With any preparation of multiple antigens, a basic question is the possibilityof immunologic interference or enhancement. This paper reports a studyon 48 infants from two to four months of age who received a multiplevaccine against diphtheria, tetanus, pertussis, and poliomyelitis.Maternal antibodies, when present, appeared to suppressboth the primary and booster response to the severalantigenic components.

SEROLOGIC RESPONSE OF INFANTS TO A MULTIPLE VAC-

CINE FOR SIMULTANEOUS IMMUNIZATION AGAINST

DIPHTHERIA, PERTUSSIS, TETANUS, AND POLIO-

MYELITIS, IN RELATION TO THE PRESENCE

OF SPECIFIC MATERNAL ANTIBODY

Gordon C. Brown, Sc.D., F.A.P.H.A., and Pearl L. Kendrick, Sc.D., F.A.P.H.A.

THERE ARE obvious advantages in theuse of a preparation of multiple anti-

gens for simultaneous immunizationagainst several diseases. Decreasing thenumber of injections, for example, be-comes an important objective with theever-increasing number of specific an-tigens available for active immuniza-tion; it is important, too, in relation tothe current emphasis on early immu-nization of infants. A growing numberof combined prophylactics have beensuggested, and some have been sub-jected to field study. Triple vaccinecontaining diphtheria and tetanus tox-oids and pertussis vaccine (DPT) hasbeen in common use in the UnitedStates for a number of years; andrecently, studies have been directed

towards the inclusion of poliomyelitisantigens to make a quadruple vaccine(DPT-polio) for simultaneous immuni-zation against all four diseases.

While the triple antigen has beenused on a large scale with apparentsuccess, the inclusion of poliomyelitisvaccine (Types I, II, and III) withthe other three antigens, introduces newfactors in the manufacture of the re-sulting multiple vaccine, and it must beevaluated as a new product. A basicquestion which needs examination, aswith any preparation of multiple an-tigens, concerns the possibility of im-munologic interference or enhancement.That is, does any one of the severalantigens interfere with the immunologicresponse to any of the others, or, is the

OCTOBER. 19960 1 529

response of any one enhanced by com-bination with the others? The questionalso arises whether the presence ofspecific antibody actively produced, orpassively acquired by the infant asmaternal antibody-either increases orsuppresses the immunologic response.In experiments in animals, using acombination of DPT and polio vaccine,Kendrick and Brown' observed thatthere was a serologic response to eachcomponent; in monkeys, there was someindication of enhancement of the re-sponse to poliomyelitis antigens in thecombined product in comparison withpoliomyelitis vaccine given alone. Schuc-hardt, Munoz, and Verwey2 comparedtriple with quadruple vaccine in potencytests in animals and observed that withthe two products the results were similarfor diphtheria and tetanus toxoids andfor the pertussis component; in mon-keys, the potency of the poliomyelitiscomponent was greater with the quad-ruple than with poliomyelitis vaccinealone except in the instance of Type Iwhich they pointed out was a weakantigen.

For immunization of children, Batson,et al.,3 mixed poliomyelitis vaccine withpertussis vaccine and also with triplevaccine in the syringe used for injectionand observed no interference among theseveral antigenic components; neitherdid they observe any influence ofmaternal antibodies on the response.Barrett and co-workers4'5 injected chil-dren from two and a half months tofive years of age with the productQuadrigen* in which the four antigeniccomponents were combined during man-ufacture. These authors found that theantibody levels following the primaryimmunization series were not as highin infants of less than six months asin older children. They stated that thepresence of maternal antibody madethe results more difficult to evaluate and

* Trade name, Parke, Davis & Company.

concluded that the response to a boosterinjection is a more meaningful criterionfor evaluating the effectiveness of vac-cine than the primary response.The official release of multiple anti-

gen preparations containing diptheriaand tetanus toxoids and pertussis andpoliomyelitis vaccines for use in theUnited States and in Canada, and therecommendation of such preparationsby the Multiple Antigens Committeeof the American Public Health Asso-ciation,6 emphasize the importance ofthe accumulation of as much basicinformation as possible on the use ofthese products. In this paper, the dataare concerned particularly with theeffect of passively acquired maternalantibody on the infant's response tothe several component antigens.

