Development of antibody following vaccination of infants and children against pneumococci

D E V E L O P M E N T OF ANTIBODY FO L LO WIN G VACCINATION OF I N F A N T S AND C H I L D R E N AGAINST PNEUMOCOCCI HORACE L . HODES,

M.D.,

J A M E S F . ZIEGLER,

Ja., M.D.,

AND

D. ZEPP BALTIMORE, MD. HELEN

the past five years 65 per cent of the cases of pneumococeus D URING pneumonia treated at the Harriet Lane Home have been caused by five types of organisms, types I, VI, XIV, XV, and XIX. I In addition, these same organisms have been the cause of very many upper respiratory refections and complications, such as otitis media, which follow them. Pneumoeoccus infections among infants and children respond so readily to treatment with sulfonamides that it does not seem advisable to attempt mass immunization of children against these organisms. However, the spread of pnemnococcus infections among infants on hospital wards 1 and in orphanages may become such a serious problem that immunization directed against the common disease-producing types of pneumoeocci might on occasion be a useful procedure in such institutions. In order to be of value immunization must be simple and harmless. The immunity induced must rise rapidly to effective levels and must remain elevated for at least several months. The available information concerning the antibody response of infants and children to vaccination with pneumococei or their products is limited. Felton 2 has included a few infants in his experiments in the use of pneumococeus capsular polysaccharide as the antigenic agent and Davies, ~ using a similar preparation, has studied antibody response in a group of sixteen infants and children. Gundel and SchMer ~ in the course of a series of studies on immunity to pneumococcus in infants and children, vaccinated a few infants with a whole cell, heat-killed vaccine. All the experiments cited gave similar results and appeared to indicate that older children respond to the whole pneumococeus or to its capsular polysaccharide by the production of antibodies in the same manner as do adults. However, this capacity is not possessed by young infants but is acquired only toward the end of the second year of life. We have studied the production of pneumococcus mouse-protective antibodies in the serum of a number of infants and children following vaccination with heat-killed type I and type VI pneumoeocei. Most of the subjects were given the vaccine by intradermal ~noculation. In a small group type I vaccine was administered by inhalation of a fine mist sprayed from a nebulizer. F r o m the H a r r i e t Lane Home, the J o h n s Hopkins Hospital; the D e p a r t m e n t of Pediatrics, the Johns Hopkins University; a n d Sydenham Hospital, Baltimore City Health Department. This study w a s supported by a g r a n t from the John and Mary R. Markle Foundation. 641

642

T H E J O U R N A L OF PEDIATRICS

The patients included in the s t u d y were children a d m i t t e d to the H a r r i e t L a n e H o m e or to S y d e n h a m Hospital. The g r o u p was made up, in part, of children who had r e c e n t l y recovered f r o m an acute infection and, in part, of those a d m i t t e d to the hospital for s t u d y of sohie neurologic or metabolic disorder. W e a t t e m p t e d to avoid choosing children who h a d h a d recent pneumoeoccus infections, but in two instances ehildreu were v a c c i n a t e d who were well along in convalescence f r o m a pneumocoecus infection Caused by an u n r e l a t e d type. The ages of the children in the s t u d y varied from 2 weeks to 13 years. METHODS

