- Email: [email protected]
Further observations on 17D-yellow fever vaccination by scarification, with and without simultaneous smallpox vaccination
493 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 54. No. 5.
FURTHER
September, 1960.
OBSERVATIONS
SCARIFICATION,
WITH
ON
AND
17D-YELLOW WITHOUT
FEVER
VACCINATION
SIMULTANEOUS
BY
SMALLPOX
VACCINATION BY
Captain P. D. MEERS, Royal Army Medical Corps*
(From the Federal Laboratory Service Headquarters, Yaba, Lagos, Nigeria) Despite the serological demonstration of the recent presence of the virus of yellow fever a few miles from Lagos, an investigation carried out by the West African Council for Medical Research, in 1958, showed a remarkably low level of immunity to the disease among Lagos school children (Macnamara, pets. commun). As a result of this survey, the M.O.H., Lagos, decided to institute a vaccination programme, using the 17D-mouse brain scratch yellow fever vaccine produced at Yaba. This decision, affecting some 60,000 children, gave rise to a most valuable opportunity for carrying out certain experiments. T h e s e are the subject of this paper. T h e use of a combined smallpox-yellow fever vaccine (the two viruses being mixed, and freeze-dried together in the same ampoule) was shown to be relatively ineffective in an experiment carried out recently (MEERS, 1959). However, as it was felt that a simultaneous vaccination with the two agents was desirable in West Africa for reasons of speed and economy, it was decided to use the present opportunity to test the simultaneous administration of smallpox and yellow fever vaccines, at separate sites. T h e dictum of the Ministry of Health (London), concerning the spacing of vaccinations with these two agents, made it necessary to watch closely for any untoward reaction to this procedure. In addition, it was decided that, in an effort to reduce the approximate 15 per cent. of failures to become demonstrably immune, found in the past among persons vaccinated against yellow fever by scratch with 17D vaccines, a group of persons would be vaccinated twice by this technique, allowing an interval to elapse between vaccinations. MATERIALS AND METHODS
(i) Smallpox vaccine This was a Lister type dried vaccine (COLLIER, 1955), prepared in the Smallpox Vaccine Laboratory, Yaba. It was applied, after reconstitution with 20 per cent. glycerine in Macllvaine's buffer, by the multiple pressure method, at a single site. Vaccine for the trial was all drawn from one batch. * Thanks are due to Dr. C. M. Norman-Williams, Chief Medical Adviser to the Federal Government of Nigeria, for permission to publish. I am grateful to Dr. O. Adeneyi-Jones, M.O.H., Lagos, Dr. F. N. Macnamara, West African Council for Medical Research, and Dr. D. W. Horn, Medical Statistician, Federal Ministry of Health, Lagos, all of whom gave valuable assistance. Major-General G. T. L. Archer, Director of Pathology, the War Office, London, was responsible for the suggestion that led to the trial of the " double scratch " yellow fever vaccination technique.
494
FURTHER OBSERVATIONS ON 1 7 D - Y E L L O W FEVER VACCINATION
(ii) Yellow fever vaccine T h e 17D mouse brain vaccine prepared by the Yellow Fever Vaccine Laboratory, Yaba, was used. This-was reconstituted with 2 per cent. gum arabic solution, and applied by Messrs. Down Bros. " vaccinostyle." Th e blade of this, when dipped into the reconstituted vaccine, has been found to pick up approximately 0.02 ml. By placing the two sides of the blade in succession at two sites about one inch (2.5 cm.) apart, this volume of vaccine was transferred to the skin of the arm over the deltoid. Four scratches, arranged in a cross hatched manner, were made through each of the drops, using the point of the vaccinostyle, and finally, the vaccine was rubbed into the scratches with the flat of the blade. Once the vaccine had been reconstituted, any remaining unused after one hour was discarded. Vaccine for the trial was all drawn from a single batch, sample ampoules of which were titrated after all the vaccinations had been completed, to estimate the dose of virus applied to each child. (iii) Treatment of the skin Before either vaccination, the skin was cleansed with a swab barely moistened with water. dressings were applied to the smallpox or to the yellow fever vaccination sites.
No
(iv) Design of the trial Four schools were selected, as being easily accessible, with a good disciplinary standard, and having a total of between 1,600 and 2,000 children over the age of 10 years, this lower age-limit being chosen solely with an eye to the collection of blood samples by venepuncture. Each class involved in each of the schools was divided into four groups, as nearly as possible equal in size, allocation being determined by the initial letter of the surname. These groups were called " A " , " B , " " C , " and " D " . Thus, between 400 and 500 children were allotted to each group. T h e groups were treated as described below. Just before vaccination, 59 children, chosen at random, were bled, the serum obtained being used to confirm that the low incidence of immunity found during the survey carried out by the West African Council for Medical Research applied to the schools selected for the trial.
Group " A " T h e children in this group were vaccinated simultaneously on opposite arms with the two vaccines. ~I'he smallpox reaction was examined 7 days later. Twenty-eight days after vaccination, as many as possible of the children were bled, t o obtain serum for protection testing.
Group "B" T h e children in this group were vaccinated by the standard scarification technique, with yellow fever vaccine alone. This was repeated after 14 days. Twenty-eight days after this second vaccination senarn was obtained from as many as possible, for protection testing.
Group "C" These children were vaccinated as for group " B , " but without the second vaccination. Blood specimens were coUected from half the group after 28 days, for comparison of protection test results with those from group " A , " and from the other half after 42 days, for comparison with group " B . "
Group " D " T h e children in this group were vaccinated with an inert substance, resembling vaccine. Blood specimens collected from this group were used for estimating the pre-vaccination level of immunity in the " population " used for the trial.
