THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 2, NO.4 163

The potency, toxicity and immunogenicity ofglutaraldehyde inactivated pertussis vaccineR. K. GUPTA, S. N. SAXENA, S. B. SHARMA, S. AHUJA

ABSTRACTWhole cell pertussis vaccine was prepared by inactivationof Bordetella pertussis organisms with glutaraldehydeunder optimal conditions (0.05% glutaraldehyde at roomtemperature for 10 minutes). This glutaraldehyde inacti-vated pertussis vaccine-1 (GIPV-1) was compared to theroutinely produced heat inactivated (56°C for 30 minutes)pertussis vaccine (HIPV) for potency, toxicity andimmunogenicity. Three batches of GIPV-1 and HIPVwere prepared from B. pertussis strains 134, 509 and10536. Other batches of GIPV were also prepared byinactivating the organisms with 0.05% glutaraldehyde atroom temperature for 30 minutes (GIPV-2). The potencyof both the GIPV preparations was slightly less than thatof HIPV but no statistically significant differences werefound. The toxicity of GIPV preparations evaluated bythe mouse weight gain test (MWGT) and tests for histaminesensitizing factor (HSF) and leucocytosis promoting factor(LPF) was lower than that of HIPV. The average weightgain of mice on day 7 and HSF and LPF activity of boththe GIPV preparations were different from those ofHIPV preparations (p

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copies after subculturing the original strains on theBordet-Gengou medium.

Purified pertussis toxinThe purified PT preparation, JNIH-534was a gift from DrsYuji Sato and Hiroko Sato of the National Institute ofHealth, Tokyo, Japan. The PT preparation was in thefreeze-dried state and contained 62.5lJ.g of total protein.It was reconstituted in 1 ml of sterile normal saline andstored at 4-8°C till used.

Monospecific pertussis typing seraDried monospecific pertussis typing sera for factors 1, 2and 3 were supplied by Dr I. A. Lapaeva, Gamaleya Insti-tute of Epidemiology and Microbiology, Moscow, USSR.These were reconstituted in normal saline and usedaccording to the instructions provided.

National standard of pertussis vaccineThe national standard of pertussis vaccine containing 54I.U. per vial, standardized against the second internationalstandard for pertussis vaccine." was used for determiningthe potency of the pertussis preparations. The contents ofeach vial were reconstituted to 8 I.U. per ml in 6.8 ml ofsterile normal saline and stored at 4-8°C.

Chinese hamster ovary cellsThe Chinese hamster ovary (CHO) cell line was receivedat the seventy-eighth passage from the Measles VaccineSection of the Institute. The CHO cells were maintainedby serial passage after every 3 days in Eagle's minimumessential medium (MEM) containing 50 lJ. g/ml of tetracy-cline, 25 mM HEPES buffer and 10% calf serum.

AnimalsSwiss albino mice of the DACA strain weighing 13 to 15gwere used in tests for potency and leucocytosis promotingfactor (LPF), those weighing 14 to 16 g for the mouseweight gain test (MWGT) and 20 to 25 g for the test forhistamine sensitizing factor (HSF). All these animals werefrom the Experimental Animal House of the Institute.

Pertussis suspensionsThree bacterial harvests from each of the B. pertussisstrains 10536, 134 and 509 were prepared by growing theorganisms in Verwey medium= at 35°C for 42 to 48 hoursin 10 litre flasks loaded on shakers. The bacterial mass ineach flask was separated by centrifugation and suspendedin 250 ml of normal saline without preservative. Each har-vest was examined for purity, opacity and agglutinogens.The capacity of each harvest was adjusted to 200 Interna-national Opacity Units (200x 109 organisms per ml) innormal saline. The organisms of each harvest were inacti-vated separately by heat and by glutaraldehyde underoptimal conditions. The inactivation conditions aredescribed below.

Heat: One portion of each batch, in a volume of 150 ml,was heated at 56°C for 30 minutes in a water bath. The sus-pension was shaken frequently during heating. After

THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 2, NO.4

heating, the bottle was cooled in running water as quicklyas possible and the contents centrifuged at 4000 rpm for60 minutes at 4°C. The bacterial mass was resuspended in150 ml of normal saline containing 0.02% thiomersal.

Glutaraldehyde: For GIPV suspensions, a 0.05% con-centration of glutaraldehyde (Wako Pure Chemical Co.,Osaka, Japan) was obtained by adding 7.5 ml of a 1%aqueous solution to 150 ml of suspension in a glass bottle.The suspension was held at room temperature for 10minutes to make the GIPV-l preparations. 50 ml aliquotsfrom 7 batches were allowed to remain at room tempera-ture for 30 minutes to make GIPV-2 preparations. Afterinactivation for 10 or 30 minutes the suspensions werecentrifuged at 4000 rpm for 60 minutes at 4°C. The super-natants were discarded and the bacterial masses resus-pended in normal saline containing 0.02% thiomersal.The volume of the normal saline was the same as that ofthe bacterial suspension used for inactivation.

