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

Preparation and Laboratory Tests of Oil-adjuvant Cholera Vaccine*

HIROSHI OGONUKI, SO HASHIZUME & BENZO TAKASHASHI

In a search for cholera vaccines of improved efficacy, agar-grown strains of classical Vibrio 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 ofInaba 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 times that of the International Reference Preparations of Cholera Vaccine. A new potency test, the " maternal-immunity" test in infant rabbits, did not give quantitative results but suggested a somewhat higher potency for the vaccine. The protective effect 6 months after vaccination, as determined by agglutinin titres in 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 of vaccinees in the field trial. Histopathological examination of tissue from the site of the reactions revealed them to be due to a combination offoreign-body reactions and local hypersensitivity.

PREPARATION OF OIL-ADJUVANT CHOLERA VACCINE This vaccine, prepared at the Chiba Serum Insti- intraperitoneally. The LD50 of agar cultures for tute, Japan, is a water-in-oil emulsion of classical infant rabbits on intraintestinal injection as de- cholera vaccine in light mineral oil, containing -cribed by Dutta & Habbu (1955) was about 4000 Arlacel A as an emulsifier. Unless stated otherwise, organisms for strain 35A-3 and 300 organisms for the recommendations of the WHO Study Group on strain 41. Requirements for ... Cholera Vaccine (1959) were observed. Seed cultures A lyophilized culture of seed strain was rehydrated PREPARATION OF THE AQUEOUS PHASE with 5 ml of brain-heart infusion (BHI, Difco) and OF THE VACCINE incubated for 5 hours at 37°C. A loopful of the growth was transferred to a BHI agar plate and Seed strains and their virulence incubated overnight at 37°C. The 35A-3 (Inaba) and 41 (Ogawa) strains of Vibrio cholerae were used. They were stored by Preparation of monovalent bulk product freeze-drying and their bacteriological and sero- About 15 ml of the seed culture described above logical properties proved to be typical. Their LD50 were transferred to a Kolle flask containing about for the stock strain (DDN) of Swiss mice main- 200 ml of BHI agar. After 6 hours' incubation tained in the Institute was around 300 organisms at 37'C, the free fluids were decanted off and the for strain 35A-3 and less than 10 organisms for culture on the surface of the agar was washed off strain 41 when suspended in 5% mucin and injected with about 20 ml of phosphate buffered saline (PBS) (1/75 M, pH 7.2), to give thick suspensions of * From the Chiba Serum Institute, Chiba, Japan. vibrios which were collected by aspiration into

2103 -729 730 H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

1-litre bottles through a nylon filter to remove the Pennsylvania Refining Company, USA, was coarse materials. A small portion of the suspension used as the mineral oil and Arlacel A (mannide in each bottle was removed for the tests of purity, mono-oleate), manufactured and purified by Atlas vibrio concentration, and other bacteriological and Chemical Industries, Ind., USA, as emulsifier serological properties. The concentration of vibrios (Ogonuki et al., 1964). was determined by viable cell counts and also by They were stored separately in airtight containers. comparing the opacity of the suspension with that For the preparation of the oil phase of the oil- of the International Opacity Reference Preparation adjuvant vaccine, 9 parts of mineral oil and 1 part (WHO Study Group on Requirements for ... Cholera of Arlacel A were mixed and sterilized by passing Vaccine, 1959). then through an ST3 Seitz filter under compressed The harvested vibrio suspension was mixed with nitrogen gas to avoid the risk of oxidation. neutral formol to a final concentration of 0.4% and kept at 4°C overnight to kill the vibrios. It was then centrifuged at 10 000 rev/min for 15 min and the PREPARATION OF THE OIL-ADJUVANT VACCINE supernatant fluids were decanted off. The vibrios Equal parts of the divalent aqueous vaccine and were resuspended in PBS with thiomersal (1 :10000) the oil-phase preparation were mixed in a Waring ;s preservative. blender for about 5 minutes to obtain a stable Preparation of divalent bulk product homogeneous emulsion. The concentration of vibrios in the emulsion thus prepared was 1 x 10"0 per ml. The concentration of the monovalent Inaba or The dose of the oil-adjuvant vaccine was 0.2 ml Ogawa suspension was adjusted to 2 x 1010 vibrios for an adult, 0.1 ml for children 5-9 years of age per ml. The monovalent suspensions were combined and 0.05 ml for an infant under 4 years, correspond- in equal parts to give a divalent aqueous-phase ing to 2 x 109, 1 x 109 and 5 x 108 vibrios per dose, vaccine containing 1 x 1010 vibrios of each type respectively; 15-ml lots of the emulsion were per ml. dispensed into 50-mi vials. MINERAL OIL AND EMULSIFYING AGENT The mixing and dispensing of the vaccine was carried out under a continuous flow of filter- Drakeol 6-VR, a light liquid petrolatum (National sterilized nitrogen gas. The recommended tempera- Formulary, 1965), manufactured and purified by ture of storage of the vaccine is 2°C-5°C.

