* FIELD TRIAL O F TWO BIVALENT TRACHOMA VACCINES IN

CHILDREN O F PUNJAB INDIAN VILLAGES

SANITTAR P. DHIR, M.D., LALIT P. AGARWAL, M.D., ROGER DETELS, M.D., SAN-PIN WANG, M.D., AND J. THOMAS GRAYSTON, M.D.

Successful growth of the trachoma agent by T'ang and his colleagues1 in 1957 pro­vided the possibility of̂ developing a vaccine for the prevention "of*;trachoma. Although trachoma is a disease' of the conjunctiva affecting only the superficial layers and has been shown to produce few antibodies in the natural setting,2 many groups have at­tempted to produce a vaccine which could confer protection.

Prevention of trachoma with experimen­tal vaccine has been reported by Grayston and associates,3·4 Collier and associates,5'6

Bietti and associates,7 Bell and associates,8

and others, both in experimental animals and human volunteers. The present status of trachoma vaccines has been recently re­viewed by Collier.9

Encouraging results of vaccine field trials have been reported in Taiwan,3 an area of low trachoma prevalence. Such trials require a considerable period of follow-up. The length of this period can be diminished by choosing an area of high prevalence and, therefore, of high attack rates. Unfortunate­ly, the follow-up of children in these areas is difficult because they are also the under­developed areas of the world.

The present study reports the results of a field trial of two trachoma vaccines in an

From the Department of Ophthalmology, All-India Institute of Medical Sciences, New Delhi, India, and the Department of Preventive Medi­cine, University of Washington School of Medicine, Seattle, Washington 9810S. This study was support­ed in part by a research grant from the Indian Council of Medical Research, by United States Public Health Service research grant No. NB-03144 from the National Institute of Neurologi­cal Diseases and Blindness, and United States Public Health Service training grant No. TI-AI-206 from the National Institute of Allergy and Infectious Diseases.

Reprint requests to: Dr. Sanittar Paul Dhir, Department of Ophthalmology, All India Insti­tute of Medical Sciences, New Delhi 16, India.

area of high trachoma prevalence in North­ern India.

METHODS AND MATERIALS

Preparation of-voecines. Two trachoma vaccines were prepared at the University of Washington: a genetron-purified vaccine and a gradient-purified vaccine. Each vac­cine contained approximately equal amounts by particle count of two Taiwan trachoma prototype strains (TW-3 and TW-5) . The trachoma agent was grown in yolk sacs of embryonated chick eggs. The harvested yolk sacs were partially purified by trypsin diges­tion, omnimix blending and differential cen-trifugation and then subjected to further purification using either trifluorotrichloro-ethane* (a fluorocarbon which inactivates the agent) or 0 to 40% sucrose-1 molar KC1 con­tinuous density gradient followed by high speed centrifugation through 40% sucrose. Methods used for purification of trachoma antigens are described in greater detail else­where.10 Each of the two vaccines was inac­tivated by suspension in 0.02% formalin in 0.1 M phosphate buffer and 4°C storage for one month. The final genetron vaccine con­tained 2.2 X 109 particles and the gradient vaccine contained 1.2 X 109 particles per ml. Nitrogen content for the genetron vaccine was 0.29 mg per ml and for the gradient vaccine was 0.34 mg per ml.

At each of the steps in the production of the vaccines the material was tested aerobi-cally and anaerobically for bacterial contam­ination. The safety of the vaccines was confirmed by tests in animals and in human volunteers. In addition, potency tests were run in mice and monkeys.10'11 Two injec-

* Genetron or Genesolv-D Allied Chemical Com­pany. We and others have referred to trachoma antigens treated with trifluorotrichlóroethane as "genetron-purified."

1639/613

1640/614 AMERICAN JOURNAL OF OPHTHALMOLOGY MAY, 1967

TABLE 1 TRACHOMA PREVALENCE IN CHILDREN

IN RURAL PUNJAB

Age (mo)

No. Children

Examined

No. Children Having Active

Trachoma

Prevalence Rate

0- 5 6-17

18-29 30-41 42-53 54-65

159 191 248 233 222 198

48 125 203 207 198 . 175

30.2% 65.4% 81.9% 88.8% 89.2% 88.4%

tions of the genetron vaccine provided about 600 potency units of TW-3 and 50 units of TW-5 while the gradient vaccine provided 300 units of TW-3 and 500 units of TW-5 based on the mouse toxicity potency test.10·11

Of the 14 isolations typed thus far (from the area of the field trials) all have fallen into group C (prototype TW-3) by the mouse toxicity prevention test.12

Selection of field study areas. From pre­vious surveys13'14 the Punjab area of India was selected as an area of high prevalence of trachoma. Villages in the rural area of Ballabgarh, 40 kilometers southwest of Delhi, were selected for the field trials. The prevalence of trachoma in children under five years in this area was greater than 85%. The field trial was limited to uninfect-ed children under five.