Preparation of Vaccine.Type I: A p p r o x i m a t e l y 15 m]. of a twenty-four-hour blood broth ( p H , 7.8) culture of a virulent pne(lmococeus type I were seeded into a p p r o x i m a t e l y 300 ml. of 5 per cent rabbit plasma infusion broth (pH, 7.8) and allowed to grow at 37.5 ~ C. for eight hours. A t this time a portion of the broth culture was removed for dilution plate counts and the remainder was heat killed by exposure for thirty-five minutes in a water bath previously adjusted to 60 ~ C. A f t e r rapid cooling the vaccine was stored overnight at icebox temperatnre. The following morning the killed culture was centrifuged, the s u p e r n a t a n t discarded, and the organisms resuspcnded in a p p r o x i m a t e l y one-tenth the original volume of infusion broth ( p i t , 7.8). The vaccine was cultured aerobically and anaerobically for forty-eight hours before use and was ordinarily not used more than forty-eight hours a f t e r this. Occasionally, because of contamination of a fresh lot, vaccine was used for as long as ten days. A f t e r this period of time the organisms still showed good capsular swelling in homologous serum and retained their gram-positive characteristics. Before use, the volume of the vaccine was so adjusted that 1 c.c. represented approximately 1,000 million killed organisms. Type VI: The procedure was identical with that for type I, but the organism employed was much less virulent for mice t h a n was the type I. The type V I vaccine also contained a p p r o x i m a t e l y 1,000 million heatkilled organisms per milliliter. Vaccination by Intradermal Inoculation.--Blood was d r a w n f r o m each child before vaccination. The vaccine was given i n t r a d e r m a l l y in the f o r e a r m on two occasions a week apart. Blood was again drawn for mouse protection test a week after the second inoculation of vaccine. In most cases the amount of vaccine given was 0.15 m]. at each injection, but occasionally the first dose was 0.1 ml. The total dosage represented a p p r o x i m a t e l y 300 million killed organisms, except in the case of two children, Ri. P. and Ra. P. (Table I ) , who received 1,000 million organisms in a single dose. Vaccination by Nasal Spra~j.--Seven children received t y p e I vaccine sprayed as a fine mist f r o m a nebulizer applied to the nasal orifice. Amounts of vaccine v a r y i n g f r o m 0.5 to 3.0 ml. were administered at

H O D E S E T AL. :

each spraying.

PNEUNIOCOCCUS VACCINATION

643

This procedure was carried out once or twice weekly for

a total of from three to seven inhalations. The total amount of vaccine

sprayed into the nose of each child varied from 2,000 million to 20,000 million organisms (Table I I I ) , but it is impossible to estimate the effective dose. As in the ease of i n t r a d e r m a l vaccination, bleeding was done before s p r a y i n g was begun and at v a r y i n g intervals during and following the course of vaccination. Reactions: No reaction of a n y k i n d was observed a m o n g the children vaccinated b y nasal spray. None of the children inoculated intrad e r m a l l y exhibited a n y rise of t e m p e r a t u r e or a systemic reaction, but n e a r l y all of t h e m showed a local reaction at the site of inoculation which resembled a m o d e r a t e l y positive Schick test. I n each ease there was e r y t h e m a and i n d u r a t i o n of v a r y i n g extent which p e r s i s t e d for about f o r t y - e i g h t hours a f t e r vaccination, and a small central papule at the site of injection r e m a i n e d for three or four days. The center of this papule occasionally became necrotic and crusted. A small area of p i g m e n t a t i o n was still visible at the end of several weeks. In no instance did the local reaction cause g r e a t discomfort, and none was extensive enough to cause alarm.

Mouse Protection Tests.-Type I: W h i t e Swiss mice weighing f r o m 18 to 22 Gin. were used. The pneumococeus t y p e I was the same organism as t h a t used to prep a r e the vaccine and was of such virulence t h a t 0.25 ml. of a 10 -7 dilution of a t w e n t y - f o u r - h o u r culture was r e g u l a r l y f a t a l f o r mice. Three mice were used for each culture dilution, the serum dilution being constant at 1:2. Rarely, w h e n the a m o u n t of serum available was small, the test serum was diluted 1:10. E a c h mouse received 0.25 ml. of the test s e r u m i n t r a - a b d o m i n a l l y , followed i m m e d i a t e l y b y the same volume of culture in the dilution being tested. N o r m a l rabbit s e r u m was used as control. The mice were observed for seventy-two hours; mice r e m a i n i n g a l i v e and well at the end of t h a t period were considered survivors. Type VI: The pneumococcus t y p e V I was much less virulent for mice. A sixteen-hour culture, 0.25 ml. of a 10 -s dilution, killed control mice with r e g u l a r i t y , although the end point was often not sharp. I n an a t t e m p t to compensate for the low virulence of the organism, mice a b o u t 3 weeks old and weighing f r o m 7 to 12 Gm. were used, and the test sera were diluted 1:10 in some of the tests. W i t h these exceptions the tests were carried out as for t y p e I. I n calculating the mouse-protective t i t e r of the scra, no difficulty was e n c o u n t e r e d in d e t e r m i n i n g the end point o f those tests carried out against the t y p e I pneumocoecus. I n tests done with the t y p e V I organisms, however, much more s c a t t e r e d results were f r e q u e n t l y obtained. I n order to clarify the i n t e r p r e t a t i o n of the data, the 50 per cent m o r t a l i t y end point f o r m u l a of Reed and Mueneh 5 was applied.

