 4608 2304

4 HoaLtia

5

70 200 840 280

" 7 '

B

"

~ N.D . i N.D . i .D . 1G24 2554 2554 3658 1 2554 5108 7296 2554 i

912 400 710 426 140 150 304 70I 75 1°24 608 1824 426 2554 ~ 304 1824 200! 912 14C! 5108 56U1 7296 400 2554 . ~ 29n1 I i

lsc 210 114 220 420 63= 157 319

eoD 1200 1680 912 1200 Sd00 11680 ~ 1(,80

304 ! 213 040 1200 852 I 2432 24]2 I 121fi I I . 1

~ 2'J 38 7U 38 3B 3B

38 140 280 100

'

i j

N.G . N.D, : : .D . 27 114 46E 628 38 114 114 114

xonkey

Prs

Titre" after eoo"t"r Do"e(") Unlt~/ml

16%k"

i

lö7 188 189 313 314 315 316 317 318 319 320

Aesponce to booeter doee(")

~

I

70 400 100 560 280 230 2(M 290 28U 1216 140 840

~

I

lnn

35 ~ 426 I A2G ~ 304' 052 4261 ~ 852 1 1 I

I

38 107 53 53 210 53

210 119 113

214 53 76 38 38 106 214 4zu 3e 38 75 175 75 400 ~ 75 I 38

I I

1 ~ 1 i '

I

I ' I

I

75 280 75 75 1+G 75

I I 1 I ~

<19 76 27 <19 38 <19 j

Adsorbed toxoid was used : FTS contained 2~of the original lethal titre in toxoid used for the final vaccine while FTSF contained < 0"03 ~. Two doses of 0"5 ml each were given, subcutaneously, 1 month apart for the basic immunization .

'Single booster only . When two booster doses, each 0~5 ml subcutaneously, were given, they were given 1 month apart. N.D . = not determined.

It was decided to determine if repeated boosting of basically immunized animals at 2 monthly intervals would maintain protective antibody levels . Animals were given 3 booster doses, and the antibody levels monitored (Table 4), Responses were good to each booster dose, most animals were consistent in their response and antibody levels appeared to be maintained at levels consistent with the likelihood of resisting a reasonable lethal challenge. This was confirmed by lethal challenge at the end of the experiment. No attempt was made to titrate the resistance level. Several animals resisted challenge although circulating antibody levels were below the limits shown in Table 2. A comparison of the effect of one or two basic immunizing doses on antibody titres and resistance of the animals to challenge was studied (Tables 5 and ~. As could be anticipated, a single immunizing

dose

produced

little

or

no

circulating antibody, whereas two im-

two-dose munizing doses produced titres of similar magnitude to those found in previous immunizations. It is of interest, however, to note that both groups responded similarly to a booster dose .

Resistance

to

challenge was consistently

good,

failure

being boost-titre

related. Toxoid FT10F, a fluid preparation without adjuvant, was lacking in antigenicity" As

it might be anticipated that clinical practice would require a booster dose at the

beginning of each season, after an interval of some months, response to a further booster 710XICON 197J VoJ. I3

42R

E. H . BAXTER and A . d. M . MARR

TABLE 4. EFFECT OF REPEAT BOOSTING AT 2 MONTHLY INTERVALS ON ANTIBODY TITRES OF RABBITS AND MONKEYS IMMUNIZED WITH SEA WASP TOXOID ~n1.ma1 vo .

.wa-

:np~1L

eooet titre

]

Units/ml

Na]ibit I lsc I 190 i 191 +276 +277 +z7s +2p2 +283 313 315 316 318 319 320 +343 +345 "347 , 6Wnkey +A 2 +A 4 ;. 5 A 6

I

+A _ +alt +;.la +A16

140 280 100 420 G00 1?Do

uoo lOJ 214 7, 70 306 214 42r? 214 420 3J0 11V 75 7i 42J 38 4^'~

6

]to

7at S:.~stcr

. .n. __ . .s -- .^eb.ranac .r, 14 date 3 mont:,s r .i"" s "" :_ .~- nr`.:r;~ ! :~. ".,,~~.~. , m : .t! :~ mite/m] Units/nl U.~ :`^,^.L ;;..^. :a,'t. ~ '._~tw :'~" ._  . . . _~_-.-_._ ~_ _--_-r--

~ i 1,. " v ,

~_

1Gilr, G720 631C 33G0 1200 GDis ssG azo 85G 840 840 600 3369 1G8n? 12DC 240U 1G90 ~1~: :GD .'.1G 15 :~

~,

_ LD'__ I : LDSD 5r? I ~

I .^.

