Bull. Org. mond. Sante 1967, 37, 729-736Bull. Wid Hith Org. j

Preparation and Laboratory Tests of Oil-adjuvantCholera Vaccine*

HIROSHI OGONUKI, SO HASHIZUME & BENZO TAKASHASHI

In a search for cholera vaccines of improved efficacy, agar-grown strains of classicalVibrio cholerae were killed with formol and emulsified with Arlacel A in mineral oil;the final vibrio concentration was adjusted to 2 x 109 vibrios per adult dose (equal propor-tions of Inaba and Ogawa types).

There was an unexplained discrepancy between determinations of the vibrio content,by cell counting and opacity measurements, and ofthe antigen content, by nitrogen measure-ments and the complement-fixation test.

The potency of the vaccine, estimated by the mouse-protection test, was about S timesthat of the International Reference Preparations of Cholera Vaccine. A new potency test,the " maternal-immunity" test in infant rabbits, did not give quantitative results butsuggested a somewhat higher potency for the vaccine.

The protective effect 6 months after vaccination, as determined by agglutinin titresin the sera of volunteers, was still high, confirming the results ofa field trial of the vaccine.However, local reactions (indurations) were observed in a considerable proportion ofvaccinees in the field trial. Histopathological examination of tissue from the site of thereactions revealed them to be due to a combination of foreign-body reactions and localhypersensitivity.

PREPARATION OF OIL-ADJUVANT CHOLERA VACCINE

This vaccine, prepared at the Chiba Serum Insti-tute, Japan, is a water-in-oil emulsion of classicalcholera vaccine in light mineral oil, containingArlacel A as an emulsifier. Unless stated otherwise,the recommendations of the WHO Study Group onRequirements for ... Cholera Vaccine (1959) wereobserved.

PREPARATION OF THE AQUEOUS PHASEOF THE VACCINE

Seed strains and their virulenceThe 35A-3 (Inaba) and 41 (Ogawa) strains of

Vibrio cholerae were used. They were stored byfreeze-drying and their bacteriological and sero-logical properties proved to be typical. Their LD50for the stock strain (DDN) of Swiss mice main-tained in the Institute was around 300 organismsfor strain 35A-3 and less than 10 organisms forstrain 41 when suspended in 5% mucin and injected

* From the Chiba Serum Institute, Chiba, Japan.

intraperitoneally. The LD50 of agar cultures forinfant rabbits on intraintestinal injection as de--cribed by Dutta & Habbu (1955) was about 4000organisms for strain 35A-3 and 300 organisms forstrain 41.

Seed culturesA lyophilized culture of seed strain was rehydrated

with 5 ml of brain-heart infusion (BHI, Difco) andincubated for 5 hours at 37°C. A loopful of thegrowth was transferred to a BHI agar plate andincubated overnight at 37°C.

Preparation of monovalent bulk productAbout 15 ml of the seed culture described above

were transferred to a Kolle flask containing about200 ml of BHI agar. After 6 hours' incubationat 37'C, the free fluids were decanted off and theculture on the surface of the agar was washed offwith about 20 ml of phosphate buffered saline (PBS)(1/75 M, pH 7.2), to give thick suspensions ofvibrios which were collected by aspiration into

2103 -729

H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

1-litre bottles through a nylon filter to removecoarse materials. A small portion of the suspensionin each bottle was removed for the tests of purity,vibrio concentration, and other bacteriological andserological properties. The concentration of vibrioswas determined by viable cell counts and also bycomparing the opacity of the suspension with thatof the International Opacity Reference Preparation(WHO Study Group on Requirements for ... CholeraVaccine, 1959).The harvested vibrio suspension was mixed with

neutral formol to a final concentration of 0.4% andkept at 4°C overnight to kill the vibrios. It was thencentrifuged at 10 000 rev/min for 15 min and thesupernatant fluids were decanted off. The vibrioswere resuspended in PBS with thiomersal (1 :10000);s preservative.

Preparation of divalent bulk productThe concentration of the monovalent Inaba or

Ogawa suspension was adjusted to 2 x 1010 vibriosper ml. The monovalent suspensions were combinedin equal parts to give a divalent aqueous-phasevaccine containing 1 x 1010 vibrios of each typeper ml.