Prevalence survey. A prevalence survey of trachoma in children under five was un­dertaken in five of the seven villages se­lected for inclusion in the field trials15 (table 1). Each of the children was examined by an ophthalmologist with a torch and loupe and by hand slitlamp whenever necessary. A total of 1,251 children was examined. From the surveys a prevalence rate for the various age groups was derived.

Selection of individuals for vaccine trials. Due to the high attack rate of trachoma in these villages it was necessary to select the individuals to participate in the study at the time of the first vaccination. Children were selected according to the following criteria:

1. No clinical evidence of trachoma. 2. Normal conjunctivas. 3. Age at the time of initial vaccination

between three months and five years. 4. Good general health. 5. Permission of parent. By necessity, examinations were done by

the ophthalmologist in the homes of the in­dividuals.

Assignment of vaccine. From the pre­dicted incidence derived from the prevalence survey, it was estimated that a minimum of 100 children would be necessary for valid statistical comparisons between the two vac­cines and placebo groups at the conclusion of the study. Therefore, a minimum of 150 children were recruited for each of the three groups to allow for loss over the course of the study. As each child was admitted to the stud)', he was assigned one of the three ma­terials according to a previously randomized list of vaccines. The two vaccines and one placebo material were coded and their iden­tity was unknown to the ophthalmologist and other workers.

Techniques of recording. For each exam­ination a separate card was made with name, address, code number and clinical findings. At the initial vaccination the name and code number of the child was affixed to the random list of vaccines. The data for each examination were transferred from the cards to the master sheets which were kept in New Delhi. The master sheets were never referred to by the ophthalmologist. At the second vaccination all sheets with vaccine designation were handled by the health workers and vaccine was not given until ex­amination had been completed by the oph­thalmologist. Vaccines were referred to only by their codes, and their identity was un­known to the ophthalmologist and the field workers. In subsequent examinations, vac­cine designation and previous findings were not available to those working in the field, making it impossible for the examiner to be prejudiced by either previous findings or vaccine group.

TRACHOMA AND ALLIED DISEASES 1641/615

TABLE 2 BOOSTER VACCINATION OF CHILDREN

Trachoma Vaccine

Gradient

150 2 S

143 107

Genetron

151 1 3

147 103

150 4 8

138 100

lOUU

451 7 16 428 310

Initially vaccinated Died before booster given Removed from study* Eligible for booster Received booster

* Misidentified, left village, inadvertently treated

Evaluation of examinations. The results of the examinations were evaluated at the end of each examination period. The chil­dren were classified from the original cards (which contained only clinical findings and code designation of the child) into the fol­lowing groups :

1. Normal conjunctiva. 2. Definite trachoma (applying W H O

criteria, Expert Committee on Tra­choma, Geneva, 1956).

3. Could not be definitely diagnosed. 4. Diagnosis obscured (children having

severe mucopurulent conjunctivitis).

Criteria of definite trachoma diagnosis. Those children who displayed any two of the following criteria were considered to have definite trachoma :

1. Follicles—conjunctival or limbal. 2. Epithelial keratitis most marked on the

upper part of the cornea. 3. Pannus—upper part of the cornea. 4. Typical scars.

RESULTS

Initial vaccination. A total of 451 children were vaccinated in several villages (Chand-avli, Unchagon, Machagarh, Sihi, Dayalpur, Tigaon and Josaintly) in the rural area of Ballabgarh, Punjab, during February and early March, 1965. One hundred fifty-one of the children were vaccinated with gene-tron-purified vaccine, 150 with gradient-purified vaccine, and 150 with placebo (aqueous diphtheria tetanus vaccine). The

sex ratio of the children was 11:10 (males: females).

Booster vaccinations. The booster in­jections were given approximately three months after the first vaccinations (May-June, 1965). A total of 310 children were revaccinated, 72% of those eligible (table 2 ) . Only those children who were ill at the time of the second vaccination were inten­tionally not vaccinated. Eye examinations were performed prior to vaccination.

Examinations. Additional examinations of children were done approximately eight and 12 months after the first vaccination (October, 1965, and February-March, 1966).

Residts of examination of children receiv­ing booster vaccination. Of the 310 children who received booster vaccinations, 11 died before the subsequent examination and four more died before the last examination. In all, 22 children of the original group died.