Observation for reactions As it was not thought to be desirable to suggest, to the children or to their teachers, the possibility that reactions might follow vaccination, it was decided that their occurrence would be investigated, in the first instance at least, by comparing the attendance records for the 21 days following vaccination, of the children in Groups "A," " C , " and " D . " This was done retrospectively, by examining class registers. (v) Protection tests T h e test used to detect the presence or absence of yellow fever antibodies in the sera collected was Smithburn's intraperitoneal test in immature mice, (SMITrlBURN, 1945). T h e results were interpreted as positive, inconclusive, or negative, according to the convention laid down by the World Health Organization Expert Committee (WHO, 1957). All inconclusive results were re-tested as part of a normal test " run," they were not tested specially using a reduced challenge virus dose. T h e m i n i m u m challenge dose employed in any " run " was 220 LDs0, though the virus titre (titrated by the intraperitoneal route in mice of the same age as those used for the test) was usually far in excess of this.
P. D. MEEBS
495
RESULTS
(i) Vaccinations A total of 1,653 children were vaccinated in all four groups in the four schools visited. Details of this total, broken down according to group and school, are given in Table I. The schools involved are numbered one to four. TABLE I. Details of children vaccinated, divided according to Group, and school. 4
Group School
A
]
B C (For definition of Groups, see p. 494)
D
1
85
88
84
84
2
77
81
78
81
3
128
127
135
126
4
125
116
118
120
415
412
415
411
Totals 1,653
(ii) Blood specimens collected A total of 1,204 blood specimens were collected. This total was made up by 357 samples from Group "A," 321 from Group "B," 292 from Group " C " and 175 from Group " D . " To the total for Group " D " were added the 59 specimens collected at the outset of the trial, as described above. None of the 175 specimens from Group " D " was obtained from among the 59 individuals bled earlier. Group "C" was divided into two sub-groups, according to whether the samples were collected 28 or 42 days after vaccination. Group "C1" (28 day specimens) numbered 165, and Group "C~" (42 day specimens) numbered 127. (iii) Yellow fever vaccine titration results In order to estimate the dosage of yellow fever vaccine applied per child, four ampoules of the vaccine batch used were taken, and each reconstituted in gum arabic solution, as for use in vaccination. The contents of two of the ampoules were pooled, and titrated at once, the other two were pooled, allowed to stand at room temperature for one hour, and then titrated. Titration was by intracerebral inoculation of tenfold dilutions of the vaccine in bovine albumin-saline solution into 35-day-old Swiss albino mice. Six mice were used at each dilution, and the end-point calculated by the method of R~ED and MUENCH (1938). By taking the geometric mean of the results of the two titrations, the average dose applied per child was found to be 16,000 LDs0. (iv) Attendance records In order to determine whether the vaccination procedures caused any serious reactions, the attendance registers for the children involved in the trial were examined for the 21 days following vaccination, and any absences recorded. By comparing the average number of missed attendances over the period for Groups "A" and "C" with the number in control
496
FURTHER OBSERVATIONS ON 17D-YELLOW FEVER VACCINATION
group " D , " it was clear that there was no increase in absenteeism in the former two groups. Where necessary, enquiry was made to determine the cause of prolonged absence of any individual child. (v) Smallpox vaccination results Readings of the results of smallpox vaccinations, after 7 days, in Group " A , " were recorded either as positive (presence of a definite " take "), or negative. Of the 415 children vaccinated in Group " A " , 17 were absent from school, or could not be found, one week later, when readings were made. Of the 398 remaining, 375 (94.5 per cent.) showed a positive reaction. (vi) Yellow fever protection test results The results of the serological tests for the presence of yellow fever antibodies in the sera collected are given in Table II. The results of the tests on Group " A " sera are given in greater detail than the rest, for reasons which will appear later. The number of sera tested shown differs slightly from the total specimens collected, given in section (ii) above. The discrepancies are accounted for by sera rejected due to bacterial contamination, insufficiency of the specimen, and, in one case, loss by accidental spilling. Only 200 specimens, selected at random, were tested from Group " D , " as a sufficiently accurate estimate of the occurrence of natural immunity among the children could be obtained by examining this number. TABLE II.
Results of yellow fever protection tests on the sera collected. indicate percentages.
Group
The figures in brackets
Results Total sera tested
i School 70
+
4-
50 (71.4)
(1.4)
1
19 (27.2)
2
70
48 (68.6)
3
108
104 (96.3)
(0.9)
3 (2.8)
95 (90.5)
2 (1.9)
(7.6)
22 (31.4)
A
4
105
1
8
B
309
303 (98.1)
2 (0.6)
4 (1.3)
Cl
165
159 (96.4)
1
(0.6)
5 (3. o)
4 (3.2)
6 (4.7)
C2
127
117 (92.1)
D
200
10 (5. o)
K e y : + = Positive (protective) result. protective) result.
:k = Inconclusive result.
190 (95.0) -- = Negative (non-
P. D. MEERS
497
The results of the protection tests given were compared by statistical analysis, to show whether the differences observed between the various groups could have occurred by chance, or whether the differences are significant. This was done by applying the g 2 test to the results obtained in the various groups, taking the inconclusive and negative results together, as vaccination failures. Yates's correction for discontinuous distributions was applied. A superficial examination of the results in Group "A," showed that they varied considerably, schools one and two giving poor results compared with schools three and four. The possible reasons for this will be discussed later, but the application of the g s test revealed that the differences were very highly significant (Zs = 33.003 ; p ---- 40.001), and for this reason Group "A" has been divided into two sub-groups, "AI" comprising the poor results o b tained in schools one and two, and "As" the better results from schools three and four. Application of the Zs test to Groups "C1" and "Cs" revealed that the difference between them was not significant (gs = 1.737 ; 0 . 1 < p < 0 . 2 ) , and that, therefore, the results may be combined, to give a single set of figures for Group "C." Next, the g s test was used to compare the results obtained in Groups "At," "As," and "B" with those obtained in " control " Group "C." This revealed that, as might be expected, Group "AI" differed very significantly from Group "C," while Group "As" did not (gs = 0.104 ; 0 . 7 ~ p < 0 . 8 ) . The results from Group "B" were significantly better than those obtained in Group " C " (Zs = 4.371 ; 0 . 0 2 < p < 0 . 0 5 ) . Lastly, to calculate the true conversion rate, from the non-immune to the immune state, produced by each of the procedures, it was necessary to correct each of the results obtained in the Groups "A" to "C" for the pre-existing immunity rate, found in Group "D." This was allowed for, and the actual conversion rates calculated, Observed conversion rate °/o - - Basal immunity °/o x 100) 100 - - Basal immunity % the results are given in Table III. Inconclusive results have again been regarded as vaccination failures in preparing this table. (True conversion rate
TABLE I I I .