MINERAL OIL AND EMULSIFYING AGENT

Drakeol 6-VR, a light liquid petrolatum (NationalFormulary, 1965), manufactured and purified by

the Pennsylvania Refining Company, USA, wasused as the mineral oil and Arlacel A (mannidemono-oleate), manufactured and purified by AtlasChemical Industries, Ind., USA, as emulsifier(Ogonuki et al., 1964).They were stored separately in airtight containers.

For the preparation of the oil phase of the oil-adjuvant vaccine, 9 parts of mineral oil and 1 partof Arlacel A were mixed and sterilized by passingthen through an ST3 Seitz filter under compressednitrogen gas to avoid the risk of oxidation.

PREPARATION OF THE OIL-ADJUVANT VACCINE

Equal parts of the divalent aqueous vaccine andthe oil-phase preparation were mixed in a Waringblender for about 5 minutes to obtain a stablehomogeneous emulsion. The concentration of vibriosin the emulsion thus prepared was 1 x 10"0 per ml.The dose of the oil-adjuvant vaccine was 0.2 ml

for an adult, 0.1 ml for children 5-9 years of ageand 0.05 ml for an infant under 4 years, correspond-ing to 2 x 109, 1 x 109 and 5 x 108 vibrios per dose,respectively; 15-ml lots of the emulsion weredispensed into 50-mi vials.The mixing and dispensing of the vaccine was

carried out under a continuous flow of filter-sterilized nitrogen gas. The recommended tempera-ture of storage of the vaccine is 2°C-5°C.

LABORATORY TESTS OF THE VACCINE

ASSESSMENT OF ANTIGENIC CONTENT

Measurement of nitrogen content

The nitrogen content of 1 ml of vaccine was

determined by the semimicro-Kjeldahl method.

Complement-fixation (CF) test

The amount of antigenic substances to be incor-porated into the cholera vaccine is regulated in themanufacturing laboratories by measuring the opacityof bulk vaccine preparations within 24 hours ofharvesting by comparison with the InternationalOpacity Reference Preparation (WHO Study Groupon Requirements for... Cholera Vaccine, 1959).However, the measurement of opacity is not a suit-.able method for the comparison of products manu-

factured by different laboratories, because vibriosin the vaccine have already undergone autolysis.

For this purpose, the complement-fixation (CF) testwith anticholera rabbit sera was used.

Antigen. The vibrio suspensions to be testedwere treated ultrasonically to liberate antigenic sub-stances from the vibrio cells. The aqueous phaseonly of the oil-adjuvant vaccine was used for thetest. The treated suspension was centrifuged at10 000 rev/min for 30 minutes, and the supernatantused as the antigen.

Antiserum. Rabbits were immunized with formol-killed suspension of several different strains ofInaba-type or Ogawa-type classical cholera or El Torvibrios by repeated intravenous injections. The serawere inactivated by heating at 56°C for 30 minutes.

Test procedure. The dilution of sera to be usedin the tests was determined by box-titrations. Forthe measurement of antigenic substances, 0.1 ml of

730

PREPARATION AND LABORATORY TESTS OF OIL-ADJUVANT CHOLERA VACCINE 731

TABLE IESTIMATION OF ANTIGENIC CONTENT BY NITROGEN DETERMINATION AND COMPLEMENT-FIXATION TEST

Complement-fixation titre

Vaccine No. of vibrios/ml Total N Inaba serum Ogawa serumby opacity (mg/ml)

V. cholerae El Tor V. cholerae El Tor124 C-5 1073 JE-4

Aqueous phase ofoil-adjuvant vaccine 2 x 10' 0.068 128 256 128 258

Cholera vaccine 8 x 10' 0.146 512 512 256 512

El Tor vaccine 8 x 109 0.161 512 512 256 512

serial twofold dilutions of antigen was mixed with0.1 ml of antiserum and 4 units of complement(0.2 ml). The mixture was shaken well and leftat 4°C overnight. It was then kept for 10 minutesat room temperature, 0.2 ml of the haemolyticsystem added, and incubated for another 20 minutesto 30 minutes at 37°C before the results were read.The haemolytic system comprised 3 units of

haemolysin and 3% of washed sheep red-cell suspen-sion, sensitized by standing for 10 minutes in awater-bath at 30°C.

Results of the nitrogen determination and the CF testAs may be seen from Table 1, both the nitrogen

determination and the CF test showed that theaqueous phase of 1 adult dose of the oil-adjuvantvaccine contained about half as much antigen as2 other vaccines (a classical cholera and an El Torvaccine used in the field trial; Pesigan et al., 1967) 1

tested for the sake of comparison, while the numberof vibrios in the aqueous phase of the oil-adjuvantvaccine was only about a quarter of that in theother 2 vaccines, as determined by opacity meas-urements. This discordance could not be explained,but the differences in the methods of cultivationmight have some bearing on it.