Tests on concentrated pertussis suspensionsAfter making pertussis suspensions (2 types of GIPV pre-parations and one heat inactivated pertussis vaccine(HIPV) preparation for various harvests), each prepara-tion was subjected to quality control tests in accordancewith WHO recornmendations.F-" An aliquot of eachpreparation was diluted in normal saline to 40 Interna-tional Opacity Units (40 x 109 organisms per ml) for thesetests. The concentration of thiomersal in the dilutedaliquot was 0.01 %.

Test for opacityThe opacity of bacterial harvests was determined beforeand after inactivation.P Briefly, the samples to be testedwere diluted with normal saline and their absorbance wasrecorded in a spectrophotometer at a wavelength of 440nm. The opacity of the sample was then determined withthe help of a standard curve showing a linear relationshipbetween absorbance and opacity.

Test for potencyThe potency (intracerebral mouse protection) test wasperformed on various types of pertussis preparations inaccordance with recommendations of the WH038 and asdescribed earlier. 40The ImDsovalues for the test and stan-dard preparations and the relative potency were deter-mined by probit analysis using a Sharp PC 1500computer.The computer programme for determination of ImDsovalues, potency and 95% fiducial limits was developed byMr Yoshinobu Horiuchi and Mr Shigekatsu Yoshizawa ofthe Chiba Serum Institute, Chiba, Japan.

Mouse weight gain testThe MWGT was performed according to the guidelines ofthe WHO.38,41For each sample, 10 randomly selectedmice were used. The deaths and group weights of mice inthe test and the control groups were recorded daily forseven days. All pertussis preparations made from a singleharvest were tested simultaneously.

GUPTA et at. : GLUTARALDEHYDE INACTIVATED PERTUSSIS VACCINE

Test for histamine sensitizing factorThe HSF activity of each preparation was determined bythe method described earlier. 33.42 The results were expres-sed as the number of organisms of each sample equivalentto one HSDso. As there is no reference preparation ofpertussis vaccine for HSF estimation, the HSDso of HIPVwas taken as reference and the relative HS activities ofGIPV-1 and GIPV-2 preparations were calculated. Tominimize variations in the HSF assay, the variouspreparations from one batch were assayed in a singleexperiment.

Test for leucocytosis promoting factorThe LPF activity of various types of pertussis preparationswas assayed by inoculating 0.5 ml of 20 InternationalOpacity Units diluted suspensions intraperitoneally toeach of five mice. Another group of five mice was inocu-lated with normal saline as a control. On day 4 post-inoculation, each mouse was bled by cutting the tip of thetail and 0.05 ml blood was mixed with 0.95 ml dilutionfluid for total leucocyte counts (TLC).43 The leucocyteswere counted in a Neubauer chamber and TLC values percu mm blood were calculated as described in the WHOmanual." The relative LPF activities for various sampleswere calculated by dividing the mean TLC values of testmice with the mean TLC values of control mice.

lmmunogenicity testThe immunogenicity of all batches of HIPV, GIPV -1 andGIPV-2 preparations was evaluated in mice with regardto production of agglutinins and neutralizing antibodiesagainst PT according to the method described already. 44The mice used for the MWGT (dose: 10 InternationalOpacity Units per mouse intraperitoneaIly) were given asecond dose of the same amount, 14 days after the first.The mice were then bled and the serum separated. Thesera of mice belonging to same group were pooled inequal volumes and inactivated at 56°C for 30 minutes.These sera were stored at - 20°C till tested by the mic-roagglutination test and PT neutralization test in CHOcells. 4~

RESULTSControl tests on bacterial harvestsAll the bacterial harvests exhibited the morphological,staining and cultural characteristics of B. pertussis. Thesuspensions made from B. pertussis strain 10536 hadagglutinogens 1, 2, 3; and those from strain 134 showedthe presence of agglutinogens 1, 3; while suspensionsfrom strain 509 contained agglutinogens 1,2. After inacti-vation of the organisms with heat or glutaraldehyde, allthe suspensions were sterile and did not contain live B.pertussis organisms.

Opacity testThe opacities of pertussis suspensions before and afterinactivation are shown in Table I. The effect of 0.05%glutaraldehyde for 10 and 30 minutes was very similar.The mean loss in opacity after inactivation with glutaral-

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TABLE I. Effect of heat and glutaraldehyde on the opacity ofdifferent strains of B. pertussis suspensions

Batch No. Number of organisms (x 10Y)

Before HIPV GIPV-l GIPV-2inactivation

1110536 200 172 1772110536 200 i72 1833/10536 200 177 187 18741134 200 177 187 1835/134 200 172 183 1836/134 200 172 187 1877/509 200 180 185 1858/509 200 177 187 1859/509 200 172 183 185

Mean 200 174.5 184.3, 185% Loss 12.8 7.9 7.5

,. Not prepared

TABLE II. Potency of different strains of B. pertussis suspensions

Batch No. Potency (I. U. per 40x 109organisms)