Plan of Study

The clinical portion of this studywas undertaken by Drs. Watson andGiammona, Department of Pediatrics,University of Michigan Medical School,who are reporting these aspects else-where.7

Forty-eight healthy infants who wereregistered at the Well Baby Clinic ofthe University of Michigan Hospitalwere from two to four months of ageat the time of their first injection with0.5 ml of a multiple vaccinet contain-ing diphtheria, tetanus, pertussis, andtrivalent poliomyelitis antigens (DPT-polio). Second and third injections of0.5 ml each were given at intervalsof one month. Thirty-one of the chil-dren received a fourth or booster dose ofthe same material, most of them fromsix to eight months later, a few aftera year. Blood specimens were obtainedfrom all 48 children at the start of thestudy and again two weeks after the

t The vaccine (Quadrigen) was kindly sup-plied by its manufacturers, Parke, Davis andCompany, who also supported financially theclinical portion of the study.

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MULTIPLE ANTIGENS-DPT AND POLIO

primary series of injections. Preboosterspecimens were obtained from only fiveof those receiving the additional injec-tion but all 31 were bled from threeto four weeks following it. The serawere maintained at 4°C until tested.

Laboratory Methods and Results

Diphtheria and Tefanus

The diphtheria and tetanus antitoxinlevels were determined in the labora-tories of Parke, Davis and Company,Detroit. Serum specimens for thesetests were available on 42 of the 48infants. The antitoxin levels have beengrouped in terms of units as shownin Table 1. It will be seen that 31 ofthe 42 infants tested for diphtheria anti-toxin had prevaccination levels of lessthan to the diphtheria, tetanus, andmunization, 28 of these showed an in-crease to levels of between 0.1 and oneunit and two had increased to morethan one but less than 10 units. Of the11 infants with preimmunization levelsbetween 0.1 and one unit only twoshowed an increase following the pri-mary series.

Thirty-seven of the 42 infants testedfor tetanus antitoxin had preimmuniza-tion levels of less than 0.1 unit. Allshowed an increased level and mostof them had more than one unit follow-

ing vaccination. Of the five infants withdetectable levels prior to immunization,two showed an increase.

Postbooster diphtheria and tetanusantitoxin levels were available for only19 of the 42 infants. Thirteen and 17of these, respectively, had less than 0.1unit of diphtheria and tetanus antitoxinprior to primary vaccination; and afterthe booster injection, all showed a fur-ther increase over the postprimary level.Only 6 of the 19 infants had detectablediphtheria antitoxin before primary vac-cination; following booster, three re-mained at the same level, and threeincreased. The two infants with detec-table tetanus antitoxin before vaccineshowed increased levels following booster.

Pertussis

Pre- and postprimary vaccinationspecimens from 47 of the 48 infantswere tested for serologic response tothe pertussis component of the quad-ruple vaccine, in terms of specific agglu-tination, using a rapid, reduced volumeprocedure.8 Briefly, 0.1 ml of eachserum dilution was mixed with 0.1 mlof a killed suspension of B. pertussis of20 opacity units by NIH standard, themixtures were shaken for three minutes,salt solution added, the tests read, andthe results expressed in reciprocal titersof the highest final serum dilution at

Table 1-Diphtheria and Tetanus Antitoxin Levels in the Sera of 42Infants Before and Following Vaccination with DPT-Polio

Number of Infants at Each Level of AntitoxinPrevaccination Postvaccination

Units Numbers <0.1 0.1 to 1.0 1 to 10 >10

Diphtheria <0.1 31 1 28 20.1 to 1.0 11 2 7 2

<0.1 37 2 28 7Tetanus 0.1 to 1.0 3 2 1

>l to lO 2 1 1

OCTOBER. 1960 1531

Table 2-Pertussis Agglutinin TitersVaccination with DPT-Polio

in 47 Infants Before and Following

Postvaccination TitersPrevaccination Number of Infants in Each Range Geometric

Titers Number Negative 4-16 32-128 Mean

Negative 43 14 13 16 10.3516 1 1 0 0 Negative32 3 1 2 0 4.00

which definite agglutination was ob-served.*

In Table 2, the results with post-vaccination sera have been arrangedaccording to the titers of samples takenbefore vaccination. Of 43 infants with-out demonstrable prevaccination anti-bodies, 14 (approximately one-third) re-mained negative-a lower response thanwould be expected following a stronglyantigenic vaccine. There were only 4of the 47 infants who had positiveagglutination tests before vaccination,that is, who had measurable maternalantibody in their serum; and all fourhad lower titers after primary vacci-nation than before. This suggests thatthere had been progressive eliminationof maternal antibody without active sero-logic response of the infant. The ques-tion immediately arises as to whetherthe apparent depressive effect of circu-lating maternal antibody on postprimaryresponse would still be observed aftera later booster injection. In Table 3,the postprimary and postbooster resultsin 30 infants suggest that the increasedtiter expected in a secondary responsewas obtained only in those infants with-out detectable agglutination before vac-cination. In the four infants with meas-