%
A c t u a l yellow fever c o n v e r s i o n rates f r o m t h e n o n - i m m u n e to t h e i m m u n e state, p r o d u c e d by each of the vaccination procedures described.
Per cent, vaccination successes 68.4 Group A 93.1 Group B
98.0
Group C
94.2
DISCUSSION
The trial described had, as its objective, the solution of the three problems, summarized below. 1) Does the simultaneous application of smallpox and yellow fever vaccines, at different sites, result in any reduction in the efficacy of either as an immunizing agent? 2) Do serious reactions commonly follow simultaneous vaccination with smallpox and yellow fever vaccines?
G
498
FURTHER OBSERVATIONS ON 17D-YELLOW FEVER VACCINATION
3) Do two scratch vaccinations, spaced in time, with yellow fever vaccine, produce a higher rate of immunity than that produced by a single scratch on one occasion only? In this discussion, an attempt is made to answer these three questions in the light of the results of the trial, and to suggest an explanation for a further important and somewhat unexpected finding. (i) Simultaneous smallpox and yellow fever vaccination The reaction to the smallpox component of the double vaccination in Group " A " was most satisfactory, the 94.5 per cent. " take " rate being scored in a population well covered by previous vaccinations, particularly during the smallpox epidemic of 1957. Most of the reactions were of the " accelerated " variety, only a very few typical primary vaccinial lesions being seen. This result reflects very closely previous experience with Yaba-produced dried smallpox vaccine. As has already been pointed out, the results of yellow fever protection tests on the sera collected from Group "A" are not uniform. The unfortunate and very significant difference observed between Groups "AI" and "As," both of which were, at first sight, treated similarly, requires elucidation. Many possible explanations for this are ruled out at once by the fact that the results from all other groups were uniform throughout for all four schools. If the results obtained in Group "AI" were to be taken as correct, it must be postulated that this was caused by interference (in its widest sense) between the two vaccines, and, in this case, to explain the higher result in Group "A2" (which agrees with the result found after yellow fever vaccination alone in Group "C"), it follows that many individuals in this group could not have been vaccinated at all with smallpox vaccine, or were vaccinated with vaccine that was not potent. This is disproved by the high smallpox vaccination " take " rate, and the fact that the few failures which did occur were spread evenly through all four schools. If, on the other hand, the high rate in Group "As" were to be accepted as correct, the low Group "AI" result can be explained by assuming either that non- or marginally-potent yellow fever vaccine was used in this group, or that some individuals in the group escaped vaccination. The former assumption can be ruled out, as the vaccinators performing the yellow fever vaccination in Group "A" also used the same vaccine for Groups "B" and "C." The second assumption is almost certainly the correct one, as shown by the following explanation. In carrying out the vaccinations, in order to disturb the schools as little as possible, all four groups were vaccinated concurrently. An entire class was called at a time, divided into four groups, and vaccinated. Children in three of the Groups ("B," "C," and "D") were each vaccinated on one arm only, the fourth group, Group "A," being vaccinated on both arms. On the first 2 days of the trial, when the schools numbered one and two were dealt with, supervisory activity was mainly directed towards watching for, and correcting, errors in technique among the vaccinators, who had not previously carried out yellow fever vaccinations by the scratch method. On the next 2 days, when schools three and four were visited, the vaccinators by this time being proficient, supervisory activity was transferred to controlling the children. It was then noticed that there was a tendency, among some children allocated to Group "A," (all of whom received the smallpox vaccination first), to assume that they could go after one vaccination, as did the children in all the other groups, and to avoid the second vaccination, against yellow fever. This tendency was checked on days 3 and 4, but almost certainly occurred undetected on days 1 and 2, a certain number of children, thus, not being vaccinated at all on these days. This would, of course, explain
P. D. MEERS
499
the lower conversion rate in Group "AI," and it is suggested that reasonable justification exists for the acceptance of the results obtained in Group "As" as the true conversion rate for the combined vaccination procedure. This result does not, as has already been pointed out, differ significantly from that obtained in Group "C." Thus, it can be stated that the performance of smallpox and yellow fever vaccinations simultaneously, at different sites, produces no significant reduction in the efficacy of either immunizing agent. (ii) Occurrence of reactions No significant difference in missed attendances was observed between the groups inoculated simultaneously with smallpox and yellow fever vaccines, with yellow fever vaccine alone, and with an inert substance. It can, therefore, be stated that there was no large scale incidence of reactions following simultaneous vaccination with the two vaccines. Enquiry amongst the teachers at the end of the trial failed to elicit any complaint. This having been established for the small group involved in the trial, it was felt to be justifiable to proceed with the rest of the mass vaccination programme, and simultaneous vaccination with the two vaccines was performed on some 60,000 children between the ages of 5 and 18 years, without any reported ill effect. (iii) The " double scratch" technique with yellow fiver vaccine The results in Group "B," the group vaccinated twice with 17D scarification vaccine at an interval of 2 weeks, are, as has been pointed out, significantly better than those in Group "C," where vaccination was by scarification on one occasion only. The draft requirements for yellow fever vaccine for international travellers (WHO, 1957) require that not less than 95 per cent. of non-immune persons shall become immune as a result of vaccination by scarification. Two spaced vaccinations, as described, fulfil this requirement. While probably not of great interest to eentres inoculating a few casual travellers, organizations requiring to immunize large numbers of persons at about the same time, such as bodies of troops going overseas, pilgrims bound for Mecca, etc., might well welcome this innovation, using, as it does, a cheap alternative to injection vaccine, and removing the risk of syringe-transmitted hepatitis inherent in carrying out mass inoculations by injection. It must be pointed out, however, that the regulations still in force make no provision for the recognition of any 17D scarification vaccine for international travellers. Further work will be required