~ .

- "D'

'These animals had received double strength vaccine for immunizing doses . Adsorbed toxoid (Batch No . FTS) containing in the toxoid used for its formulation a residual lethal titre equal to 2% of the original titre was used in these studies . Each booster dose was 0'S ml, given subeutaneously . These animals had received various basic immunisation courses and booster doses several months before being included in these studies . Challenge carried out at time of last bleed shown for the animal . LD b p repreSentS the LD4 p dose for the species challenged, determined in controls concurrently (sce text). L = lived . D = died .

dose after periods varying from 4 to 8 months was studied (Table 7). Antibody response was good. Challenged 2 months after a repeated booster dose monkeys resisted well, and rabbits poorly. The rabbit group which had been repeatedly boosted and then rested for 7 months showed reasonable resistance to challenge (Table 8). Rabbit No. 315, which had a history of unusually high antibody titre, resisted a challenge of 16 rabbit LD6o doses of venom . These results provided support for the possibility that, if adequate boosting was practised, resistance to challenge would result . In contrast with the findings of Table 2, which had been based on basic immunizations only, the circulating antibody levels did not correlate with resistance to lethal challenge . Groups of animals were therefore given 1 and 2 dose basic immunizations, and single booster doses, and challenges some months later. ('This would correspond to winter immunization, spring pre-season booster dosing and maintenance of challenge resistance to the end of the season.) A fluid toxoid (FT10F) was also included in the experiment. Results are shown in Table 9. These confirmed the hypothesis that long-term challenge resistance is not residual titre related . There is also evidence that 2 dose basic immunization is more effective in establishing a long-term challenge resistance following booster dosing . A group of animals had been dosed with a single dose of 5 ml of toxoid in the course of safety testing of the vaccines . Opportunity was taken to TOXICON 1975 Vol . 13

Sea Wasp Toxoid

429

TABI,H S . ANTIBODY Tr11tES AND rrrrrr~r. CHALiSNOH RESIS'rANCH AFTER HOOSIER IMMUNIZATION OF MONSEYS BASICALLY IMMUNIZBD WITH A SINGLE DOSE OF SEA WASP TOXOII) Tozold

Nonkay

Tiatch t ;o .

^ry

:.dsot~

FP10A Adsorbed

FT10? P1u1d

ao .

~

I

d2 4J 40 43 4i 60 G8 64 68 67 7y Bl 77 78 7f, 30

~

I I

~ ~

I ~ ~ Î

I

i

! :^1te/ml

Cn1te/ml

21 day poetbcoeter titre ~ llnlte/ml

;3 <38 <~;7 <38 <38

75 63 lOG 1
2000 '.00 2000 2000 :50

B7D50 9L0 16I37 60 16I375 ~ 16LD50

L L L L D

<26 <25 <28 <25 <28

"air, <160 1J !160 <160

!