At the second, third, and fourth examina­tions, there were 16, 20, and 22 children in all groups showing clinical conversion to trachoma. The distribution of the conver­sions to trachoma according to type of vac­cine given is shown in Table 3. During the three-month period between the initial vacci­nation and the booster vaccination only one child among the group receiving the gra­dient vaccine and four children among the group receiving genetron vaccine contracted trachoma, while 11 children who received the placebo vaccine developed trachoma. The rate of conversion to trachoma among the vaccinated groups increased at each of the last two examinations. At the end of one year

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TABLE 3 RESULTS OF EXAMINATIONS 2, 3 AND 4 IN CHILDREN RECEIVING

TWO INJECTIONS OF VACCINE OR PLACEBO

Examined May-June, 1965 Total (received booster vaccination)

Trachoma-free Converted to trachoma Not classified Severe MPC*

Gradient

107 86

1(0.9%) 11 9

Vaccine

Genetron

103 71 4(3.9%)

13 15

Placebo

100 63 11(11%) 15 11

Total

310 220

16 39 35

Examined—October, 1965 Previous conversion excluded) Trachoma-free Converted to trachoma Not classified Severe MPC

95

60 3(3.2%)

24 8

88

57 6(6.8%)

16 9

81

46 11(13.6%) 20 4

264

163 ,20

60 21

Examined February, 1966 (Previous conversions excluded) Trachoma-free Converted to trachoma Not classified Severe MPC

86

64 5(5.8%)

13 4

82

63 7(8.5%) 7 5

65

47 10(15.4%) 7 1

233

174 22 27 10

* Mucopurulent conjunctivitis

the cumulative rate of conversion to trachoma (table 4) was significantly lower in the group receiving gradient vaccine (10.0%, P < 0.001 ) and the group receiving genetron vac­cine (18.5%, P < 0.01) than in the group receiving placebo vaccine 36.8% ).

The effectiveness in preventing trachoma (obtained by applying the conversion rate of the placebo group to the vaccine groups) of the gradient vaccine was 73% and of the genetron vaccine 50%. The age distribution of the children examined at one year is shown in Table 5. In addition, the age dis­tribution of those children who contracted trachoma is shown. The highest attack rate was in the oldest children. Vaccine protec­tion was present in all age groups. The age-adjusted effectiveness was 70% for the gra­dient vaccine and 47% for the genetron vac­cine.

In the unlikely event that the 27 children whom we were unable to classify at the end of one year went on to develop trachoma the effectiveness of the vaccines would still be 46% for gradient vaccine and 42% for the

genetron vaccine. The difference in the rate of conversion between the two vaccine groups is not statistically significant.

A qualitative comparison of the trachoma contracted in each of the three groups re­vealed no difference in severity of disease between the vaccine groups and the placebo group.

Results of examinations of children not receiving a booster vaccination. Of the 118 children who did not receive a booster injec­tion 69 were examined at the third examina­tion and 78 at the fourth examination. The

TABLE 4 EFFECTIVENESS OF TRACHOMA VACCINE

Converters to trachoma (cumulative total)

Examined at one year (including cumulative converters)

Cumulative conversion rate

Effectiveness

Gra­dient

9

90

10.0% 73%

Gene­tron

17

92

18.5% 50%

Pla­cebo

32

87

36.8%

TRACHOMA AND ALLIED DISEASES 1643/617

TABLE 5 AGE DISTRIBUTION OF DOUBLE-VACCINATED SUBJECTS WITH TRACHOMA CONVERSION RATE

Age (year) <2 2 & 3 4 & 5 Total

Total Population 129 90 SO 269

Population 45 26 19 90 Gradient Converted 4 3 2 9

% converted 9 12 11 10

Population 50 26 16 92 Genetron Converted 8 4 5 17

% converted 16 15 31 18

Population 34 38 15 87 Placebo Converted 9 16 7 32

% converted 26 42 47 37

effectiveness of preventing trachoma of the gradient vaccine was 50% at the third ex­amination and 29% at the fourth examina­tion. The figures for the genetron vaccine were 50% effectiveness at the third exam­ination and 38% at the fourth examination.

DISCUSSION

The examinations were carried out by the ophthalmologist with a binocular loupe and a flashlight. Though the method is satisfac­tory for well-developed cases of trachoma, very early cases may have been missed. Slit-lamp examination could not be carried out in the study because the children were very young, and examinations had to be done in the homes.

The number of children who would ac­cept booster vaccinations was disappointing (310 or 69%) but the subsequent follow-up rate of this doubly vaccinated group was quite high (90.3% for the third examination and 86.8% for the fourth examination). Al­though the only reaction noted to the vac­cine injections was a febrile reaction of short duration, Indian village mothers are not fully educated to the rationale of im­munization and, therefore, many could not be convinced to allow a second injection of their children. In addition, the time of the second injection fell toward the end of the hot dry season when some of the children were ill and could not be revaccinated. For­tunately, the mothers were willing to have

their children's eyes examined and so those children not followed up at the third and fourth examinations represented mainly children who had died or had moved from the village either temporarily or permanently.