to determine whether spacing periods other than 14 days would be satisfactory, and to discover if the two yellow fever vaccinations could be fitted in with other standard immunization procedures, such as, for instance, T.A.B. or cholera inoculations, or, indeed, whether the two vaccinations could be performed within i or 2 days of each other. The choice of the 14-day period here was primarily for convenience, though it was considered that, for the purpose of the trial, the two vaccinations should not be so close as to mimic vaccination with insertions at multiple sites on the same day, or so far apart as to risk useless neutralization of the virus at the site of inoculation on the second occasion by antibodies produced as a result of the first vaccination. The results obtained in Groups "As" and "C," after making allowance for the basal pre-vaccination immunity level, giving a conversion rate following a single vaccination by scarification with 17D vaccine of 93.1 and 94.2 per cent., respectively, came as something of a surprise. Previous work done from Yaba has always given a conversion rate of about 85 per cent. (MEERS, 1957), though this figure was recently brought into question by the
500
FURTHER OBSERVATIONS ON 1 7 D - Y E L L O W F E V E R VACCINATION
findings of DE ROEVER-BONNETet al. (1958), who obtained a 93.0 per cent. conversion rate by 17D scratch vaccination among Europeans who had never lived in a yellow fever endemic area. The present result agrees closely with this figure, and, as an explanation of the difference (which is statistically significant) an attractive theory is available. It is suggested that, in the areas in which trials have been done in the past, there has been a high rate of immunity to viruses antigenically related to the yellow fever virus (principally other arthropod-borne viruses of Casals' group "B"). This, there is some experimental evidence to suggest, results in the development of cross reacting antibodies, which might prevent or modify an infection with the 17D virus (MACNAMARAet al., 1959). In retrospect, both the large trials of 17D vaccination by scarification with which I have been associated, were carried out in lowlying forest belt areas, the very places where high infection rates with group " B " viruses might be expected. Lagos, on the other hand, almost certainly has a low group "B" virus immunity level (which may well be due to the same cause as the low incidence of immunity to yellow fever), as was demonstrated by the result of the less specific protection test (using the Asibi virus) done on the sera of the school children involved in the 1958 West African Council for Medical Research survey (MAcN~ARA, pers. commun.). The population covered in the trial reported here, therefore, probably corresponds fairly closely in this respect with De Roever-Bonnet's European group. A further pointer in this direction may be the low incidence of "inconclusive " results obtained throughout the present trial, compared with previous experience. It is suggested that an inconclusive result might well follow a modified or partially suppressed infection with the 17D virus, due to the presence of related antibodies. This might lead to an increase in the titre of these, sufficient to give an inconclusive result in the specific test, without the development of specific antibodies. Further work to clarify the question of cross-immunity in relation to yellow fever immunization is obviously required. As a result of the trial, it has been recommended that the simultaneous combined vaccination with smallpox and yellow fever vaccines at different sites should be provisionally adopted for mass immunization in West Africa, with the proviso that a watch must be maintained for reactions following vaccination, and that, at least until further experience has been gained, children below school age should not be included. It is also to be hoped that an early decision on the adoption of the proposed recommendations on minimum requirements for yellow fever vaccine (WHO, 1957) will be taken, now that the 95 per cent. conversion rate has been achieved. Before adoption, however, the requirement for scarification vaccine that 1 ml. of vaccine prepared for use should contain not less than 500,000 LDso should be modified. This high virus content is unnecessary, and would go a long way towards cancelling out one of the major advantages of scarification vaccine--its low price. A minimum titre of 100,000 LDs0 per ml., or 2,000 LDs0 per dose, would be quite adequate. It might be considered, too, that insistence upon complete bacterial sterility is unnecessary for a scratch vaccine. A maximum of 10 non-pathogenic bacteria per ml. of vaccine is infinitely better than the allowable contamination of smallpox vaccine, and this figure has been set as the maximum for the mouse-brain vaccine produced at Yaba. The inclusion of all inconclusive protection test results with the negative results for the calculation of the final conversion rates following yellow fever vaccination is taking a deliberately pessimistic view, as most, if not all, inconclusive results could probably be considered as vaccination successes. As the inclusion of inconclusive results with positive results would inevitably invite criticism of the results as a whole, it is not proposed to make any change ; but it should be pointed out that the conversion rates given may be regarded as minima.
P. D. MEERS
501
SUMMARY
A trial among Nigerian school children is described, in which simultaneous vaccination with scratch 17D yellow fever and smallpox vaccines at separate sites, and two vaccinations with scratch yellow fever vaccine at an interval of 14 days, were performed. For comparison, a group vaccinated by a single yellow fever scratch vaccination, and a group vaccinated with an inert substance, were included, the latter for comparison with the other groups, to investigate the occurrence of reactions. The results showed that both yellow fever and smallpox vaccines could be applied at separate sites, without any reduction in the efficacy of either immunizing agent, and without detecting any reactions among over 60,000 children involved. A positi-Ce reaction to the smallpox component occurred in 94.5 per cent. of cases, and to the yellow fever component in 93.1 per cent. of previously non-immune cases. The double yellow fever vaccination fully protected 98.0 per cent. of children previously non-immune. The control, single scratch yellow fever vaccinated group gave a result (94.2 per cent. conversions from negative to positive protection), somewhat better than previously obtained--a possible reason for this is discussed. REFERENCES COLLma, L. H. (1955). y. Hyg., 53, 76. DE ROEV~t-BoNN~T,H. & HO~V~STRA,J. (1958). Doc. Med. geog. trop., 10, 289. MACNAMARA,F. N., HORN,D. W. & PORTERFIELD,J. S. (1959). Trans. R. Soc. trop. Med. Hyg., 53, 202. MEmos, P. D. (1957). Ibid., 51, 338. - (1959). Ibid., 53, 196. REED, L. J. & MUENCH,H. (1938). Amer. ft. Hyg., 27, 493. SMITHntmN, K. C. (1945). y. Immunol., 51, 173. World Health Organization (1957). Tech. Rep. Set. No. 136.