POTENCY TESTS

Mouse-protection testThe method of Feeley & Pittman (1963) was used

to compare the potencies of the aqueous phase ofthe oil-adjuvant vaccine and the 2 comparison vac-cines with that of the International Reference Pre-parations of Cholera Vaccine (WHO Study Group

1 See the paper on p. 816 of this issue.

on Requirements for ... Cholera Vaccine, 1959).The results are shown in Table 2.

In both the Inaba and Ogawa tests, considerabledifferences in relative potency were observed betweenthe aqueous phase of the oil-adjuvant vaccine andthe 2 comparison vaccines, which could possibly beexplained in terms of the smaller number of vibriosin the former. Thus, we might say that the poten-cies of the 3 vaccines as determined by the mouse-protection test were comparable when related to thesame concentration of vibrios.

Rabbit maternal-immunity testThe potency of a vaccine should be measured by

appropriate animal experiments in which the specificdisease is reproduced by the respective organism.et al. reported their very important study on experi-mental cholera in various animals. In 1955, Duttaet al. reported their very important study on experi-mental cholera in infant rabbits by direct intra-intestinal challenge.The clinical and pathological signs in these infected

infant rabbits resembled closely those of humancholera. When challenged with sufficiently virulentstrains these animals died, usually within 24 to48 hours, with profuse rice-water stools.

Stimulated by this work, we tried to apply thismethod for the measurement of the potency ofcholera vaccines. At first, we tried the method of directimmunization of infant rabbits but failed, becausethe animals were not able to produce antibodies.We then tried passive immunization of infant rab-bits by intracardiac or intravenous injection. Theywere challenged 6 or more hours later. This indirectmethod gave promising results in some instances,but seemed to need more study.

H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

TABLE 2

RESULTS OF THE MOUSE-PROTECTION TEST

Challenge VcieNo. of Dos Suvvl 0 SD b Relativestrain lby opacity m) (%) potency

Aqueous phase ofoil-adjuvantvaccine

Cholera vaccine

El Tor vaccine

International Refer-ence Preparationof CholeraVaccine (Inaba)

Aqueous phase ofoil-adjuvantvaccine

Cholera vaccine

El Tor vaccine

International Refer-ence Preparationof Cholera Vaccine(Ogawa)

2 x 109

8 x 109

8 x 109

1.6 x 10'

2 x 109

8 x 109

8 x 109

1.6 x 10'

20

4

0.8

20

4

0.8

20

4

0.8

100

20

4

10

2

0.4

10

2

0.4

10

2

0.4

100

20

4

10

4

2

14

9

5

14

8

3

9

2

2

20

13

7

23

19

13

25

17

2

28

17

9

0.001196

0.000235

0.000314

1-

0.009776

0.0003996

0.00008819

62-1 62

66-1 51

70-1 44

56-1 78

65-1 54

56-1 79

0.0000501 80-132

0.001356 76-1 32

8.2

41.1

31.3

1.0

3.39

15.4

27.1

1.0

a Out of 16 for the Inaba tests, and out of 32 for the Ogawa tests.b Log EDso has a normal distribution. SD represents the antilogarithm of the standard deviation of log EDso, expressed as a

percentage of the mean value of EDso.

Attempts were then made to immunize femalerabbits before or after mating and to challengesuckling rabbits delivered by them. This methodwe called the " maternal-immunity " method.

Agglutinin titres of 1 :100 or more were foundfor the sera of these infant rabbits, and in success-

fully conducted experiments they survived intra-

intestinal challenge. This suggested that the pro-

cedure could afford a method for assay of theprotective effects of vaccines. These experimentshave been reported briefly by Ogonuki et al. (1964).

Procedure. Adult female rabbits were given 1 sub-cutaneous dose of serially diluted vaccine. Theywere usually mated within I week after vaccination,

732

El TorInaba V86

El TorOgawa 17

PREPARATION AND LABORATORY TESTS OF OIL-ADJUVANT CHOLERA VACCINE

and delivered litters after about 25 days. The suck-ling rabbits were challenged intraintestinally underlight ether anaesthesia at ages between 10 and16 days. The infected infant rabbits were left withtheir mothers and observed. The surviving rabbitswere sacrificed on the fourth or fifth day of infection,and several specimens of their intestines were culturedfor vibrios.