>10 >10

fl6J 8;0 15u 1900 2400

BLD 6I3)5~ 16LD60 167.u50 ~ 16LD60

L L D L L

<2 i <26 <26 !25 <26 <25

<1J <16^. >10 <]50 >10 !30 <1 : <10

<26 70 850 <26 <15 <26

473780 4IL50 flIU flID50 16LD60 1613)50

D L L D 0 D

21 dey titre

~

Pta-boostez tltte (3 months)

~~~

i >1C :" 1J

~

!

I ~ (

Chellenge Level

Result

Adsorbed or fluid toxoid as shown was used : all preparations contained < 005 % of the original lethal titre in the toxoid used for tho final vaccine . One doso of 1 ml was given, subcutaneously, for the basic immunization . A single booster dose of 0~5 ml was given, subcutaneously. Challenge carried out at time of last bleed shown for the animal . LD represents the LD dose for the species challenged, determined in controls concurrently (see text). L = lived. D = died .

include them in the potency test group, but such a large primary dose would probably be undesirable . Fluid toxoid again appears inferior to adsorbed toxoid. Challenge resistance immediately following basic immunization was titre-related, however, in the light of long-term challenge findings following boosting, it was important to know if similar non-titre-related resistance developed following basic immunization . Groups of monkeys which had received only two basic immunizing doses and no boosts were bled and challenged after 3 and 5 month rest intervals, respectively. Results are shown in Table 10. Appreciable challenge resistance was evident, possibly better after the longer interval, which was not titre-related . Toxicity of the vaccine

In preparing the toxoids to be used in the formulation of batches FTS, FT6 and FTIP2, duration of action of the formalin was restricted to retain some toxicity. When converted to the adsorbed form, no toxicity (either lethal or dermonecrotic) could be detected in the supernatant, when it was tested following separation by centrifugation from the aluminium phosphate carrier. On the other hand, the toxoids used for batches FTSF, FTI, FT10A and FT10F were prepared by allowing the formalin to act until no lethal or dermonecrotic activity was detectable in them. When guinea pigs, rabbits and monkeys were dosed subcutaneously with up to 5 ml of any of the whole final vaccines there was consistently no apparent systemic effect evident in them after many months . Guinea pigs and rabbits exhibit little or no local reaction to the vaccine, but a residual nodule, self-resolving after varying periods of time, appears in some monkeys. Such nodules are no more prominent in partly detoxified materials than in fully detoxified preparations . Vaccines produced using aluminium hydroxide gel adjuvant proved to be as prone to nodule formation as preparations using aluminium phosphate as adjuvant. It was observed that pre-incubation of fully detoxified TorrcoN rsrrs vo<.

ie

E. H . BAXTER and A . G. M . MARK

430

TABLE G. ANTIBODY TI7RES AND LETHAL CHALLENGE RESISTANCE AFTER HOOSIER IMMUNIZATION OF MONREY3 BASICALLY IMMUNIZED WITH TWO DOSES OF SEA WASP TOXOID

To :oid Batch No .

Yo :Jcey No .

FT6 adacrbed

21 day titra

Psehooater titre (3 mthel

21 day postbooster titre

Unite/ml

Da1ta/ml

Dnits/ml

16 17 13 16 14 18

1270 1824 636 1270 2544 912

36 37 39 34 35 38

FT9 adsorbed

tT10F 1'luid

FT9P2 adsorbed

I

Challenga Level

eesult

100 200 100 200 140 7~

296 1692 74 2400 N.D . N .D .

4LD 4LD50 BLD50 8LD50 16LD"0 16L05~

L L D L L D

600 1200 1694 2400 420 2400

160 106 600 600 106 106

376 1060 1600 750 750 1050

BLD OLD50 8L0 16LD5° 16LD50 16LD50

0 L L L L L

52 63 sa 66 6G 67

400 1120 loD 200 <60 50

76 152 <3e <38 <38 <38

840 750 12ao 600 375 376

BLD 8LD50 einso BLD60 16LD60 16LD5~

L L L L L D

89 89 90 52 91 53

<19 <19 <19 <21
<19 <19 19 27 <19 <19

6LD50 6LD 6LD5~ 6LD 12LD~ 12LD~

D D D D 0 D

~ ~

~

<12.G <12.6 <12 .6 <12.6 <12.6 <12.6