* As explained elsewhere,9 if these titers areto be compared with those obtained in theone-ml volume tests, the titers obtained in therapid test should be multiplied by five, inorder to assure comparison of reactions be-tween equivalent amounts of serum and anti-gen.

urable prevaccination antibodies thepostprimary titers showed a decrease;and the response was still depressedafter the booster injection six monthslater. The preprimary, postprimary, andpost booster titers of these four infantswere, respectively: 32, 8, 4; 16, 4, 8;32, 4, 4; 32, 4, 4. The numbers aresmall and do not warrant firm con-clusions, but the results are consistentwith the hypothesis that the presenceof maternal antibody depressed theserologic response of the infant to pri-mary vaccination, and also that thisdepressive effect may still be presentafter a booster injection six monthslater. The effect of a still later boosterinjection under these conditions was notstudied.

Poliomyelitis

Poliomyelitis antibody levels were de-termined by performing neutralizationtests against standard strains of thethree types of virus in plastic paneltissue cultures of monkey kidney cells.'0Pre- and postvaccination sera were avail-for all 48 infants and were always testedtogether on the same day. Antibodytiters are expressed in terms of thereciprocal of the highest original serumdilution which neutralized 100 tissueculture doses of virus. Since most ofthe mothers had received poliomyelitisvaccine during pregnancy or within thetwo years preceding, many of theirinfants had detectable antibodies at the

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MULTIPLE ANTIGENS-DPT AND POLIO

time they received the first injection. childrenAs indicated in Table 4, only 18 had three typno detectable antibodies to Type I virus other; tiat the lowest dilution tested (1:4), ten types mi~were negative for Type II, and 22 for a thitdType III. Only four were "triple nega- cine, partive." The geometric mean antibody prevaccintiters of the prevaccination specimens this phenwere 7, 19, and 7 for Types I, II, andIII, respectively. The postprimary vac-cine mean titers for the same three Prevaccinetypes were 7, 24, and 22, indicating Postprimarthat except for Type III very little Postboosteiantibody development had been inducedby the vaccine. However, examinationof the postprimary vaccination titers Prevaccinein relation to their corresponding pre- Postprimarvaccination titers reveals a startlingfact. Of the 18 negative for Type I These rEbefore vaccination, seven developed anti- passivelybodies, as did nine of ten negative for bodies, ifType II, and 18 of 22 for Type III. greater, iIn contrast, only 5 of the 30 with pre- active imvaccination antibodies to Type I, 8 of vaccine.38 for Type II, and 7 of 26 for Type In TabIII showed a rise in antibody titer infants wafter primary vaccination! Furthermore, from six r

most of the infants with prevaccine anti- third dosbodies that showed a subsequent rise arrangedin titer had very low titers (either four maternalor eight) before immunization. When time of t]the prevaccine titers were 16 or greater, without athe titers of the vast majority of them geometricdecreased following the three injections, maternal;many to such an extent that antibodies for Typeswere no longer detectable. In some The mea]

Table 3-Pertussis Agglutinin Titers in the Sera of 30Primary and Postbooster Vaccination with DPT-Polio

the titers of antibody to thees varied independently of eachhe titer of antibodies for twoght decrease while the titers ofwould develop following vac-rticularly if not present in theie specimen. Two examples ofiomenon are shown:

Iyr

ry~r

R.H.I II16 <4<4 8<4 256

S.K.I II<4 256256 256256 8

III64<4<4

III<4

10241024

esults suggest strongly thattransferred maternal anti-

f present in titers of 1:16 orinterfere with the response tomunization with poliomyelitis

ble 5, the results for the 31rho received booster injectionsmonths to one year following the;e of the primary series wereaccording to those infants withantibodies (by type) at the:heir first injection, and thosentibodies. The prevaccinationmean titers of those with

antibodies were 24, 35, and 22sI, II, and III, respectively.n titers of these same indi-

D Infants Before and Following

PostboosterPostprimary Nega-

Prevaccination Negative Geometric tive GeometricTiter Number at 1 :4 4-16 32-128 Mean at 1:4 4-16 32-128 mean