LABORATORY TESTS OF THE VACCINE

ASSESSMENT OF ANTIGENIC CONTENT For this purpose, the complement-fixation (CF) test with anticholera rabbit sera was used. Measurement of nitrogen content Antigen. The vibrio suspensions to be tested The nitrogen content of 1 ml of vaccine was were treated ultrasonically to liberate antigenic sub- determined by the semimicro-Kjeldahl method. stances from the vibrio cells. The aqueous phase Complement-fixation (CF) test only of the oil-adjuvant vaccine was used for the test. The treated suspension was centrifuged at The amount of antigenic substances to be incor- 10 000 for and the into the cholera vaccine is in the rev/min 30 minutes, supernatant porated regulated used as the antigen. manufacturing laboratories by measuring the opacity of bulk vaccine preparations within 24 hours of Antiserum. Rabbits were immunized with formol- harvesting by comparison with the International killed suspension of several different strains of Opacity Reference Preparation (WHO Study Group Inaba-type or Ogawa-type classical cholera or El Tor on Requirements for... Cholera Vaccine, 1959). vibrios by repeated intravenous injections. The sera However, the measurement of opacity is not a suit- were inactivated by heating at 56°C for 30 minutes. .able method for the comparison of products manu- Test procedure. The dilution of sera to be used factured by different laboratories, because vibrios in the tests was determined by box-titrations. For in the vaccine have already undergone autolysis. the measurement of antigenic substances, 0.1 ml of PREPARATION AND LABORATORY TESTS OF OIL-ADJUVANT CHOLERA VACCINE 731

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

Complement-fixation titre No. of vibrios/ml Total N Inaba serum Ogawa serum Vaccine by opacity (mg/ml) V. cholerae El Tor V. cholerae El Tor 124 C-5 1073 JE-4

Aqueous phase of oil-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 with on Requirements for ... Cholera Vaccine, 1959). 0.1 ml of antiserum and 4 units of complement The results are shown in Table 2. (0.2 ml). The mixture was shaken well and left In both the Inaba and Ogawa tests, considerable at 4°C overnight. It was then kept for 10 minutes differences in relative potency were observed between at room temperature, 0.2 ml of the haemolytic the aqueous phase of the oil-adjuvant vaccine and system added, and incubated for another 20 minutes the 2 comparison vaccines, which could possibly be to 30 minutes at 37°C before the results were read. explained in terms of the smaller number of vibrios The haemolytic system comprised 3 units of in the former. Thus, we might say that the poten- haemolysin and 3 % of washed sheep red-cell suspen- cies of the 3 vaccines as determined by the mouse- sion, sensitized by standing for 10 minutes in a protection test were comparable when related to the water-bath at 30°C. same concentration of vibrios. Results of the nitrogen determination and the CF test Rabbit maternal-immunity test As seen the may be from Table 1, both nitrogen potency a determination and the CF test showed that the The of vaccine should be measured by aqueous phase of 1 adult dose of the oil-adjuvant appropriate animal experiments in which the specific vaccine contained about half as much antigen as disease is reproduced by the respective organism. 2 other et al. reported their very important study on experi- vaccines (a classical cholera and an El Tor mental cholera in various animals. In Dutta vaccine used in the field trial; Pesigan et al., 1967) 1 1955, tested for the sake of comparison, while the number et al. reported their very important study on experi- of vibrios in the aqueous phase of the oil-adjuvant mental cholera in infant rabbits by direct intra- vaccine was only about a quarter of that in the intestinal challenge. other 2 vaccines, as determined by opacity meas- The clinical and pathological signs in these infected urements. This discordance could not be explained, infant rabbits resembled closely those of human but the differences in the methods of cultivation cholera. When challenged with sufficiently virulent might have some bearing on it. strains these animals died, usually within 24 to 48 hours, with profuse rice-water stools. POTENCY TESTS Stimulated by this work, we tried to apply this method for the measurement of the potency of Mouse-protection test cholera vaccines. At first, we tried the method ofdirect The method of Feeley & Pittman (1963) was used immunization of infant rabbits but failed, because to compare the potencies of the aqueous phase of the animals were not able to produce antibodies. the oil-adjuvant vaccine and the 2 comparison vac- We then tried passive immunization of infant rab- cines with that of the International Reference Pre- bits by intracardiac or intravenous injection. They parations of Cholera Vaccine (WHO Study Group were challenged 6 or more hours later. This indirect method gave promising results in some instances, 1 See the paper on p. 816 of this issue. but seemed to need more study. 732 H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