HIPV GIPV-l

1110536 10.3 (4.7-21.0) 8.1(4.5-14.8)2/10536 11.7 (4.5-50.7) 9.9(6.5-.D3.1)3/10536 19.7(11.4-31.7) 17.7(7.5-39.1)4/134 9.3 (4.2-21.0) 6.8(3.9-13.4)51134 7.7 (5.5-16.3) 8.1(3.5-17.5)61134 9.7 (4.9-20.1) 7.3(3.8-14.1)7/509 13.3 (6.2-28.1) 23.9(8.9-35.8)8/509 17.5 (9.fr35.5) 14.4(6.1-32.1)9/509 13.3 (6.1-32.7) 12.1(4.8-31.0)

GIPV-2

19.1(8.3-37.6)5.6(3.1-13.7)7.3(3.4-16.7)5.8(4.2-12.0)

21.2(8.8-43.5)13.9(8.4-32.0)11.5(7.1-24.4)

Figures in parenthesis show 95% fiducial limits of potency.• Not prepared

dehyde for 10 minutes was 7.9%; for 30 minutes it was7.5% while the mean loss was 12.8% after inactivation ofthe organisms with heat.

Potency testTable II shows the potency values of the HIPV, GIPV-1and GIPV-2 preparations. The potency of all prepara-tions were almost similar and there were no statisticallysignificant differences. However, the potency values ofGIPV-1 and GIPV-2 preparations were slightly lowerthan those of HIPV preparations for most of thebatches.

Mouse weight gain testTable III shows the average weight gains of mice inocu-lated with HIPV, GIPV-1 and GIPV-2 preparations. Theaverage weight gains on post-inoculation days 3 and 7 forGIPV-1 and GIPV-2 inoculated mice were higher thanthose inoculated with HIPV preparations. The mean percent weight gains on post-inoculation day 7 when comparedwith the control group were 77, 104 and 104 for the HIPV,GIPV-1 andGIPV-2 preparations respectively. Therewere significant differences between weight gains on day

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TABLE III. Mouse weight gain of vaccines prepared from differentstrains of B. pertussis

Batch No Average weight gain (g) per mouse

HIPY GIPY-l GIPY-2Day3 Day7 Day3 Day7Day3 Day7

1110536 0.2 0.7 0.3 1.02/10536 0 1.0 0.2 1.33/10536 0 1.2 0.2 1.5 0.34/134 0 0.6 0.7 1.3 0.551134 0 0.7 0.4 1.0 0.46/134 0.1 0.9 0.5 1.1 0.67/509 0.4 1.2 0.5 1.2 0.38/509 0.2 0.8 0.6 1.2 0.59/509 0 0.8 0.3 1.0 0.2

Mean 0.1 0.9 0.4 1.2 0.4%wt.gain 77 104

• Not prepared

7 of mice inoculated with HIPV and GIPV-l preparations(p

GUPTA etai.: GLUTARALDEHYDE INACTIVATED PERTUSSIS VACCINE 167

TABLE VI. Agglutinin production in mice immunized with 2 doses (each dose of lOx 109 organisms permouse) of pertussis vaccines

Batch Type of Agglutinin titre against B. pertussis suspensionsNo. pertussis

vaccine Before adsorption After adsorption

10536 GL353 134 360E Tohama 10536 GL353 134 360E Tohama

1110536 HIPY 800 640 960 160 320 720

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findings are similar to those found earlier. 33,52Both types ofGIPV preparations were much less toxic than the HIPVpreparations for all the batches when assayed for MWGT,HSF and LPF activities. The average weight gains on day7, HSD50values, HS activities, total leucocyte counts andLPF activities of GIPV-l and GIPV-2 preparations weresignificantly different from those of HIPV preparations.The residual PT in th~ HIPV preparations is mainlyresponsible for the toxicity and reactogenicity of pertussisvaccine while the dermonecrotic toxin is destroyed duringheating. The lipopolysaccharide or endotoxin ispyrogenic and causes weight loss in mice." The role ofother toxins of the pertussis vaccine such as cytotoxin,adenylate cyclase and haernolysin has not been clearlydefined. 12.20

. ,..In an earlier study we found no reversal of toxicity of/", Ptirl GIPV preparations which did not show an increase

in HSF activity at 25 or 35°C for three months'? Pertussistoxoid prepared with glutaraldehyde did not show reversionof the tmlbid whereas the .toxoid prepared with formal-dehy~did.53 The potency values of all the three types ofpreparations were almost similar. These results supportour earlier findings with GIPV preparations.29,32,33Thereare a few reports on the preparation of whole cell pertussisvaccine by inactivation of the organisms with glutaral-dehyde.22,23,27,54,55However, glutaraldehyde has beenextensively studied for the production of safe and potenttetanus and diphtheria toxoids.P

GUPTA et al. : GLUTARALDEHYDE INACTIVATED PERTUSSIS VACCINE

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