Two doses of toxoid, each of 0~5 ml, were given subcutaneously 1 month apart for the basic immunization . A single booster dose of 0~5 ml was given subcutaneously. Adsorbed or fluid toxoid as shown was used ; FT6 contained 1 ~7 ~ of the original lethal titre in the toxoid used for final vaccine, FT91?2 contained 007 ~, and FT9 and FT10F contained < 005 ~. Challenge carried out at time of last bleed shown for the animal . LD, o represents the LDs o dose for the species challenged, determined in controls concurrently (see text) . L = lived . D = died . N.D . = not determined . type vaccine with sea wasp antivenene before injection abolished nodule formation . Microscopic examination of excised nodule sites revealed no essential histologic differences between tissues injected with the vaccine and similar sites receiving tetanus vaccine prepared with the same adjuvant. It was the histologist's opinion that there appeared to be no contra-indication to the vaccine being clinically used . Adjuvant alone produced a lesser reaction, and a mock vaccine prepared by adsorbing boiled sea wasp venom to aluminium phosphate produced reactions similar to the vaccines . No vaccine preparation has yet been found to revert to toxicity, although at the time of reporting some have been stored at 4° C for up to 22 months . DISCUSSION Protection against envenomation by the box jellyfish throughout the season of its appearance in tropical waters (usually November-April) requires a lasting immunity over this period . If this is to be achieved by an appropriate immunizing routine, it would be expected that an annual pre-season booster dose would be required to reinforce this immunity . The work in experimental animals reported in this paper shows that after basic immunization and booster dosing immunity lasts over a simulated season, and that recall boosting after some months' interval produces a regenerated resistance . No circulating TOXICON l97S YoJ. !3

43 1

Sea Wasp Toxoid

. TABLE 7. SEA WAS TOXOII) IMMUNIZATION : ANTIBODY RPSPONSE AND 73rrc7s3 CHALtFNOS RESISTANCE OF ANIMAIS REPEAT BOOSTER D(~9ED AFTER A LONO INI~VAL

7osoid Datoh lao .

Titza 11 4Ya altar bowter

~ainal lb .

Ortitshl Aahblt ri'S

i

185 187 188 316

408 917 476 7]04

pzar"p"at booster titr" lt"

I

I

Units/el

Tltra altar rapaat baostsr 11 aaya

7 m11tb "

Cballenya Level

RaNlt

Unit"/al

Onit "%al

B-ths 38 710 113 105

478 180 1B0 7100

I

107 600a 100~ 157

1 .3LD 30 li .C . ]1 .0 . 1 .3LD50

D L

4 ntha

!TS

~

i

780 317 192 314

608 917 713 1874

SO 38 730 53

150 711 1680 178

79 76 157 710

1 .3LD 1.3LD50 7 .6LD 2.6LD50

D D D D

7~3 ~13 àl7 A70

713 7437 608 1716 ]01 BS7

38 75 38 38 7B 75

840 856 1717 1716 )04 478

151 600 170 600 100 787

7.6LD50 7.6LD~ S.7LD50 S.7LD~ lO .ILD 10 .4LD~

L L L L L L

u A8

Labbit !TS!

!

43 H 43 46

n 48

,

_7 mt}u 100 560 780 780 171s 810

I

<19 76 77 <19 3e <19

170 710 600 117 l70o 117

i

I j

N.C . s.C . 11 .C . N.C .

n.c .

B.C .

Adsorbed toxoid was used : FTS contained 2 ~ of the original lethal titre in toxoid used for the final vaccine while FTSF contained < 003~. Two doses of 0~5 ml each were given for the basic immunization, two booster doses each of 0~5 ml for the first booster course (one only for rabbit 18~, and a single repeat booster dose of 0~5 ml following the 4-8 months post-booster rest. All doses were given subcutaneously . 'Rabbits 187 and 188 had titres of 420 and 210 at 4 months and 53 and 53 at 8 months . Challenge carried out at time of last bleed shown for the animal . ~"o rep ts the ~6 " dose for the species challenged, determined in controls concurrently (see text). L = lived. D = died . N.C . = not challenged .

antibody advantage seems to result from using two boosting doses, although it may well be that better confidence could be placed in long-lasting lethal challenge resistance if this regime were adopted. In choosing a detoxifying agent, preliminary investigations of the use of formalin, EDTA, glutaraldehyde, thioctic acid, and hydrogen peroxide~opper sulphates--formalin mixture by direct addition to the venom indicated that none of the other reagents was superior to formalin . It appeared, however, that concentrations of formalin sufficiently strong to destroy the toxicity of the venom also destroyed its antigenicity. We therefore adopted the method of dialysing the venom