Experiments on monkeys with cholera toxin partially purified and detoxified with formol and glycine*

CHOLERA Experiments on monkeys with cholera toxin partially purified and detoxified with formol and glycine* M. SALETTI I & A. RICCI 2 Cholera toxoid partially purified and detoxified with formol and glycine and inoculated in monkeys proved safe. Histological examination revealed no changes in the skin of the animals treated. Blood samples taken from monkeys immunized with 2 doses of toxoid 12 days after the second inoculation revealed appreciable levels of antitoxin in the animals that had received two 100-,ug doses of toxoid. The monkeys vaccinated with toxoid, when inoculated with challenge doses of cholera toxin, had the capacity to neutralize the toxin. Detoxification with formol and glycine may be the first step towards the preparation of a highly purified, innocuous, and antigenic cholera toxoid. Northrup and Chisari (1) showed that cholera toxin detoxified with formol and inoculated in rhesus monkeys produced reactions owing to the reversion of the toxoid to toxin in vivo. Lingood (2) showed that the detoxification of purified diphtheria toxin with formol resulted in products that tended to become toxic again; reversion could, however, be inhibited by the addition of certain amino acids during the detoxification process. Tests were conducted to determine whether chol- era toxin partially purified with aluminium hydrox- ide and detoxified with formol in the presence of glycine remains harmless and retains its capacity to stimulate antitoxins when inoculated in monkeys. Previous studies (3) have shown that highly purified toxin detoxified with glutaraldehyde loses its immu- nizing capacity. MATERIALS AND METHODS The cholera toxin used in this study was prepared according to the technique described by Spyrides & Feeley (4). Vibrio cholerae strain 569 B Inaba was grown in Syncase medium for 18 h, and the vibrios were then killed with 0.01 % thiomersal and the * From Centro Richerche, Istituto Sieroterapico e Vac- cinogeno Toscano " A. Sclavo ", Siena, Italy. I Associate Director. 2Chief, Microbiology Unit. culture was centrifuged. The clear supernatant was treated with aluminium hydroxide at pH 6.4 and the eluted toxin with 0.1 mol/litre sodium citrate at pH 8.0. The toxin was filtered with a Millipore 0.45 filter and concentrated by ultrafiltration on an Ami- con cell with a PM 20 membrane. The characteristics of the toxin before detoxification were as follows: Proteins 1.49 mg/ml Polysaccharides 0.192 mgfml Blueing dose (rabbit) 14 200 BD/ml Safety test (monkey) reaction Limit of blueing 1 473 LB/ml The toxin was then inactivated with a 0.37% solution of formaldehyde with the addition of 0.5 mol/litre glycine (7 000 mol of formol per mol of toxin), maintained at 37°C for 6 days, and then dialysed in phosphate-buffered saline at pH 7.2 and filtered with a Millipore 0.22 filter. This material, when tested on the shaved dorsal skin of New Zealand rabbits, showed no residual toxic activity and the vascular permeability factor was absent. Experimental animals Rhesus monkeys each weighing 2-3 kg were used. The monkeys were divided into 7 groups and inocul- ated subcutaneously in the deltoid area with 1-ml doses of toxoid (10, 50, or 100 ,ug), toxin (50 or 100 ,ug), cholera vaccine, or saline. On the fourth day 3310 - 641 BULL. WORLD HEALTH ORGAN., Vol. 51, 1974 M. SALETrI & A. RICCI of inoculation a biopsy was done under general anaesthesia on one monkey from each group; the monkey used for the biopsy was not used later. One month after inoculation a sample of blood was taken from the animals followed by a second dose of toxoid. Twelve days after the second inoculation another blood sample was taken. Tests Protein was determined by the Lowry micro- method (5), and polysaccharides by the method of Winzler (6). Titration of the blueing dose The vascular permeability of the toxin was assayed by the intracutaneous injection of serial dilutions in a total volume of 0.1 ml into the shaved skin of New Zealand white rabbits, according to the technique of Craig (7, 8). Limit of blueing Swiss Serum and Vaccine Institute antitoxin con- taining 4 470 antitoxic units (AU) per ml was used for the titration according to the technique described by Craig (7, 8). A volume of serum containing 1 AU/ml was incubated with an equal volume of serial dilutions of toxin (in phosphate buffer saline, pH 7.5, containing 0.02% gelatin) for 1 h at 37'C, and 0.1-ml doses were then inoculated into the shaved dorsal skin of New Zealand rabbits. An intravenous