644

THE

JOURNAL

OF

PEDIATRICS

The logarithmic difference b e t w e e n the n u m b e r of 50 p e r cent mort a l i t y doses protected against by the control serum as c o m p a r e d with the test serum has been expressed (Table I) as the n e x t lowest r o u n d number. F o r example, in Table I the serum-protective p o w e r of patient Ri. P. a f t e r vaccination is r e c o r d e d as 300,000 50 p e r cent mort a l i t y doses. This means t h a t 300,000 times the n u m b e r of organisms were required to kill 50 per cent of the mice t r e a t e d with the p a t i e n t ' s s e r u m as were necessary t o kill 50 per cent of those t r e a t e d w i t h the control serum. That is, the p a t i e n t ' s serum e x h i b i t e d 300,000 times the mouse protective capacity shown b y the control serum. 9R E S U L T S

Type / . - - T h e titers of mouse-protective antibody against type I pneumococcus following i n t r a d e r m M vaccination were determined' one week a f t e r the completion of vaccination and are recorded in Table I. The occurrence of significant amounts of a n t i b o d y in the s e r u m was uncommon before vaccination. Such titers were d e m o n s t r a t e d in only three sera obtained f r o m children over 2 years of age. W i t h i n one week following vaccination, however, there was a sharp rise in titer a m o n g the children over 2 y e a r s of age. A m o n g this g r o u p the sera of seven of the fifteen children p r o t e c t e d mice against 100,000 or more 50 p e r cent m o r t a l i t y doses of t y p e I pneumoeocei. Seven others were effective against f r o m 10,000 to 50,000 50 per cent m o r t a l i t y doses, while the one r e m a i n i n g serum afforded protection against 5,000 doses. As recorded in Table I, none of the sixteen children u n d e r 2 years of age showed a significant titer of mouse-protective a n t i b o d y before vaccination , and f o u r t e e n of these children failed to develop a significant increase in titer a f t e r vaccination. The serum of one of the two remaining' children gave p r o t e c t i o n against 10,000, the other against 150,000 50 per cent m o r t a l i t y doses. The sera of four of the children over 2 years of age were also tested a n u m b e r of months a f t e r vaccination. In each ease the mousep r o t e c t i v e titer was still at a high level. The sera of one child, C.L., p r o t e c t e d against 50,000 50 p e r cent m o r t a l i t y doses one w e e k a f t e r vaccination and showed a t i t e r of 100,000 two months later. The titer of the serum of C. K. was 20,000 one week after vaccination and 50,000 a f t e r an i n t e r v a l of tire months, while tile serum of D. 3/[. rose f r o m 15,000 one week a f t e r vaccination to 100,000 three months later. The f o u r t h child, S.T., exhibited a decline in titer f r o m 50,000 one week a f t e r vaccination to 5,000 w h e n the s e r u m was tested a f t e r a period of four months. This l a t t e r figure, however, represents a significant increase over his prevaceination serum protective titer which was zero. The three children of the older g r o u p whose sera possessed a high titer of mouse-protective a n t i b o d y to t y p e I pneumoeoeeus before vaccination showed quite different reactions f r o m the rest of the group. The serum of one of these children showed only a twofold increase in

HODES ET AL. : antibody, A month

645

PNEUMOCOCCUS V&CCINATION

and the antibody levels in the other two were following vaccination, these children were bled

decreased. again and

mouse-protective antibody had returned to its original prevaccination l e v e l o r t o s l i g h t l y a b o v e it. I t is p r o b a b l e t h a t t h e s e v a r i a t i o n s a r e within the limits of error of the method of measuring antibody and are themselves of no significance except to suggest that wiih antibody levels in this range existing before vaccination the antigenic stimulus administered had little effect. TABLE I DEVELOPIVIENT OF SERUM ~r

M0~JSE-I~ ANTIBODY ~OLLOWING W I T H ]-[EAT-~4~ILLEDTYPE ] PNEUMOCOCCI PROTECTIVE

NAME

AGE

Ri. P.* Ra. P.* D.L. M.L. D.R. I~.K. M. Mc. S.T. C.L. C.K. D.M. V. tt. K.B. B.B.