against ten volumes of weak formalin solution, thereby treating the venom with an adequate amount of formaldehyde without subjecting it to an unduly high concentration of the reagent. Early studies in mice using $uid toxoid produced by direct addition offormalin revealed the desirability of detoxifying only to the point where a small residuum of toxicity remained (unpublished results) . Reduction of lethality below a level of 1 ~ residual caused a marked fall in antigenicity . It was possible to show that toxoid produced response levels equivalent to those resulting from doses of unmodified venom of the same original lethal dose content. Unmodified venom may be used for experimental antigenicity studies if given subcutanTOXICON 1975 Yol. !3

432

E . H . BAXTER and A. G . M . MARR

TABLE H . SEA WASP TOXOID IMMUNIZATION : LETHAL CHALLENGE OP MULTIPLE ROOSTED RAHHTIS AFTER SEVERAL MONTHSRE3TINTERVAL Nntt,it N4 .

Ira-chnllerwle TStre 7 montl,a iStOr la9t d:,Se'

~

ICVPl

xc-~~hallen. -, " fr.y'vit

-

Cnits;sl

i

1?I ]vl 276 277 29t 313 ls6 316 319 319 320 343 345 347 t315

21C 21U T]C 19ti 21U 75 lD5 z13 105 4T0 305 150 105 15U >1200

I

~ ~

4LD 4LD5J 4I~ç U 4LD5~ 4LD . 4LD5~ aLn ° &LD5° 81~50

I

:. L~ 1 .. .. L

9LDSÔ 9LD 0yD50

:. L

9LD'D 9ID50 15LD5U 5U

u .. -~-

Adsorbed toxoid (Batch No . FI'S) containing in the toxoid used for its formulation a residual lethal titre equal to 2 ~ of the original titre was used in these studies . Challenge carried out at time of last bleed shown for the animal . Sao represents the Lnao dose for the species challenged, determined in controls concurrently (see text) . L = lived . D = died . " These animals had been rested for a further 5 months following multiple boosting, resting 2 months and challenging (see Table 4) . TThis animal, having a history of high titre (see Table 4), allocated to high challenge level . All other challenges `blind' (see text) .

eously, as it is not lethal when given by that route (BA,XTF.R and MARK, 1969). When doses of venom alone of a level per dose equivalent to the residual venom in a satisfactory toxoid dose were used, responses were low. A detoxifying level of 1-2 ~ residual original lethal content was adopted. This concept was continued into the dialysis procedure, and only after the adoption of casamino acids in lieu of peptone as a stabilizing agent (Batches 9 and 10) was complete detoxifying again investigated . It became evident as the work progressed that the earlier held view, that detoxification carried to the point of abolition of lethality abolished antigenicity, was erroneous for two reasons. Firstly, the higher concentrations (per se) of formalin needed to `fully' abolish toxicity of venom were harmful to the antigen when formalin was added directly to the venom, whereas dialysis detoxifying using low concentration of formalin preserved the antigenicity . Secondly, as indicated by the later experiments, adjuvant was required for effective antigenicity. The preliminary work (unpublished) employed fluid preparations . The occurrence of local injection reactions resulting from injection of the earlier partdetoxified preparations caused a revival of interest in `fully' detoxified preparations . The fully detoxified, adjuvant adsorbed preparations FTSF, FT9 and FT10A appear to be at no potency disadvantage compared with the preparations retaining some residual in vivo toxicity before adsorption ; however, the minor nodule formation in monkeys persisted. The presence of minimal local reactions to these fully detoxified preparations gave reason to investigate again a fluid preparation without adjuvant. This toxoid (FTlOF) proved quite innocuous at the injection site, but unfortunately it was not as antigenic as a preparation containing adjuvant (FT10A) from the same parent toxoid . The preparation with adjuvant was satisfactorily potent . It is apparent that only preparations containing adjuvant should be considered. TOXICON 7975 Vol. l3

43 3

Sea Wasp Toxoid irai- CFIALi.EDiOE wrrx sEA wASF zoxow

TABLE 9 . PROGRFSSIV6 ANI7HODY TI7RES AND iozui~l