Negative 26 7 7 12 10.44 0 11 15 23.8716 1 1 0 0 Negative 0 1 0 8.032 3 1 2 0 4.0 2 1 0 2.57

Total 30 9 9 12 2 13 15

OCTOBER. 1960 1533

Table 4-Effect of Vaccination with DPT-Polio on Poliomyelitis Neutraliz-ing Antibody Titers in the Sera of 43 Infants

Prevaccination PostvaccinationType Titer Number Increase Same Decrease

<4 18 (7) 114-8 9 (4) 1 416-64 20 1 1 (18)

>64 1 0 0 1

<4 10 (9) 14-8 7 (6) 0 1

16-64 22 2 4 (16)>64 9 0 1 (8)

<4 22 (18) 44-8 12 (6) 0 6

16-64 13 1 2 (10)

>64 1 0 0 (1)

Negative 50 (34) 164-8 28 (16) 1 11

All types 16-64 55 4 7 (44)

>64 11 0 1 (10)

NOTE: numbers in parentheses are for special emphasis.

viduals were only 4, 17, and 12 follow-ing the primary series and an additionalbooster injection six months later. Incontrast, the postbooster means of thosewith no detectable antibody before vac-cination were 27, 161, and 268 for thethree types, respectively. It will beseen that of eight infants with nodetectable antibodies to Type I virusbefore vaccination, five were still in the<4 category after the primary series;all but one, however, had developedantibodies as a result of the boosterinjection. In contrast, of 17 infantswith prevaccination titers of 16 or 64,most were lower in titer following pri-mary and 11 were <4 following thebooster injection. This pattern of anti-body titer shift is essentially the samefor all three types. Thus it is clearthat the presence of maternal antibodiesinhibited not only the primary response,but, under the cover of maternal anti-bodies in high titer, vaccine did notcondition the infants to the booster

phenomenon. This is further illustratedin Table 6, which shows the results ofall tests on the only five infants whowere bled immediately prior to thebooster injection.

Discussion

It was not the primary purpose ofthis study to measure the efficacy ofthe multiple vaccine or to investigatethe antigenicity of its individual com-ponents in comparison with the sameantigens given separately. By the strict-est of the requirements for measurementof antigenicity of a vaccine, namely,the response of children with no detec-table specific antibodies before vaccina-tion, the components measured up well.In fact, the conversion of "negatives"to "positives," in terms of antibodywas very good for the diphtheria andtetanus but was somewhat less than ex-pected for the pertussis and poliomye-litis. Nor is it the purpose of the study

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MULTIPLE ANTIGENS-DPT AND POLIO

to interpret serological responses in and none of four with pertussis agglu-terms of actual protection against given tinins before vaccine. These results arediseases. It is recognized that measur- entirely in keeping with the more ex-able antibodies, especially those deter- tensive data observed in the study ofmined by neutralization tests, provide poliomyelitis antibody response. Duean index of probable protection, but to the recent vaccination of most ofimmunity can exist in the absence of the mothers against this disease a verydetectable antibodies. Conversely, the high proportion of the infants had highpresence of antibodies in high titer is levels of maternal antibody at delivery.no guarantee of absolute protection Thirty of 48 had demonstrable anti-against invasion and infection with any bodies for Type I, 38 for Type II, andof these agents. 26 for Type III. As mentioned pre-

Granted, then, that immunity is rela- viously only four infants failed to showtive, the investigator should be con- antibodies for at least one type of virus.cerned with any factor which interferes Only 16 per cent, 21 per cent, and 26with the development of antibodies fol- per cent of the infants with antibodieslowing experience with an antigen. One showed an increased titer to the threesuch factor appears to be evident in types, respectively, following vaccination.this study, namely, the suppressive effect Perhaps more pertinent to the questionof maternal antibodies on the antibody of suppression is the fact that antibodyresponse of infants to vaccination. Al- titers actually decreased in the majoritythough only a small number of infants of such infants. This was true of per-had persisting maternally acquired anti- tussis as well as poliomyelitis and to abodies to diphtheria, tetanus, and per- lesser extent of diphtheria and tetanus.tussis the data were sufficient to illus- Suppression of active immunizationtrate this phenomenon. The vaccine by passive antibodies is not newlywas successful in stimulating an in- recognized. Talmage"' states that trans-crease in measurable antibody in only fer of serum from an immunized to2 of 11 such infants with diphtheria a normal animal has been shown toantibodies, two of five with tetanus, suppress rather than accentuate the sub-