injection of Evans blue was given 23 h later and one hour later the diameter of the coloured area was measured. One limit of blueing was deter- mined as that amount of toxin that in the presence of 1 AU gives an area of blueing 4 mm in diameter, provided the toxin-antitoxin mixture is injected in a total volume of 0.1 ml (9). Toxoid units The technique described by Rappoport (10) was used. For the titration, a volume of serum contain- ing 2 AU/ml was incubated with an equal volume of serial dilutions of toxoid at 37°C for 30 min. An- other series of tubes containing a volume of serum and equal volumes of buffer were incubated under the same conditions. After incubation an equal volume of serial dilutions of toxin was added to both series of tubes. The subsequent procedure was the same as that reported for the blueing limit. One toxoid unit is the amount of toxoid that neutralizes one AU. Mouse protection test The technique described by Feeley & Pittmann (11) was carried out. The US National Institutes of Health cholera vaccine Inaba lot IN 12 was used. Titration of the antitoxin Titration was carried out on serum from the immunized monkeys according to a modification of the technique of Craig (7, 8) as described previ- ously (3). Reactions to two different challenge doses of cholera toxin Toxin (50 or 100 ,ug) was injected intracutaneously in the shaved arms of the monkeys (7, 12, 13). The monkeys were observed daily for 21 days. Titration of the vibriocidal antibodies The technique described by Verwey et al. (14) was used. RESULTS Clinical and histological observations The results of subcutaneous inoculation of the monkeys with cholera vaccine, toxin, toxoid, or saline solution are shown in Table 1. In the monkeys inoculated with toxin, a local reaction characterized by erythema and oedema appeared on the second or third day after inoculation. The reaction reached a peak on the fourth day and lasted for 10-12 days. The animals inoculated with toxoid, vaccine, or saline solution did not give any macroscopic reac- tion. Preparations of skin were stained with haematoxy- lin-eosin and examined by light microscopy. Toxin given at 100 or 50 ,ug caused a diffused oedematous swelling and at the perivascular site lymphocytic elements were present. The most important changes were seen in the reticular dermal zone where oedema was apparent together with phlogistic cellular infil- trates composed of diffused lymphocytes or perivas- cular accumulation of lymphocytes. Toxoid in all 3 concentrations did not cause any change in the structure of the dermis and epidermis and there was no evidence of oedema or haemorrhage. The cholera vaccine produced visible oedema in the entire dermis. In the intermediate layers of the dermis there was a dense infiltration of neutrophil granulocytes, while the basal layers appeared very swollen, with lack of 642 TREATMENT OF CHOLERA TOXIN WITH FORMOL AND GLYCINE Table 1. Reactions to cholera vaccine, toxoid and toxin in inoculated monkeys Toxoid (Mg) Toxin (9g) Reaction Vaccine 10 50 100 50 100 induration no no no no yes yes erythema no no no no yes yes day of onset - - - - 3 2 dayof maximum - - - - 4 4 duration (days) - - - - 8-10 10-12 maximum erythema (mm) - - - - 8 x 8 35 x 60 Table 2. Cholera antitoxin and anti-Inaba vibriocidal levels in monkeys that had received 2 doses of cholera toxoid Toxoid First inoculation Second inoculation Monkey No. dose antitoxin antivibriocidal antitoxin antivibriocidal ( g) units/ml units/ml units/ml units/ml 1 10 x 2 <20 25 <20 2500 2 10 x 2 <20 50 <20 5000 3 50 x 2 <20 50 <20 1 250 4 50 x 2 <20 200 <20 5 000 5 100 x 2 <20 1 600 160 10000 6 100 x 2 20 1 600 250 10000 7, 8 saline <20 <5 <20 <5 structural detail and purulent fusion. Perivascular cuffings of the adventitial swollen cells were present. The skin of the animals treated with saline solution did not show any structural changes. Antitoxins The toxoid doses inoculated and the results of serum tests are shown in Table 2. After the first inoculation only one monkey injected with 100 tsg of toxoid had a titre of 20 AU/ml, the minimum value detectable by this test. After the second inoculation the 2 monkeys treated with 2 x 100 ,ug toxoid showed total antitoxin levels of 160-250 AU/ml. The small doses (10 and 50 ,ug) of toxoid inoculated produced no rise in antitoxin levels. Challenge dose The results of inoculating the monkeys with chal- lenge doses of toxin are shown in Table 3. Of all the monkeys vaccinated only one, inoculated with 2 x 50 tsg toxoid, showed a slight skin reaction to the challenge dose of 100 ,tg toxin at the inoculum site. No reaction was observed in any of the other monkeys and no structural changes in the epidermis were revealed by histological examination. The his- tological changes in the monkey that reacted to the inoculation of 100 jug toxin were slight and consisted of considerable diffused oedema together with small groups of mononuclear cells and perivascular cuffing. There was no granulomatous inflammation, no ne- crosis, and no suppuration. Vibriocidal antibodies All the animals inoculated with partially purified toxoid showed vibriocidal antibodies 1 month after the first vaccination. The vibriocidal antibodies in- creased considerably after the second inoculation. 