Vol. 54. No. 5.
FURTHER
September, 1960.
OBSERVATIONS
SCARIFICATION,
WITH
ON
AND
17D-YELLOW WITHOUT
FEVER
VACCINATION
SIMULTANEOUS
BY
SMALLPOX
VACCINATION BY
Captain P. D. MEERS, Royal Army Medical Corps*
(From the Federal Laboratory Service Headquarters, Yaba, Lagos, Nigeria) Despite the serological demonstration of the recent presence of the virus of yellow fever a few miles from Lagos, an investigation carried out by the West African Council for Medical Research, in 1958, showed a remarkably low level of immunity to the disease among Lagos school children (Macnamara, pets. commun). As a result of this survey, the M.O.H., Lagos, decided to institute a vaccination programme, using the 17D-mouse brain scratch yellow fever vaccine produced at Yaba. This decision, affecting some 60,000 children, gave rise to a most valuable opportunity for carrying out certain experiments. T h e s e are the subject of this paper. T h e use of a combined smallpox-yellow fever vaccine (the two viruses being mixed, and freeze-dried together in the same ampoule) was shown to be relatively ineffective in an experiment carried out recently (MEERS, 1959). However, as it was felt that a simultaneous vaccination with the two agents was desirable in West Africa for reasons of speed and economy, it was decided to use the present opportunity to test the simultaneous administration of smallpox and yellow fever vaccines, at separate sites. T h e dictum of the Ministry of Health (London), concerning the spacing of vaccinations with these two agents, made it necessary to watch closely for any untoward reaction to this procedure. In addition, it was decided that, in an effort to reduce the approximate 15 per cent. of failures to become demonstrably immune, found in the past among persons vaccinated against yellow fever by scratch with 17D vaccines, a group of persons would be vaccinated twice by this technique, allowing an interval to elapse between vaccinations. MATERIALS AND METHODS
(i) Smallpox vaccine This was a Lister type dried vaccine (COLLIER, 1955), prepared in the Smallpox Vaccine Laboratory, Yaba. It was applied, after reconstitution with 20 per cent. glycerine in Macllvaine's buffer, by the multiple pressure method, at a single site. Vaccine for the trial was all drawn from one batch. * Thanks are due to Dr. C. M. Norman-Williams, Chief Medical Adviser to the Federal Government of Nigeria, for permission to publish. I am grateful to Dr. O. Adeneyi-Jones, M.O.H., Lagos, Dr. F. N. Macnamara, West African Council for Medical Research, and Dr. D. W. Horn, Medical Statistician, Federal Ministry of Health, Lagos, all of whom gave valuable assistance. Major-General G. T. L. Archer, Director of Pathology, the War Office, London, was responsible for the suggestion that led to the trial of the " double scratch " yellow fever vaccination technique.
494
FURTHER OBSERVATIONS ON 1 7 D - Y E L L O W FEVER VACCINATION
(ii) Yellow fever vaccine T h e 17D mouse brain vaccine prepared by the Yellow Fever Vaccine Laboratory, Yaba, was used. This-was reconstituted with 2 per cent. gum arabic solution, and applied by Messrs. Down Bros. " vaccinostyle." Th e blade of this, when dipped into the reconstituted vaccine, has been found to pick up approximately 0.02 ml. By placing the two sides of the blade in succession at two sites about one inch (2.5 cm.) apart, this volume of vaccine was transferred to the skin of the arm over the deltoid. Four scratches, arranged in a cross hatched manner, were made through each of the drops, using the point of the vaccinostyle, and finally, the vaccine was rubbed into the scratches with the flat of the blade. Once the vaccine had been reconstituted, any remaining unused after one hour was discarded. Vaccine for the trial was all drawn from a single batch, sample ampoules of which were titrated after all the vaccinations had been completed, to estimate the dose of virus applied to each child. (iii) Treatment of the skin Before either vaccination, the skin was cleansed with a swab barely moistened with water. dressings were applied to the smallpox or to the yellow fever vaccination sites.
No
(iv) Design of the trial Four schools were selected, as being easily accessible, with a good disciplinary standard, and having a total of between 1,600 and 2,000 children over the age of 10 years, this lower age-limit being chosen solely with an eye to the collection of blood samples by venepuncture. Each class involved in each of the schools was divided into four groups, as nearly as possible equal in size, allocation being determined by the initial letter of the surname. These groups were called " A " , " B , " " C , " and " D " . Thus, between 400 and 500 children were allotted to each group. T h e groups were treated as described below. Just before vaccination, 59 children, chosen at random, were bled, the serum obtained being used to confirm that the low incidence of immunity found during the survey carried out by the West African Council for Medical Research applied to the schools selected for the trial.
Group " A " T h e children in this group were vaccinated simultaneously on opposite arms with the two vaccines. ~I'he smallpox reaction was examined 7 days later. Twenty-eight days after vaccination, as many as possible of the children were bled, t o obtain serum for protection testing.
Group "B" T h e children in this group were vaccinated by the standard scarification technique, with yellow fever vaccine alone. This was repeated after 14 days. Twenty-eight days after this second vaccination senarn was obtained from as many as possible, for protection testing.
Group "C" These children were vaccinated as for group " B , " but without the second vaccination. Blood specimens were coUected from half the group after 28 days, for comparison of protection test results with those from group " A , " and from the other half after 42 days, for comparison with group " B . "
Group " D " T h e children in this group were vaccinated with an inert substance, resembling vaccine. Blood specimens collected from this group were used for estimating the pre-vaccination level of immunity in the " population " used for the trial.