4 yr. 8 yr. 7 yr. 8 yr. 7 yr. 5 yr. 8 yr. 4 yr. 6 yr. 7 yr. 5yr. 7 yr. 3 yr. 9 yr. 7 yr.

S. Ta.

TITER OF SERUM

BEFORE VACCINATION

INTRADEI%IVfAL

(510% ~[ORTALITY DOSES) 01",~.E ~'EEI'~ AFTER, VACCINATION I l

Children Over 2 Years of Age 300,000 <]o 150,000 o 150,000 o 150,000 o 150,000 ]o 100,000 o 50,000 o 50,000 o
4 I/1o. 0 0 M.A. 9 too. 0 0 R.M. 12 mo. 0 0 ]0 too. W. Wa.
Type VI.--The

results of the study

on mouse-protective

antibody

in

infants and children before and after vaccination with type VI pneu~ mococci were quite inconclusive. It is apparent that the type VI pneumococcus vaccine was less regular in its effect than was that made with the type I organisms. The results obtained with the type VI vaccine are shown in Table II. Of the fifteen children over 2 years of

646

THE JOURNAL OF PEDIATRICS

age, n i n e p o s s e s s e d s e r a w h i c h p r o t e c t e d a g a i n s t 1,000 or m o r e 50 p e r c e n t m o r t a l i t y doses of t y p e V I p n e u m o e o c e i b e f o r e v a c c i n a t i o n . Th e s e r a of t w o of t h e t w e l v e i n f a n t s s t u d i e d s h o w e d p r o t e c t i v e p o w e r in a similar range before vaccination. O n l y f o u r of t h e f i f t e e n indiv i d u a l s in t h e g r o u p o v e r 2 y e a r s o f a g e w e r e v a c c i n a t e d . O f t h e s e o n l y one s h o w e d a s i g n i f i c a n t i n c r e a s e in s e r u m a n t i b 0 d y - - f r o m a t i t e r Of 500 to 4,000 50 p e r c e n t m o r t a l i t y doses. T e n of t h e t w e l v e i n f a n t s u n d e r 2 y e a r s o f a g e w e r e v a c c i n a t e d , b u t a s i g n i f i c a n t r i s e of m o u s e - p r o t e c t i v e a n t i b o d y t i t e r f o l l o w i n g v a c c i n a t i o n was d e m o n s t r a t e d in o n l y t w o . I n one i n s t a n c e (S. B.) s e r u m d r a w n a f t e r v a c c i n a t i o n f a i l e d to p r o t e c t m i c e a g a i n s t 100 50 p e r c e n t m o r t a l i t y doses, w h i l e s e r u m d r a w n b e f o r e v a c c i n a t i o n p r o t e c t e d a g a i n s t 3,000 s u c h doses. T A B L E II

DEVELOPMENT

Oli~ SEEUM WITII

MOUSE-PROTECTIVE ~IEAT-I~ILLED TYPE

ANTIBODY FOLLOWING "VI PNEUMOCOCCI

"~rACCINATION

PROTECTIVE TITER

DOSE OF VACCINE

NAME

G.S. B.W. J.M. P.B. W. Me. J.P. P.W. L.M. Mu. W. C.B. A.N. W.S. E.J. J. tt. M.G.

AGE

8 yr. 13 yr. 7 yr. 9 yr. 12 yr. 13 yr. 3 yr. 6 yr. 7 yr. 10 yr. 13 yr. 5 yr. 13 yr. 2 yr. 7 yr.