It is reassuring to note that those infec­tions of trachoma which break through the vaccine are no more severe than the tracho­ma seen in the placebo groups. Experimen­tal infections in monkeys immunized with these vaccines and other similar prepara­tions often result in more severe disease than in nonvaccinated monkeys even though good protection is observed.11 Our data ap­pear to indicate that this is not true for hu­mans.

These preliminary results suggest that the protection conferred by the vaccines appears to wear off with time, although a booster vaccination prolongs the duration of protec­tion. At each successive examination a larger proportion of the children had developed tra­choma (table 3) . Therefore, it would seem reasonable that with the present vaccine a series of two injections spaced about three months apart followed later by booster in­jections would give the best results.

SUMMARY

A field trial of two bivalent trachoma vac­cines was conducted in the Punjab state of India. Four hundred fifty-one children were randomly vaccinated with either of the two vaccines or a placebo material. Three

1644/618 AMERICAN JOURNAL OF OPHTHALMOLOGY MAY, 1967

hundred ten of these children received a second vaccination three months later. Both the sucrose-KCl gradient purified and the trifluorotrichlorethane (Genetron) purified vaccines protected against trachoma infec­tion during the course of a one year follow-up. However, protection decreased succes­sively at the 2, 8 and 12 month examination. At the end of the year, the cumulative tra­choma conversion rate was 37% in the pla­cebo group compared to 10% of those re­ceiving gradient vaccine. The conversion rate in each vaccine group is statistically different from the placebo group. The effec­tiveness in preventing trachoma infection of the gradient purified vaccine was 73% and of the genetron vaccine 50% at one year. No difference was observed in the severity of the trachoma contracted in the two groups receiving vaccine and the group re­ceiving placebo material.

R E F E R E N C E S

1. Tang, F. F., Chang, H. L., Huang, Y. T. and Wang, K. C. : Studies on the etiology of tra­choma with special reference to isolation of the virus in chick embryo. Chinese M. J. 75:429, 1957.

2. Woolridge, R. L. and Grayston, J. T.: Further studies with a complement fixation test for tra­choma. Ann. N.Y. Acad. Sci. 98:314, 1962.

3. Grayston, J. T., Woolridge, R. L., Wang, S. P., Yen, C. H., Yang, C. Y, Cheng, K. H. and Chang, I. H. : Field studies of protection from infection by experimental trachoma virus vaccine

in preschool aged children on Taiwan. Proc. Soc. Exp. Biol. and Med. 112:589, 1963.

4. Grayston, J. T., Woolridge, R. L., Wang, S. P., Yen, C. H., Yang, C. Y, Cheng, K. H., Chang, I. H. and Yang, Y. F.: Trachoma vaccine field studies on Taiwan. Orient. Arch. Ophth. 1:93, 1963.

5. Collier, L. H.: Experiments with trachoma vaccines. Lancet, 1:795, 1961.

6. Collier, L. H., Sowa, S., Sowa, J. and Blyth, W. : Experiments with trachoma vaccine. Orient. Arch. Ophth. Soc. 12:67, 1963.

7. Bietti, G. B., Guerra, P., Felici, A. and Vozza, R.: Role of trachoma vaccine. Orient. Arch. Ophth. 1:76, 1963.

8. Snyder, J. C. et al: Vaccination against tra­choma in Saudi Arabia: Design of field trials and initial results. Indust. & Trop. Health, 5:65, 1965.

9. Collier, L. H. : The present status of tra­choma vaccination studies. Bull. World Health Org. 34:233, 1966.

10. Wang, S. P. and Grayston, J. T.: A potency test for trachoma vaccine utilizing the mouse tox-icity prevention test Am. J. Ophth. 63:1443, 1967.

11. Wang, S. P., Grayston, J. T. and Alexander, E. R.: Trachoma vaccine studies in monkeys. Am. J. Ophth. 63 :1615, 1967.

12. Alexander, E. R., Wang, S. P. and Grayston, J. T.: Further classification of TRIC agents from ocular trachoma and other sources by the mouse toxicity prevention test. Am. J. Ophth. 63: 1469, 1967.

13. Agarwal, L. P., Malik, S. R. K. and Mohan, M.: Prevalence study of trachoma. Ori­ent. Arch. Ophth. 1:100, 1963.

14. Das, T., Nirankari, M. S. and Chaddah, M. R.: Trachoma (epidemiology and treatment). J. All-India Ophth. Soc. 12:72, 1961.

15. Agarwal, L. P., Dhir, S. P. and Lamba, P. A.: Trachoma Eradication—a pilot study. Proc. All-India Ophth. Soc. Jan. 1965.