Observation for reactions As it was not thought to be desirable to suggest, to the children or to their teachers, the possibility that reactions might follow vaccination, it was decided that their occurrence would be investigated, in the first instance at least, by comparing the attendance records for the 21 days following vaccination, of the children in Groups "A," " C , " and " D . " This was done retrospectively, by examining class registers. (v) Protection tests T h e test used to detect the presence or absence of yellow fever antibodies in the sera collected was Smithburn's intraperitoneal test in immature mice, (SMITrlBURN, 1945). T h e results were interpreted as positive, inconclusive, or negative, according to the convention laid down by the World Health Organization Expert Committee (WHO, 1957). All inconclusive results were re-tested as part of a normal test " run," they were not tested specially using a reduced challenge virus dose. T h e m i n i m u m challenge dose employed in any " run " was 220 LDs0, though the virus titre (titrated by the intraperitoneal route in mice of the same age as those used for the test) was usually far in excess of this.
P. D. MEEBS
495
RESULTS
(i) Vaccinations A total of 1,653 children were vaccinated in all four groups in the four schools visited. Details of this total, broken down according to group and school, are given in Table I. The schools involved are numbered one to four. TABLE I. Details of children vaccinated, divided according to Group, and school. 4
Group School
A
]
B C (For definition of Groups, see p. 494)
D
1
85
88
84
84
2
77
81
78
81
3
128
127
135
126
4
125
116
118
120
415
412
415
411
Totals 1,653
(ii) Blood specimens collected A total of 1,204 blood specimens were collected. This total was made up by 357 samples from Group "A," 321 from Group "B," 292 from Group " C " and 175 from Group " D . " To the total for Group " D " were added the 59 specimens collected at the outset of the trial, as described above. None of the 175 specimens from Group " D " was obtained from among the 59 individuals bled earlier. Group "C" was divided into two sub-groups, according to whether the samples were collected 28 or 42 days after vaccination. Group "C1" (28 day specimens) numbered 165, and Group "C~" (42 day specimens) numbered 127. (iii) Yellow fever vaccine titration results In order to estimate the dosage of yellow fever vaccine applied per child, four ampoules of the vaccine batch used were taken, and each reconstituted in gum arabic solution, as for use in vaccination. The contents of two of the ampoules were pooled, and titrated at once, the other two were pooled, allowed to stand at room temperature for one hour, and then titrated. Titration was by intracerebral inoculation of tenfold dilutions of the vaccine in bovine albumin-saline solution into 35-day-old Swiss albino mice. Six mice were used at each dilution, and the end-point calculated by the method of R~ED and MUENCH (1938). By taking the geometric mean of the results of the two titrations, the average dose applied per child was found to be 16,000 LDs0. (iv) Attendance records In order to determine whether the vaccination procedures caused any serious reactions, the attendance registers for the children involved in the trial were examined for the 21 days following vaccination, and any absences recorded. By comparing the average number of missed attendances over the period for Groups "A" and "C" with the number in control
496
FURTHER OBSERVATIONS ON 17D-YELLOW FEVER VACCINATION
group " D , " it was clear that there was no increase in absenteeism in the former two groups. Where necessary, enquiry was made to determine the cause of prolonged absence of any individual child. (v) Smallpox vaccination results Readings of the results of smallpox vaccinations, after 7 days, in Group " A , " were recorded either as positive (presence of a definite " take "), or negative. Of the 415 children vaccinated in Group " A " , 17 were absent from school, or could not be found, one week later, when readings were made. Of the 398 remaining, 375 (94.5 per cent.) showed a positive reaction. (vi) Yellow fever protection test results The results of the serological tests for the presence of yellow fever antibodies in the sera collected are given in Table II. The results of the tests on Group " A " sera are given in greater detail than the rest, for reasons which will appear later. The number of sera tested shown differs slightly from the total specimens collected, given in section (ii) above. The discrepancies are accounted for by sera rejected due to bacterial contamination, insufficiency of the specimen, and, in one case, loss by accidental spilling. Only 200 specimens, selected at random, were tested from Group " D , " as a sufficiently accurate estimate of the occurrence of natural immunity among the children could be obtained by examining this number. TABLE II.
Results of yellow fever protection tests on the sera collected. indicate percentages.
Group
The figures in brackets
Results Total sera tested
i School 70
+
4-
50 (71.4)
(1.4)
1
19 (27.2)
2
70
48 (68.6)
3
108
104 (96.3)
(0.9)
3 (2.8)
95 (90.5)
2 (1.9)
(7.6)
22 (31.4)
A
4
105
1
8
B
309
303 (98.1)
2 (0.6)
4 (1.3)
Cl
165
159 (96.4)
1
(0.6)
5 (3. o)
4 (3.2)
6 (4.7)
C2
127
117 (92.1)
D
200
10 (5. o)
K e y : + = Positive (protective) result. protective) result.
:k = Inconclusive result.
190 (95.0) -- = Negative (non-
P. D. MEERS
497
The results of the protection tests given were compared by statistical analysis, to show whether the differences observed between the various groups could have occurred by chance, or whether the differences are significant. This was done by applying the g 2 test to the results obtained in the various groups, taking the inconclusive and negative results together, as vaccination failures. Yates's correction for discontinuous distributions was applied. A superficial examination of the results in Group "A," showed that they varied considerably, schools one and two giving poor results compared with schools three and four. The possible reasons for this will be discussed later, but the application of the g s test revealed that the differences were very highly significant (Zs = 33.003 ; p ---- 40.001), and for this reason Group "A" has been divided into two sub-groups, "AI" comprising the poor results o b tained in schools one and two, and "As" the better results from schools three and four. Application of the Zs test to Groups "C1" and "Cs" revealed that the difference between them was not significant (gs = 1.737 ; 0 . 1 < p < 0 . 2 ) , and that, therefore, the results may be combined, to give a single set of figures for Group "C." Next, the g s test was used to compare the results obtained in Groups "At," "As," and "B" with those obtained in " control " Group "C." This revealed that, as might be expected, Group "AI" differed very significantly from Group "C," while Group "As" did not (gs = 0.104 ; 0 . 7 ~ p < 0 . 8 ) . The results from Group "B" were significantly better than those obtained in Group " C " (Zs = 4.371 ; 0 . 0 2 < p < 0 . 0 5 ) . Lastly, to calculate the true conversion rate, from the non-immune to the immune state, produced by each of the procedures, it was necessary to correct each of the results obtained in the Groups "A" to "C" for the pre-existing immunity rate, found in Group "D." This was allowed for, and the actual conversion rates calculated, Observed conversion rate °/o - - Basal immunity °/o x 100) 100 - - Basal immunity % the results are given in Table III. Inconclusive results have again been regarded as vaccination failures in preparing this table. (True conversion rate
TABLE I I I .