. :~bnkey~

121 iay

Sitrea after booster dosaa 21 days sitar lat

PT9 Adeor!»d

PR'l0A I Adwrbsd

RESISTANCE OF MONSEYS HOOTER DOSED

45 4a 47 49 51 50

lx lx lx lx lx lx

Ssd Sad Snl lml lad Ld

19 <19 19 <14 <19 <19

848 3600 1208 848 2400 2400

56 59 60 61 63

2x 2: 2x 2x 2x

~al !g1. 7pa1 1,m1. i~,l

70 400 600 400 560

1050 525 262 525 525

70 71 72 73 74 7S

1.x~5ad 1x Sod lx lnl lx 1aS lx lad lx lad

<25 <25 <25 <25 <25 <25

1062 2120 425 750 425 S30

I

21 daye nftar 2Id

I

" "

Chellenga

i+~ntha after booster 2

" "

2100 350 700 250 380 760

" "

1000 500 53 152 212

3

4 ~

5

I

200 800 1120 100 400 200

200 140 50 50 150 ~

I

"

~

Animal aooidantslly lost " 800 42fi 150 283 304 19 564 608 1S0 100 106 26 " 100 53 <19

1

I

7

2 82 83 84 85 86 87

lx Se! 1: Ssd 1: lal lx lul lx lal lx Sml

<2S 2S <2S <2S QS Q5

S30 ZS 35 QS Q3 <13

525 38 39 <9 13 <9

94 96 98 99 95 97

2x liad 2x lid 2x7,a1 2x ilml 2x 1,ad 2x7,a1

d9
<19 13 1410 <13 54 <13

17 ~ 25 1500 3 50 <3'

, 3

426 27 106 <38 Q9 3 <2S <25 19 <25 <25 3S

Auult

22 23 140 26 35 22

5LD50 SLD lOLD50 5LD50 SLD50 lOLD50

L L D D D

35 140 18 10 18

4LD 4LD50 4LD50 4LD50 4LD5 p

L

4LD 4LD50 ~ 4LD~ 4LD50

L L L D D

lbnths alter 3 baostsrs .

lP10P lluid

Level

~5~

D L L L L

4

S

,

105 Q3 13 Q3 Q3 <13

7 dLD 4LD50 4îb50 4LDS0 4LD~ 4LDSQ

' L D L D D D

<9
21D 2LD50 2LD50 2LD~ 4LD5~ 4iD50

D L L D D D

Adsorbed or Said toxoid as shown was used : all preparations contained < 0"05 % of the original lethal titre in the toxoid used for the final vaccine. Basic immuniTation doses, given subcutaneously, as shown. Booster doses, 0"S ml subcutaneously, given 3 months after basic dose, or doses, and where two were given, 1 month apart. Challenge carried out at time of last bleed shown for the animal. X66 represents the ~6o dose for the species challenged, determined in controls concurrently (sce teat). L = lived. D = died. "Single boost only.

The nature of the immunity developed appears to change when immunized animals receive a booster dose. Challenge resistance correlates with neutralizing antibody titre on basic immunization and immediately following booster dosing, but challenge resistance remains despite decline of circulating antibody levels over subsequent months . It would appear that some other type of immunity (possibly cellular) is developed following a booster dosing routine. Even the residual antigen in basic dose depots is sufficient to develop this type of immunity (Table 10). There appears to be no problem with systemic toxicity, particularly since it has been ascertained that after adsorption fully detoxified preparations are adequately antigenic. Although no toxicity was evident in test animals injected with the final vaccine prepared from parent material of low residual toxicity, some reservation would be held in using vaccine of this type in humans . With fully detoxified preparations this reservation has been removed. There remains, in monkeys, local fibrous capsule formation which slowly resolves, but histological examination supports the view that this local reaction is no less acceptable clinically than other currently accepted vaccines of like adjuvant composition. From experimental animal evidence, it is likely that such local nodules are only evident in monkeys TOXlCON l975 Yd. !3

E. H . BAXTER and A . G . M. MARR

43 4 TABLE

lO.

ANTLBODY TITRE.4 AND RESISTANCE OF MONKEYS TO DELAYED LETHAL CHALLENGE AFTER BASIC IMMUNIZATION WITH SEA WASP TOXOII> ~n~Y lb .

Titre eftnr 21 deye Unite/ml

I

3 months Unite/ml

100 l01 103 loa 105 107 102 105

soo 75 150 2400 420 1680 1600 1680

zco <12 .5 < 12 . 5 ]an 7C 7n 50 7n

108 110 111 112 113 115 109 114

4zo 1;C 1206 16îc 10~ 2»an 21n 1640

la ".1 lns 21? 1Ss l',2 7.13

j

I

5 nonthn Unite/ml

I

Challenge j

Level SLD~ 5~50 SLD~ SO 5~50 SLD~ 1~SO

lao <25 100 70 <25 100
q~50 4~50 4~50 so 4~60 8~50 50 8LDS0

I

Pesult

n n L L 0

D