5 643 M. SALETTI & A. RICCI Table 3. Reactions to a challenge dose of cholera toxin inoculated subcutaneously in monkeys previously inoculated with cholera toxoid or saline solution Toxoid dose Saline Reaction 10x 2 50x 2 100 x 2 CH 50a CH 100b CH 50 CH 100 CH 50 CH 100 CH 50 CH 100 induration no no no yes c no no yes yes erythema no no no yes no no yes yes dayof onset - - - 2-3 - - 3 2 dayof maximum - - - 3-4 - - 4 4 duration (days) - - - 5-6 - - 8-10 10-12 maximum erythema (mm) - - - 8 x 8 - - 8 x 8 35 x 60 a CH 50 = challenge of 5Oug cholera toxin (10 BD/Ig). b CH 100 = challenge of 100 Ag cholera toxin (10 BD/Ig). c The values refer to the monkey that gave a reaction. DISCUSSION The cholera toxoid used in these studies proved to be safe in that it did not produce reactions caused by its reversion to toxin. Histological examination of monkey skin inoculated with choleia toxoid did not reveal any alteration to the structure. In these experiments there was no apparent reac- tion to the toxin detoxified with formol and glycine, and we can therefore conclude that there is no reversion to toxin after inoculation of toxoid. Blood samples from animals treated with toxoid obtained 4 weeks after the first inoculation showed antitoxin levels of less than 20 AU/ml, apart from the serum of a monkey that had received 100 ug of toxoid. Twelve days after the second inocu- lation of toxoid the blood samples showed ap- preciable levels of antitoxin in the animals that had received 2 x 100 jug of toxoid; all the others had <20 AU/ml. When the challenge dose of cholera toxin was inoculated subcutaneously into monkeys vaccinated with toxoid there appeared to be no direct correla- tion between antitoxin titre and lack of skin reaction. In fact only one monkey, treated with 2 x 50 ,ug of toxoid, had a slight reaction; in the other monkeys treated with 10 and 50 ,ug of toxoid there was neutralization of the toxic activity. Therefore, very small doses of antitoxin that are not revealed by the methods used can be sufficient to make the toxic effect negative to the toxin in vivo. In conclusion, the lack ofreactivity and the stimula- tion of antitoxins in monkeys inoculated with cholera toxin partially purified and detoxified with formol in the presence of glycine, can solve the problem of preparing a safe and antigenic cholera toxoid. UMt EXPERIENCES PRATIQUEES SUR DES SINGES AVEC UNE TOXINE CHOLERIQUE PARTIELLEMENT PURIFIEE ET DATOXIFIEE PAR LE FORMOL ET LA GLYCINE L'anatoxine cholerique partielle purifiee et detoxifi6e par le formol et la glycine a 6te inoculee au singe et s'est montree sans danger. L'examen histologique n'a revel6 aucune modification cutanee chez les animaux traites. Des singes ont ete vaccines par 2 doses de 10, de 50, ou de 100 ,ug d'anatoxine et des echantillons de sang ont ete preleves 12 jours apres la seconde inoculation. Des taux appr6ciables d'antitoxine ont et6 mis en evidence chez les animaux ayant recu 2 doses de 100 jug. Les singes vaccines par l'anatoxine ont pu neutraliser l'activite de la toxine cholerique qui leur a 6t6 inoculee a titre d'epreuve. D'apres les donnees fournies, la detoxification par le formol et la glycine peut constituer une premiere etape vers la preparation d'une anatoxine cholerique hautement purifiee, inoffensive et antigenique. 644 TREATMENT OF CHOLERA TOXIN WITH FORMOL AND GLYCINE 645 REFERENCES 1. NORTHRUP, R. S. & CHISARi, F. V. Response of monkeys to immunization with cholera toxoid, toxin vaccine: reversion of cholera toxoid. Journal of infectious diseases, 125: 471-479 (1972). 2. LINGGOOD, F. V. ET AL. The toxoiding of purified diphtheria toxin. British journal of experimental pathology, 44: 177-188 (1963). 3. SALETI, M. & Ricci, A. Experiments with cholera toxin detoxified with glutaradehyde. Bulletin of the World Health Organization, '1: 633-639 (1974). 4. 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