%
A c t u a l yellow fever c o n v e r s i o n rates f r o m t h e n o n - i m m u n e to t h e i m m u n e state, p r o d u c e d by each of the vaccination procedures described.
Per cent, vaccination successes 68.4 Group A 93.1 Group B
98.0
Group C
94.2
DISCUSSION
The trial described had, as its objective, the solution of the three problems, summarized below. 1) Does the simultaneous application of smallpox and yellow fever vaccines, at different sites, result in any reduction in the efficacy of either as an immunizing agent? 2) Do serious reactions commonly follow simultaneous vaccination with smallpox and yellow fever vaccines?
G
498
FURTHER OBSERVATIONS ON 17D-YELLOW FEVER VACCINATION
3) Do two scratch vaccinations, spaced in time, with yellow fever vaccine, produce a higher rate of immunity than that produced by a single scratch on one occasion only? In this discussion, an attempt is made to answer these three questions in the light of the results of the trial, and to suggest an explanation for a further important and somewhat unexpected finding. (i) Simultaneous smallpox and yellow fever vaccination The reaction to the smallpox component of the double vaccination in Group " A " was most satisfactory, the 94.5 per cent. " take " rate being scored in a population well covered by previous vaccinations, particularly during the smallpox epidemic of 1957. Most of the reactions were of the " accelerated " variety, only a very few typical primary vaccinial lesions being seen. This result reflects very closely previous experience with Yaba-produced dried smallpox vaccine. As has already been pointed out, the results of yellow fever protection tests on the sera collected from Group "A" are not uniform. The unfortunate and very significant difference observed between Groups "AI" and "As," both of which were, at first sight, treated similarly, requires elucidation. Many possible explanations for this are ruled out at once by the fact that the results from all other groups were uniform throughout for all four schools. If the results obtained in Group "AI" were to be taken as correct, it must be postulated that this was caused by interference (in its widest sense) between the two vaccines, and, in this case, to explain the higher result in Group "A2" (which agrees with the result found after yellow fever vaccination alone in Group "C"), it follows that many individuals in this group could not have been vaccinated at all with smallpox vaccine, or were vaccinated with vaccine that was not potent. This is disproved by the high smallpox vaccination " take " rate, and the fact that the few failures which did occur were spread evenly through all four schools. If, on the other hand, the high rate in Group "As" were to be accepted as correct, the low Group "AI" result can be explained by assuming either that non- or marginally-potent yellow fever vaccine was used in this group, or that some individuals in the group escaped vaccination. The former assumption can be ruled out, as the vaccinators performing the yellow fever vaccination in Group "A" also used the same vaccine for Groups "B" and "C." The second assumption is almost certainly the correct one, as shown by the following explanation. In carrying out the vaccinations, in order to disturb the schools as little as possible, all four groups were vaccinated concurrently. An entire class was called at a time, divided into four groups, and vaccinated. Children in three of the Groups ("B," "C," and "D") were each vaccinated on one arm only, the fourth group, Group "A," being vaccinated on both arms. On the first 2 days of the trial, when the schools numbered one and two were dealt with, supervisory activity was mainly directed towards watching for, and correcting, errors in technique among the vaccinators, who had not previously carried out yellow fever vaccinations by the scratch method. On the next 2 days, when schools three and four were visited, the vaccinators by this time being proficient, supervisory activity was transferred to controlling the children. It was then noticed that there was a tendency, among some children allocated to Group "A," (all of whom received the smallpox vaccination first), to assume that they could go after one vaccination, as did the children in all the other groups, and to avoid the second vaccination, against yellow fever. This tendency was checked on days 3 and 4, but almost certainly occurred undetected on days 1 and 2, a certain number of children, thus, not being vaccinated at all on these days. This would, of course, explain
P. D. MEERS
499
the lower conversion rate in Group "AI," and it is suggested that reasonable justification exists for the acceptance of the results obtained in Group "As" as the true conversion rate for the combined vaccination procedure. This result does not, as has already been pointed out, differ significantly from that obtained in Group "C." Thus, it can be stated that the performance of smallpox and yellow fever vaccinations simultaneously, at different sites, produces no significant reduction in the efficacy of either immunizing agent. (ii) Occurrence of reactions No significant difference in missed attendances was observed between the groups inoculated simultaneously with smallpox and yellow fever vaccines, with yellow fever vaccine alone, and with an inert substance. It can, therefore, be stated that there was no large scale incidence of reactions following simultaneous vaccination with the two vaccines. Enquiry amongst the teachers at the end of the trial failed to elicit any complaint. This having been established for the small group involved in the trial, it was felt to be justifiable to proceed with the rest of the mass vaccination programme, and simultaneous vaccination with the two vaccines was performed on some 60,000 children between the ages of 5 and 18 years, without any reported ill effect. (iii) The " double scratch" technique with yellow fiver vaccine The results in Group "B," the group vaccinated twice with 17D scarification vaccine at an interval of 2 weeks, are, as has been pointed out, significantly better than those in Group "C," where vaccination was by scarification on one occasion only. The draft requirements for yellow fever vaccine for international travellers (WHO, 1957) require that not less than 95 per cent. of non-immune persons shall become immune as a result of vaccination by scarification. Two spaced vaccinations, as described, fulfil this requirement. While probably not of great interest to eentres inoculating a few casual travellers, organizations requiring to immunize large numbers of persons at about the same time, such as bodies of troops going overseas, pilgrims bound for Mecca, etc., might well welcome this innovation, using, as it does, a cheap alternative to injection vaccine, and removing the risk of syringe-transmitted hepatitis inherent in carrying out mass inoculations by injection. It must be pointed out, however, that the regulations still in force make no provision for the recognition of any 17D scarification vaccine for international travellers. Further work will be required