TABLE 2 RESULTS OF THE MOUSE-PROTECTION TEST

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

El Tor Aqueous phase of 2 x 109 20 10 Inaba V86 oil-adjuvant vaccine 4 4 0.001196 62-1 62 8.2 0.8 2

Cholera vaccine 8 x 109 20 14 4 9 0.000235 66-1 51 41.1 0.8 5

El Tor vaccine 8 x 109 20 14 4 8 0.000314 70-1 44 31.3 0.8 3 1- International Refer- 1.6 x 10' 100 9 ence Preparation of Cholera 20 2 0.009776 56-1 78 1.0 Vaccine (Inaba) 4 2

El Tor Aqueous phase of 2 x 109 10 20 Ogawa 17 oil-adjuvant vaccine 2 13 0.0003996 65-1 54 3.39

0.4 7

Cholera vaccine 8 x 109 10 23 2 19 0.00008819 56-1 79 15.4 0.4 13

El Tor vaccine 8 x 109 10 25 2 17 0.0000501 80-132 27.1 0.4 2

International Refer- 1.6 x 10' 100 28 ence Preparation of Cholera Vaccine 20 17 0.001356 76-1 32 1.0 (Ogawa) 4 9

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 female intestinal challenge. This suggested that the pro- rabbits before or after mating and to challenge cedure could afford a method for assay of the suckling rabbits delivered by them. This method protective effects of vaccines. These experiments we called the " maternal-immunity " method. have been reported briefly by Ogonuki et al. (1964). Agglutinin titres of 1 :100 or more were found Procedure. Adult female rabbits were given 1 sub- for the sera of these infant rabbits, and in success- cutaneous dose of serially diluted vaccine. They fully conducted experiments they survived intra- were usually mated within I week after vaccination, PREPARATION AND LABORATORY TESTS OF OIL-ADJUVANT CHOLERA VACCINE 733

and delivered litters after about 25 days. The suck- to be more than 10 times that of the International ling rabbits were challenged intraintestinally under Reference Preparations of Cholera Vaccine. light ether anaesthesia at ages between 10 and Comparison was also made between the aqueous 16 days. The infected infant rabbits were left with phase of the oil-adjuvant vaccine and the El Tor their mothers and observed. The surviving rabbits vaccine mentioned above. It will be seen from were sacrificed on the fourth or fifth day of infection, Table 3 that both vaccines had a similar potency. and several specimens oftheir intestines were cultured for vibrios. SEROLOGICAL TESTS ON VOLUNTEERS VACCINATED Results. Comparative tests were made on the WITH OIL-ADJUVANT VACCINE aqueous phase of the oil-adjuvant vaccine and the International Reference Preparations of Cholera It would be interesting to know whether the Vaccine (WHO Study Group on Requirements for ... agglutinin content of the sera of immunized persons Cholera Vaccine, 1959). The results are shown in is related to the ability of the vaccine to protect Table 3. against infection with cholera. Serological surveys Although a quantitative evaluation is difficult to of people in the trial area before and after vaccina- make from these experiments, the potency of the tion were planned, but could not be realized because aqueous phase of the oil-adjuvant vaccine seems of difficult circumstances in the field. The results