Results. Comparative tests were made on theaqueous phase of the oil-adjuvant vaccine and theInternational Reference Preparations of CholeraVaccine (WHO Study Group on Requirements for ...

Cholera Vaccine, 1959). The results are shown inTable 3.

Although a quantitative evaluation is difficult tomake from these experiments, the potency of theaqueous phase of the oil-adjuvant vaccine seems

to be more than 10 times that of the InternationalReference Preparations of Cholera Vaccine.Comparison was also made between the aqueous

phase of the oil-adjuvant vaccine and the El Torvaccine mentioned above. It will be seen fromTable 3 that both vaccines had a similar potency.

SEROLOGICAL TESTS ON VOLUNTEERS VACCINATED

WITH OIL-ADJUVANT VACCINE

It would be interesting to know whether theagglutinin content of the sera of immunized personsis related to the ability of the vaccine to protectagainst infection with cholera. Serological surveysof people in the trial area before and after vaccina-tion were planned, but could not be realized becauseof difficult circumstances in the field. The results

TABLE 3RESULTS OF THE MATERNAL IMMUNITY TEST ON INFANT RABBITS

Challenge Test results a

Aqueous phase of oil-adjuvant International Reference Preparation Non-vaccine of Cholera Vaccine immunized

Strain No. ofcotlvibrios No. of vibrios No. of vibrios control

4 x 10' 1 x 10' 2.5 x 10' 1 x 10' 2.5 x 10'

InabaEl Tor Inaba 5 x 104 3/3 2/4 0/4 0/9V 86

5 x 10' 3/3 5/5 2/4 0/9 0/85 x 102 1/1 212 3/4 5/9 0/75 x 10 6/6 0/2

OgawaEl Tor Ogawa 5.7 x 104 3/3 414 2/6 0/4 0/8Ph 141 8

5.7 x 103 3/3 4/4 5/6 3/4 0/65.7 x 102 1/1 2/2 4/4 1/3 3/5

5.7 x 10 1/2

El Tor Vaccine

No. of vibrios

8x109 2x10' 5xlog

El Tor Inaba 7 x 10 D- D++++ DDD++ ++--- +++-- D++++ DDD

El Tor Ogawa 9 x 102 -_ D---- +++-- ++--- ++++- DD+17

a Expressed in the top half of the table as number surviving number tested, and in the bottom half as follows: D = died;+ = vibrios found on autopsy; - = no vibrios found on autopsy.

733

H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

TABLE 4RESULTS OF TEST VACCINATIONS WITH OIL-ADJUVANT VACCINE

tNo. General reactions Dimensions of local reactions Agglutinin titreaNo. ~~~~~~~~~(cm)

Case of vibrios Before vaccination 5 weeks laterx 109 Fever lWeakness Erythema after Induration after

48 hours 5 weeks Inaba Ogawa Inaba Ogawa

H.O. 6.0 _ + 7.5 x 6.0 1.5 x 2.0 20 <20 320 160

G.M. 6.0 - _ 9.0 x 8.0 1.5 x 1.0 20 <20 80 80

H.Y. 6.0 - + 7.5 x 6.0 2.0 x 2.0 40 20 320 160

M.M. 3.0 - 10.0 x 5.0 1.0 x 1.0 80 40 160 80

H.M. 3.0 - - 5.0 x 4.5 2.0 x 2.0 80 40 160 80

E.T. 2.0 -_ 3.5 x 4.0 0.5 x 0.5 20 <20 320 320

M.R. 2.0 -_ 0 <20 <20 320 160

T.O. 2.0 - _ 6.0 x 5.0 1.0 x 1.0 40 <20 320 160

T.U. 2.0 - - 6.5x 6.5 1.0x 1.0 20 20 160 80

T.S. 2.0 _ + 7.5 x 5.5 2.0 x 1.0 80 40 640 320

Y.M. 1.5 _ _ 2.0x2.5 80 20 320 160

T.H. 1.5 - - 6.0 x 6.5 1.5 x 2.0 20 <20 320 160

Y.S. 1.5 - - 3.0 x 3.5 0 <20 <20 160 40

N.S. 1.5 - - 6.0 x 4.0 0 20 <20 160 40

U.D. 1.0 - - 2.0 x 3.5 0 80 20 160 40

S.G. 1.0 - - 4.5 x 6.5 1.5 x 1.0 20 <20 320 160

K.K. 1.0 - - 3.0 x 2.5 0 <20 <20 160 40

I.l. 1.0 - - 6.5 x 4.5 1.5 x 2.5 <20 <20 320 160

N.S. 0.5 - - 3.0 x 3.0 0 80 20" 320 80

I.T. 0.5 _ _ 0 0 320 160

a The agglutination test was done with live suspensions of V. cholerae strains Inaba 35-A3 and Ogawa 41.

of tests on volunteers in the preliminary experimentsare summarized in Tables 4 and 5.