Adsorbed toxoid (Batch No . FI'9) containing in the toxoid used for its formulation a residual lethal titre of < 0~05~ of the original titre was used in these studies . Two doses of 0~5 ml each were given, subcutaneously, 1 month apart. Challenge carried out at time of last bleed shown for the animal . LD represents the LD so dose for the species challenged, determined in controls concurrently (see text). L = lived . D = died .

whose skin is thin and delicate ; vaccine injected into deep subcutaneous tissue of human subjects would probably be undetectable . Graded challenge envenomation was employed throughout the investigation to provide comparative information on various preparations and dosing regimes. The venom doselethality curve is quite steep, two LD ga doses invariably representing a lethal dose in control animals. Direct intravenous instillation of a challenge venom which acts so rapidly (a multilethal dose results in death of a monkey within a few min) is a very severe test of the immune state. It may well be that there is an upper limit of challenge venom dose above which neutralizing combination dynamics would not allow the immune mechanisms to be effective. Although as much as 16 LIi gU challenges and frequently 8 LD gU challenges were resisted, 4 LDgp doses would appear to be a sufficiently severe challenge for quality control potency tests of vaccine preparations . There is no obvious relationship between venom quantity per se and antibody level required to resist (here we refer to basic responses) but rather a level somewhat related to lethal dose for the particular species (see Table 2). if a venom quantity to antibody protecting level relationship held, it would be expected that some ten times the antibody level would be needed to protect a monkey against a given number of monkey lethal doses compared with the level needed to protect a rabbit against the same number of rabbit lethal doses. This is obviously not so, and gives some hope that experimental results might be translated to the human situation in terms of number of lethal doses likely to be experienced. Dr. J. Barnes of Cairns (personal communication) is of the opinion, formed from study of area of tentacle contact in fatal and non-fatal envenomations and total tentacle length of box jellyfish, that a victim might expect to experience a maximum of two, possibly three, human lethal doses. No human response trials having yet been undertaken it is not know what antibody levels might be expected to result from injection of this vaccine, but it would seem reasonable to expect that levels comparable to those seen in monkeys and rabbits would be adequate. T~OXlCON 1973 Yol. 13

Sea Wasp Toxoid

43 5

Acknowledgement.~Thanks are due to Dr . J. Bnxxrïs of Cairns for supply of venom, and to Dr. Jotnv Huxl ev for histopathology studies. Dr. Psrm Scxrns and the late Dr. W. R . L.~tve have ban most helpful in discussions during the progress of the work . Mrs . BRENDA H~,rox and Mrs . Pea Dowxer provided useful technical assistance. The first author (E.H.B .) is very grateful to Mrs . HEATHER G~ct-no for carrying out serum titrations during the illness of, and following the death of the second author (A .G .M .) . REFERENCES BARNES, J . H . (1967) Extraction of cnidarian venom from living tentacle : a symposium paper. In : Animal Toxins, pp . 115-129, (RUSSE[a., F. E . and $AUNDERS, P. R ., Eds .) . Oxford : Pergamon Ptess . BAxTER, E. H., MARK, A . G . M. and LANE, W . R. (1968) Immunity to the venom of the sea wasp . Toxicon 6, 45 . BAxTER, E. H . and MARK, A . G . M . (1969) Sea wasp (Chironex fleckeri) venom : lethal, haemolytic and dermonecrotic properties . Toxicon 7, 195 . BAxreR, E. H ., MARK, A . G . M . and LANE, W. R . (1973) Sea wasp (Chironex fleckeri) toxin--experimental immunity . In : Toxins of Animal and Plant Origin, Vol . 3, pp . 941-954, (DE VRiES, A . and KocxwA, E., Eds .) . New York : Gordon & Breach . CLELAND, J. B . and Sovrxcoz-r, R . V . (1965) Injuries to man from marine invertebrates in the Australian region. Spec. Rep. Ser . nain. Hlth med. Res. Coun ., Canberra, No . 12, 109 . REED, L . J. and MuENCH, H . (1938) A simple method of estimating 50 per cent end points . Am . J. Hyg. 27, 493 .

TOXICON 1973 Vol, l3