TABLE 3 RESULTS 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 No. Strain vibriosofcotl No. of vibrios No. of vibrios control 4 x 10' 1 x 10' 2.5 x 10' 1 x 10' 2.5 x 10' Inaba El Tor Inaba 5 x 104 3/3 2/4 0/4 0/9 V 86 5 x 10' 3/3 5/5 2/4 0/9 0/8 5 x 102 1/1 212 3/4 5/9 0/7 5 x 10 6/6 0/2 Ogawa El Tor Ogawa 5.7 x 104 3/3 414 2/6 0/4 0/8 Ph 141 8 5.7 x 103 3/3 4/4 5/6 3/4 0/6 5.7 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. 734 H. OGONUKI, S. HASHIZUME & B. TAKASHASHI

TABLE 4 RESULTS OF TEST VACCINATIONS WITH OIL-ADJUVANT VACCINE

No. Dimensions of local reactions Agglutinin titrea t GeneralNo.reactions ~~~~~~~~~(cm) Case of vibrios Before vaccination 5 weeks later x 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 experiments TABLE 5 are summarized in Tables 4 and 5. PERSISTENCE OF ANTIBODIES IN THE SERA OF VOLUNTEERS VACCINATED Table 4 shows that the agglutinin titres in the sera WITH OIL-ADJUVANT VACCINE of persons vaccinated with oil-adjuvant vaccine rose rather and reached their after 4-6 Agglutinin titre Vibriocidin titre slowly peak weeks, Case after which they remained almost unchanged for Inaba Ogawa Inaba Ogawa months. Table 5 shows that the agglutinin titre 6 months after vaccination was still above the critical level. Tests for vibriocidin were also made, and gave fairly high titres.

TOXICITY OF AND LOCAL REACTIONS TO OIL-ADJUVANT VACCINE

After the usual safety tests in animals, as required by the WHO Study Group on Requirements for... FIG. 1 INFILTRATION OF LYMPHOCYTES, PLASMA CELLS AND HISTIOCYTES a

37F AP

..

It H-E stain, magnification 100x.

FIG. 2 GRANULOMATOUS LESION, SHOWING GIANT CELLS AND INFILTRATION OF NEUTROPHILIC LEUCOCYTES FIG. 3 EMPTY CAVITY AFTER ABSORPTION OF LIQUEFIED NECROTIC MATERIAL a

(I H-E stain, magnification 40 x.