to determine whether spacing periods other than 14 days would be satisfactory, and to discover if the two yellow fever vaccinations could be fitted in with other standard immunization procedures, such as, for instance, T.A.B. or cholera inoculations, or, indeed, whether the two vaccinations could be performed within i or 2 days of each other. The choice of the 14-day period here was primarily for convenience, though it was considered that, for the purpose of the trial, the two vaccinations should not be so close as to mimic vaccination with insertions at multiple sites on the same day, or so far apart as to risk useless neutralization of the virus at the site of inoculation on the second occasion by antibodies produced as a result of the first vaccination. The results obtained in Groups "As" and "C," after making allowance for the basal pre-vaccination immunity level, giving a conversion rate following a single vaccination by scarification with 17D vaccine of 93.1 and 94.2 per cent., respectively, came as something of a surprise. Previous work done from Yaba has always given a conversion rate of about 85 per cent. (MEERS, 1957), though this figure was recently brought into question by the
500
FURTHER OBSERVATIONS ON 1 7 D - Y E L L O W F E V E R VACCINATION
findings of DE ROEVER-BONNETet al. (1958), who obtained a 93.0 per cent. conversion rate by 17D scratch vaccination among Europeans who had never lived in a yellow fever endemic area. The present result agrees closely with this figure, and, as an explanation of the difference (which is statistically significant) an attractive theory is available. It is suggested that, in the areas in which trials have been done in the past, there has been a high rate of immunity to viruses antigenically related to the yellow fever virus (principally other arthropod-borne viruses of Casals' group "B"). This, there is some experimental evidence to suggest, results in the development of cross reacting antibodies, which might prevent or modify an infection with the 17D virus (MACNAMARAet al., 1959). In retrospect, both the large trials of 17D vaccination by scarification with which I have been associated, were carried out in lowlying forest belt areas, the very places where high infection rates with group " B " viruses might be expected. Lagos, on the other hand, almost certainly has a low group "B" virus immunity level (which may well be due to the same cause as the low incidence of immunity to yellow fever), as was demonstrated by the result of the less specific protection test (using the Asibi virus) done on the sera of the school children involved in the 1958 West African Council for Medical Research survey (MAcN~ARA, pers. commun.). The population covered in the trial reported here, therefore, probably corresponds fairly closely in this respect with De Roever-Bonnet's European group. A further pointer in this direction may be the low incidence of "inconclusive " results obtained throughout the present trial, compared with previous experience. It is suggested that an inconclusive result might well follow a modified or partially suppressed infection with the 17D virus, due to the presence of related antibodies. This might lead to an increase in the titre of these, sufficient to give an inconclusive result in the specific test, without the development of specific antibodies. Further work to clarify the question of cross-immunity in relation to yellow fever immunization is obviously required. As a result of the trial, it has been recommended that the simultaneous combined vaccination with smallpox and yellow fever vaccines at different sites should be provisionally adopted for mass immunization in West Africa, with the proviso that a watch must be maintained for reactions following vaccination, and that, at least until further experience has been gained, children below school age should not be included. It is also to be hoped that an early decision on the adoption of the proposed recommendations on minimum requirements for yellow fever vaccine (WHO, 1957) will be taken, now that the 95 per cent. conversion rate has been achieved. Before adoption, however, the requirement for scarification vaccine that 1 ml. of vaccine prepared for use should contain not less than 500,000 LDso should be modified. This high virus content is unnecessary, and would go a long way towards cancelling out one of the major advantages of scarification vaccine--its low price. A minimum titre of 100,000 LDs0 per ml., or 2,000 LDs0 per dose, would be quite adequate. It might be considered, too, that insistence upon complete bacterial sterility is unnecessary for a scratch vaccine. A maximum of 10 non-pathogenic bacteria per ml. of vaccine is infinitely better than the allowable contamination of smallpox vaccine, and this figure has been set as the maximum for the mouse-brain vaccine produced at Yaba. The inclusion of all inconclusive protection test results with the negative results for the calculation of the final conversion rates following yellow fever vaccination is taking a deliberately pessimistic view, as most, if not all, inconclusive results could probably be considered as vaccination successes. As the inclusion of inconclusive results with positive results would inevitably invite criticism of the results as a whole, it is not proposed to make any change ; but it should be pointed out that the conversion rates given may be regarded as minima.
P. D. MEERS
501
SUMMARY
A trial among Nigerian school children is described, in which simultaneous vaccination with scratch 17D yellow fever and smallpox vaccines at separate sites, and two vaccinations with scratch yellow fever vaccine at an interval of 14 days, were performed. For comparison, a group vaccinated by a single yellow fever scratch vaccination, and a group vaccinated with an inert substance, were included, the latter for comparison with the other groups, to investigate the occurrence of reactions. The results showed that both yellow fever and smallpox vaccines could be applied at separate sites, without any reduction in the efficacy of either immunizing agent, and without detecting any reactions among over 60,000 children involved. A positi-Ce reaction to the smallpox component occurred in 94.5 per cent. of cases, and to the yellow fever component in 93.1 per cent. of previously non-immune cases. The double yellow fever vaccination fully protected 98.0 per cent. of children previously non-immune. The control, single scratch yellow fever vaccinated group gave a result (94.2 per cent. conversions from negative to positive protection), somewhat better than previously obtained--a possible reason for this is discussed. REFERENCES COLLma, L. H. (1955). y. Hyg., 53, 76. DE ROEV~t-BoNN~T,H. & HO~V~STRA,J. (1958). Doc. Med. geog. trop., 10, 289. MACNAMARA,F. N., HORN,D. W. & PORTERFIELD,J. S. (1959). Trans. R. Soc. trop. Med. Hyg., 53, 202. MEmos, P. D. (1957). Ibid., 51, 338. - (1959). Ibid., 53, 196. REED, L. J. & MUENCH,H. (1938). Amer. ft. Hyg., 27, 493. SMITHntmN, K. C. (1945). y. Immunol., 51, 173. World Health Organization (1957). Tech. Rep. Set. No. 136.