Table 4 shows that the agglutinin titres in the sera

of persons vaccinated with oil-adjuvant vaccine rose

rather slowly and reached their peak after 4-6 weeks,after which they remained almost unchanged formonths.

Table 5 shows that the agglutinin titre 6 monthsafter vaccination was still above the critical level.Tests for vibriocidin were also made, and gavefairly high titres.

TOXICITY OF AND LOCAL REACTIONS

TO OIL-ADJUVANT VACCINE

After the usual safety tests in animals, as required bythe WHO Study Group on Requirements for...

TABLE 5

PERSISTENCE OF ANTIBODIES IN THE SERAOF VOLUNTEERS VACCINATEDWITH OIL-ADJUVANT VACCINE

Agglutinin titre Vibriocidin titreCase

Inaba Ogawa Inaba Ogawa

734

FIG. 1

INFILTRATION OF LYMPHOCYTES, PLASMA CELLS AND HISTIOCYTES a

It H-E stain, magnification 100x.

FIG. 2GRANULOMATOUS LESION, SHOWING GIANT CELLS AND INFILTRATION OF NEUTROPHILIC LEUCOCYTES

37FAP

..

FIG. 3EMPTY CAVITY AFTER ABSORPTION OF LIQUEFIED NECROTIC MATERIAL a

(I H-E stain, magnification 40 x.

FIG. 4

GRANULOMATOUS LESION, CONSISTING OF EPITHELIOID CELLS AND INFILTRATIONOF LYMPHOCYTES AND HISTIOCYTES a

1' H-E stain, magnification 40 x.

PREPARATION AND LABORATORY TESTS OF OIL-ADJUVANT CHOLERA VACCINE 735

Cholera Vaccine (1959), preliminary test vaccinationwith oil-adjuvant vaccine of different vibrio concen-trations was carried out on a limited number ofvolunteers. As shown in Table 4, only a few com-plained of general weakness, and none of fever.Some local reactions such as erythema and mildswelling were observed at the site of injection;such reactions diminished within a few days. Fiveweeks after injection, indurations of minimal sizewere observed in most of the vaccinated persons.The production of agglutinin seemed to be fairlysatisfactory in groups who received vaccines con-taining more than 2 x 109 vibrios; this concentrationof 2 x 109 was therefore adopted as the adult dose ofthe oil-adjuvant vaccine to be used in the field.The number of volunteers for test vaccination

was then increased, and a total of 512 persons werevaccinated. The only reaction manifested was ery-thema: 32 persons (6.3%) showed erythema lessthan 4 cm in diameter, 280 (54.1 %) between 4.1 cmand 8 cm, 173 (33.8%) between 7.1 cm and 10.0 cm,and 27 (5.3%) 10.1 cm or over. These reactionsbeing judged acceptable, the mass vaccination beganin mid-May and ended early-in July 1964 (Azurinet al., 1967).1

Reports of abscess or granuloma formation atthe site of injection began to be received from thefield in June 1964. Careful bacteriological examina-tions were carried out with material taken fromabscess masses but no pathogenic agents were isolatedin most cases; these aseptic abscesses were diagnosedas due to delayed local reactions to the oil-adjuvant

1 See the paper on page 703 of this issue.

vaccine. The number of cases of abscess formationremained considerable for 17 months. The patientswere treated by aspiration of pus and in the laterstage by curetting of granulomatous masses. Oint-ments with antibiotics such as chloramphenicol wereapplied to prevent secondary infections.

Histopathological examination of the tissues fromthe site of delayed reactions. Fifteen specimens,taken from patients whose delayed local reactionswere treated by curettage, were examined histopatho-logically.