FIG. 4 GRANULOMATOUS LESION, CONSISTING OF EPITHELIOID CELLS AND INFILTRATION OF 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 vaccination vaccine. The number of cases of abscess formation with oil-adjuvant vaccine of different vibrio concen- remained considerable for 17 months. The patients trations was carried out on a limited number of were treated by aspiration of pus and in the later volunteers. As shown in Table 4, only a few com- stage by curetting of granulomatous masses. Oint- plained of general weakness, and none of fever. ments with antibiotics such as chloramphenicol were Some local reactions such as erythema and mild applied to prevent secondary infections. swelling were observed at the site of injection; such reactions diminished within a few days. Five Histopathological examination of the tissues from weeks after injection, indurations of minimal size the site of delayed reactions. Fifteen specimens, were observed in most of the vaccinated persons. taken from patients whose delayed local reactions The production of agglutinin seemed to be fairly were treated by curettage, were examined histopatho- satisfactory in groups who received vaccines con- logically. taining more than 2 x 109 vibrios; this concentration Macroscopically, the materials were soft masses of 2 x 109 was therefore adopted as the adult dose of of tissue, areas of abscess formation and necrosis. the oil-adjuvant vaccine to be used in the field. Microscopically (Fig. 1-4), they showed mixed pic- The number of volunteers for test vaccination tures of chronic productive and acute exudative was then increased, and a total of 512 persons were inflammation. There was no tendency to malignant vaccinated. The only reaction manifested was ery- change. All the specimens revealed pictures of thema: 32 persons (6.3%) showed erythema less granuloma with infiltration of lymphocytes, macro- than 4 cm in diameter, 280 (54.1 %) between 4.1 cm phage, plasma cells and frequently epithelioid cells and 8 cm, 173 (33.8%) between 7.1 cm and 10.0 cm, as well as multinucleated giants cells. In some cases and 27 (5.3%) 10.1 cm or over. These reactions there was marked proliferation of connective tissues being judged acceptable, the mass vaccination began and formation of small patchy scars. The softening in mid-May and ended early-in July 1964 (Azurin or liquefaction of necrotic tissue was associated et al., 1967).1 with marked infiltration by neutrophilic leucocytes. Reports of abscess or granuloma formation at Empty cavities were often found, resulting from the site of injection began to be received from the absorption of the liquefied necrotic material. In some field in June 1964. Careful bacteriological examina- areas fibrinoid and haemorrhagic necrosis with tions were carried out with material taken from masses of round-cell infiltration in the perivascular abscess masses but no pathogenic agents were isolated areas were seen, which might have been due to in most cases; these aseptic abscesses were diagnosed local antigen-antibody reaction. Thus, the local as due to delayed local reactions to the oil-adjuvant changes at the site of injection might have been due to the combined effects of both foreign-body 1 See the paper on page 703 of this issue. reactions 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 excipient le formol. La dose inoculee a un adulte etait de 0,2 ml, huileux, qui etait l'un des vaccins mis a 1'epreuve dans soit 2 x 109 vibrions. le cadre du projet conjoint Japon-Philippines-OMS de On a 6value I'activite de la phase aqueuse du vaccin recherche sur le cholera, en faisant une emulsion en par 1'epreuve de protection de la souris (methode de parties egales d'huile minerale (Drak6ol 6 VR avec Feeley & Pittman), et on l'a trouvee comparable a celle 10% d'Arlacel A comme emulsionnant) et d'une sus- des vaccins anticholeriques liquides, classique et El Tor, pension aqueuse de cultures de vibrions choleriques employes dans la meme epreuve sur le terrain. On a classiques (souches Inaba 35A-3 et Ogawa 41) tues par aussi 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'immunite que les titres seriques d'agglutinine etaient plus eleves maternelle inventee par les auteurs. Suivant cette methode, et plus durables que ceux obtenus aprts injection on administre a des lapins, avant l'accouplement, une de vaccins liquides. On n'a constate aucune reaction injection sous-cutanee de vaccin, de concentration en facheuse. vibrions connue, et, quelques jours apres la naissance Au cours d'une campagne de vaccination de masse, de la portee, on -injecte des vibrions virulents vivants un nombre non negligeable de personnes auxquelles par voie intra-intestinale aux jeunes lapins. Les resultats avait ete administre le vaccin 'a excipient huileux ont ont montr6 que le vaccin a excipient huileux conferait signale l'apparition d'un granulome ou d'un abces au une immunite tres elevee vis-a-vis du cholera experi- point d'injection deux semaines environ apres la vac- mental, ce qui est conforme aux resultats des essais sur cination. Les examens histopathologiques ont montre le terrain. que ces ph6nomnnes etaient probablement dus a la Au cours d'un essai limite, on a administre le vaccin combinaison d'une reaction au corps etranger et d'une a excipient huileux a 512 volontaires, et on a constate reaction d'hypersensibilite locale.

REFERENCES

Azurin, J. C., Cruz, A., Pesigan, T. P., Alvero, M., Ogonuki, H., Hashizume, S. & Takashi, B. (1964) Newer Camena, T., Suplido, R., Ledesma, L. & Gomez, C. Z. developments in cholera vaccines. In: Proceedings of (1967) Bull. Wld Hlth Org., 37, 703 the Seventh International Congresses on Tropical Dutta, N. K. & Habbu, M. K. (1955) Brit. J. Pharmacol., Medicine and Malaria, Rio de Janeiro, Brazil, vol. 3, 10, 153 p.37 Feeley, J. C. & Pittman, M. (1963) Bull. Wld Hlth Org., Pesigan, T. P., Sumpaico, J. & Sychangco, G. (1967) Bull. 28, 379 Wld Hlth Org., 37, 816 National Formulary (1965) 12th ed., Washington, D.C., WHO Study Group on Requirements for... Cholera American Pharmaceutical Association, p. 298 Vaccine (1959) Wld Hlth Org. techn. Rep. Ser., 179