~

( KILLED ORGANIS~S)

Children Over2 Years of Age Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated Not vaccinated 300 million 300 million 300 million 300 million

(50%

OF SERUM

M01~TALITY DOSES)

l

VACCINATIoNBEFORE [

300 300 1,000 ],000 3,000 3,000 3,000 10,000 15,000 30,000 500,000 10 10 10 5OO

ONE WEEK

AFTER VACCINATION

10 100 100 4,000

Children Under 2 Years of Age 300 million 0 0 C.R. 300 million 0 0 A.R. 300 million 0 0 300 million M.S. 10 0 300 million 1. D. 0 10 B.L. 300 million 0 10 H.S. 300 million 100 10 S.B. 300 million 3,000 100 300 million G.S. 500 10 C.C. 300 million 1,500 0 B. tt. Not vaccinated 100 F.S. Not vaccinated 1 mo. 1,000 *Each child vaccinated received two intradermal inoculations, one week apart, totaling approximately 300 million heat-killed pneumoeoeci. :B,C.

3 toO. 5 too. 8 too. 2 too. 7 too. 4 mo. 12 too. 30 too. 4 mo. 6 mo. 4 too.

Vaccination by Nasal S p r a y . - - S e v e n c h i l d r e n , all o v e r 2 y e a r s of age, r e c e i v e d t h e t y p e I v a c c i n e i n t r a n a s a l l y as a fine s p r a y f r o m a n e b u l i z e r ( T a b l e I I I ) . S i g n i f i c a n t t i t e r s of m o u s e - p r o t e c t i v e a n t i b o d y d e v e l o p e d

HODES

ET

AL. :

PNEUMOCOCCUS

647

VACCINATION

TABLE III DEVELOPMENT

OF

SERUM SPRAY

~/[OUSE-~PI~OTECTIVE ~xNTIBODY FOLLOWING OF ]~IEAT-I~ILLED TYPE I PNEUMOCOCCI PROTECTIVE

NAME

AGE

APPROXIMATE NUMBER TOTAL OF SPI~AYINGS ORGANISMS SPRAYED (BILLIONS)

I~I.IF). P.W. Ir

31 mo. 2 30 too. 2 8 yr. J.M. 5 yr. 2 R.B. 4 yr. 6 J.S. 8 yr. 4 E.L. 12 yr. 20 *Nine weeks after completion of spraying. tEighteen weeks after eomloletion of spraying.

INHALATION

OF

TITER OF sERuM

(50% MORTALITYDOSES) TITERBEFORE MAXIMUM TITER INHALATION RECORDED OFVACCINE FOLLOWING INIIALATION OFVACCINE ,~JO <]o 0 0 0

0

:10

0 5,0 2,000* I0,000" 30,000t

in only three of the children in this group. This is in contrast to the effectiveness of the intradermal inoculation of the same vaccine in children over 2 years of age.

DISCUSSION The results following intraderma] vaccination with whole cell, heatkilled vaccine prepared from pneumocoeeus type I agree with those of the investigators previously cited with r e g a r d to the failure to demonstrate an appreciable a n t i b o d y response in y o u n g infants. I t seems quite clear that the antigenic stimulus which was adequate for the p r o d u c t i o n of serum antibody in children over 2 years of age was gem erally ineffective when administered to infants u n d e r this age. However, a m o n g children over 2 years of age, the intradermM vaceination with the type I pneumoeoceus is regularly followed within a week b y a g r e a t rise in the mouse-protective titer of the serum. This increase in titer appears to remain at a significantly high level for at least a n u m b e r of m o n t h s and appears to be of n e a r l y the same order of m a g n i t u d e as that which is f o u n d following pneumocoecus pneumonia. F i n l a n d and associates," in a s t u d y of antibody response after type I pneumoeoeeus pneumonia treated with sulfonamides, have studied the mouse-protective a n t i b o d y level in fifty-six adult patients. Of these fifty-six patients, the sera of two (3 per cent) protected against 0 or 10 minimal lethal doses of type I pneumoeoeei; six (11 per cent} had titers of 100 or J,000 minimal lethal doses. The sera of nineteen (35 per cent) protected against 10,000 or 100,000 minimal lethal doses and twenty-nine (51 per cent), against a million or more minimal lethal doses. I n our group of twelve children over 2 years of age whose prevaeeination antibody titer was 100 or lower, six (50 per cent} protected against 100,000 or more 50 per cent m o r t a l i t y doses after vaeeination and five (42 per cent} d e v e l o p e d titers of 10,000 or mor% while