Macroscopically, the materials were soft massesof tissue, areas of abscess formation and necrosis.Microscopically (Fig. 1-4), they showed mixed pic-tures of chronic productive and acute exudativeinflammation. There was no tendency to malignantchange. All the specimens revealed pictures ofgranuloma with infiltration of lymphocytes, macro-phage, plasma cells and frequently epithelioid cellsas well as multinucleated giants cells. In some casesthere was marked proliferation of connective tissuesand formation of small patchy scars. The softeningor liquefaction of necrotic tissue was associatedwith marked infiltration by neutrophilic leucocytes.Empty cavities were often found, resulting fromabsorption of the liquefied necrotic material. In someareas fibrinoid and haemorrhagic necrosis withmasses of round-cell infiltration in the perivascularareas were seen, which might have been due tolocal antigen-antibody reaction. Thus, the localchanges at the site of injection might have beendue to the combined effects of both foreign-bodyreactions and local hypersensitivity-type reactions.

ACKNOWLEDGEMENT

Special thanks are due to Dr J. C. Feeley, Biologics Standards Division, National Institutes of Health, Bethesda,Md., USA, for carrying out tests of the vaccine in collaboration with other laboratories in France, Hungary, India,Japan and the USA.

RESUME

On a prepare le vaccin anticholerique a excipienthuileux, qui etait l'un des vaccins mis a 1'epreuve dansle cadre du projet conjoint Japon-Philippines-OMS derecherche sur le cholera, en faisant une emulsion enparties egales d'huile minerale (Drak6ol 6 VR avec10% d'Arlacel A comme emulsionnant) et d'une sus-pension aqueuse de cultures de vibrions choleriquesclassiques (souches Inaba 35A-3 et Ogawa 41) tues par

le formol. La dose inoculee a un adulte etait de 0,2 ml,soit 2 x 109 vibrions.On a 6value I'activite de la phase aqueuse du vaccin

par 1'epreuve de protection de la souris (methode deFeeley & Pittman), et on l'a trouvee comparable a celledes vaccins anticholeriques liquides, classique et El Tor,employes dans la meme epreuve sur le terrain. On aaussi contr6l6 l'efficacite du vaccin a excipient huileux

736 H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

sur de tres jeunes lapins par la methode de l'immunitematernelle inventee par les auteurs. Suivant cette methode,on administre a des lapins, avant l'accouplement, uneinjection sous-cutanee de vaccin, de concentration envibrions connue, et, quelques jours apres la naissancede la portee, on -injecte des vibrions virulents vivantspar voie intra-intestinale aux jeunes lapins. Les resultatsont montr6 que le vaccin a excipient huileux conferaitune immunite tres elevee vis-a-vis du cholera experi-mental, ce qui est conforme aux resultats des essais surle terrain.Au cours d'un essai limite, on a administre le vaccin

a excipient huileux a 512 volontaires, et on a constate

que les titres seriques d'agglutinine etaient plus eleveset plus durables que ceux obtenus aprts injectionde vaccins liquides. On n'a constate aucune reactionfacheuse.Au cours d'une campagne de vaccination de masse,

un nombre non negligeable de personnes auxquellesavait ete administre le vaccin 'a excipient huileux ontsignale l'apparition d'un granulome ou d'un abces aupoint d'injection deux semaines environ apres la vac-cination. Les examens histopathologiques ont montreque ces ph6nomnnes etaient probablement dus a lacombinaison d'une reaction au corps etranger et d'unereaction d'hypersensibilite locale.

REFERENCES

Azurin, J. C., Cruz, A., Pesigan, T. P., Alvero, M.,Camena, T., Suplido, R., Ledesma, L. & Gomez, C. Z.(1967) Bull. Wld Hlth Org., 37, 703

Dutta, N. K. & Habbu, M. K. (1955) Brit. J. Pharmacol.,10, 153

Feeley, J. C. & Pittman, M. (1963) Bull. Wld Hlth Org.,28, 379

National Formulary (1965) 12th ed., Washington, D.C.,American Pharmaceutical Association, p. 298

Ogonuki, H., Hashizume, S. & Takashi, B. (1964) Newerdevelopments in cholera vaccines. In: Proceedings ofthe Seventh International Congresses on TropicalMedicine and Malaria, Rio de Janeiro, Brazil, vol. 3,p.37

Pesigan, T. P., Sumpaico, J. & Sychangco, G. (1967) Bull.Wld Hlth Org., 37, 816

WHO Study Group on Requirements for... CholeraVaccine (1959) Wld Hlth Org. techn. Rep. Ser., 179