648

THE

JOURNAL

OF P E D I A T R I C S

the remaining child developed a titer above 1,000. In comparing the two groups it is apparent that approximately one-third of the adult patients recovering from type I pneumoeoecus pneumonia developed mouse-protective antibody titers which were of the same order of mugnitude as those induced in the children by vaccination, while the titers of one-half of the convalescent adults reached somewhat higher levels than those attained by the children. It is not clear whether or not the presence in the serum of vaccinated infants of mouse-protective antibody in amounts comparable to those of patients convalescent from pneumonia is in itself a guarantee of immunity, either complete or partial. However, it has been demonstrated that such levels o f antibody may be induced against type I pneumoeocci in children over 2 years of age with some regularity by the intradermal inoculation of an amount of antigen which does not cause systemic reactions. The administration of type I pneumocoeeus vaceine by intranasal spray apparently results in some instances in the development of mouseprotective antibody of significant titer. However, this method is not reliable and in our experienee failed more often than it suceeeded. In addition, it was very cumbersome and time-consuming and required a very mueh larger amount of antigen. A considerable portion of the vaccine administered in the spray collects in the nasopharynx and is swallowed. It is possible that the number of bacteria reaching the gastrointestinal tract is large enough to cause a rise in antibody titer, sider Ross 7 has shown that immunity may be induced in rats by feeding 5 ml. of a culture of pneumococci (probably 500 million organisms) by stomach tube. It is impossible to determine what role was played by absorption of antigen through the mueosa of the nose and throat. In the group of children over 2 years of age whose sera were tested for antibody against type VI pneumococeus, more than oDe&all showed protective titers of 1,000 50 per eent mortality doses before vaeeination. This is in striking contrast with the lack of prevaeeination antibody against type I pneumoeocei in the same age group. Only three of the twenty-two children (14 per cent) over 2 years of age whose sera were tested before vaccination showed a titer higher than 100 against the type I organism. The significance of this findiDg is not dear, but it is noteworthy that in our experience amoDg children over 2 years of age 4 1 p e r cent of the eases of pneumonia are eaused by type I pneumoeoeei and only 6 per cent by type VI. It is conceivable that the lower incidence of type VI pneumonia among children over 2 years of age is dependent, in part, upon the relatively common presence of specific antibody. There is insufficient data to judge the efficacy of the type VI vaccine empIoyed. Only four children over 2 years of age were vaccinated, and of these only one developed a high titer of antibody. Only two of the twelve infants vaccinated showed a definite response, but this finding is in keeping with that observed with the type I vaccine.

IIODES

ET

AL.

:

PNEUMOCOCCUS

VACCINATION

649

CONCLUSIONS

1. Among children over 2 years of age, intradermal inoculation of a dose of heat-killed type I pneumococei which did not cause systemic reactions or serious local reactions was followed in nearly every instance by an abrupt rise in serum mouse-protective antibody titer. The level of antibody attained appears to be of the same order of magnitude as has been reported in adults following type I pneumoeoccus pneumonia treated with su]fonamides. 2. The rise in antibody titer following vaccination persisted for at least several months. 3. With few exceptions, no rise in antibody titer following intradermal vaccination with type I pneumococci was demonstrated among infants under 2 years of age. 4. hlhalation by children over 2 years of age of a spray of killed type I pnemnococci was followed in a few instances by a significant rise in mouse-protective antibody titer. However, this method of vaccination was unreliable and cumbersome. 5. Intradermal vaccination with type VI pneumocoeeus vaccine yielded equivocal results. 6. Among children over 2 years of age, a high prevaceination titer of mouse-protective antibody against type VI pneumococeus was found much more often than against type I pneumococei. REFERENCES 1. 2. 3. 4. 5. 6. 7.

Hodes, I-I. L , Ziegler, J. E , and Zepp, H . D . : To be published. Felton, L . D . : Pub. }Iealth Rep. 53: 1855, 1938~ Davies~ J-. A.: J. ImmunoL 33: 1, 1937. Gunde], M., and Sehifer, 0.: Arch. f. iqyg. 108: 9~j 1932. Reed~ L. J., and Muench, l-I.: Am. J. ISyg. 27: 493j 1938. ]~'inland, ~f., Strauss, E., and Peterson, O . L . : Ann. Int. 3~[ed. 16: 1, 1942. Ross, u J. Exper. 1Ked. 5[[: 585, 1930.