Table 5-Poliomyelitis Neutralizing Antibody Titers in the Sera of 31 Infants Beforeand Following Primary and Postbooster Vaccination

Prevaccination Postprimary PostboosterType Titer No. <4 4 8 16 64 256 1,024 <4 4 8 16 64 256 1,024

<4 8 5 1 1 1 1 2 1 2 1 1I 4-8 5 2 1 1 1 2 1 2

16-64 17 6 2 4 5 11 2 3 1>64 1 1 1

<4 6 1 1 1 1 1 1 3 2 1

II 4-8 6 1 1 1 2 1 1 1 1 316-64 14 3 2 4 4 1 3 1 4 4 2>64 5 1 3 2 2 1

<4 15 1 3 2 1 6 2 1 4 3 74-8 6 2 1 1 2 1 3 2

16-64 9 4 1 3 1 6 1 1 1>64 1 1 1

OCTOBER. 1960 1535

Table 6-Individual Poliomyelitis Neutralizing Antibody Titers of Five Infants by Type

Serum J.C. R.N. N.J. J.R. T.W.Specimen I II III I II III I II III I II III I II III

Preprimary 64 8 16 4 64 <4 16 8 64 8 64 4 8 8 <4Postprimary <4 <4 <4 <4 16 <4 <4 16 4 <4 8 <4 16 64 16Prebooster <4 <16 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 4 <4 <4Postbooster 8 64 16 64 <4 64 <4 256 <4 16 16 64 64 8 64

sequent antibody response. di Sant'Agnese12 found that newborn childrendid not respond as well as older childrento diphtheria immunization but observedthat this condition was corrected follow-ing a booster dose. However, he addedthat the final response was better whenthe booster was given a year laterrather than after six months. Addi-tional examples of this phenomenonare cited by Edsall13 in a recentreview. The inferior response of younginfants to poliomyelitis vaccine hasbeen observed recently by several au-thors.45"4-'9 Some of these authorsassociated this inhibition with the pres-ence of maternally transferred passiveantibodies.520 Since there is ampleevidence that older children and adultswith actively produced antibodies re-spond well to vaccination, the obviousconclusion is that the difference whichexists between the two types of responseis not related to the presence or absenceof antibodies per se, but to whetherthey were passively acquired or activelyproduced by the individual himself. Inthe latter case the antibody formingmechanism has been activated by eithernatural exposure or previous vaccina-tion and additional stimuli are there-fore capable of enhancing the outputof antibodies.

In the case of passively acquiredcirculating antibodies, no host mecha-nism is operating and the combinationof antibody with antigen may representthe phenomenon of the neutralization

test in vivo. This theory is supportedby the present data which show thatinterference with active immunization ismore likely to occur in the presenceof higher concentrations (titer of 1:16or higher) of passively acquired anti-bodies. A previous study21 has shownthat children respond very well to polio-myelitis vaccine administered three daysfollowing gamma globulin in double theusual prophylactic dose. This amount ofgamma globulin, however, was shown tobe detectable only in very low titer (1:2or 1:4) in the circulating blood andfor a limited time not exceeding threeweeks.22 The titer of passive maternalantibodies acquired by the infant canbe very high depending upon the levelin its mother, and can persist for manymonths.'7 Thus the level of mother'santibodies appears to be the governingfactor in the serologic response of herinfant to vaccine.

In a study beginning in 1954, polio-myelitis vaccine was reported to bereasonably effective in stimulating anti-bodies in two- to four-month-old in-fants.23 Very few of the babies hadantibodies at this age since the practiceof routine vaccination of pregnantwomen had not yet been inaugurated.During recent years, however, manymore women have poliomyelitis anti-bodies at the same time of delivery.Since it has been shown that the polio-myelitis antibody titer of mother's bloodand cord blood are very similar,2324"14and of mother's blood, cord, and new-

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MULTIPLE ANTIGENS-DPT AND POLIO

born infant's blood,17 and since persist-ence of these passive antibodies inbabies is a function of the originaltiter,23,17,14,25 it seems clear that inter-ference with active immunization of theinfant is becoming more rather thanless of a problem. This is illustratedby the findings in a study undertakenin 1956 in which 30 per cent of unvac-cinated and 71 per cent of vaccinatedwomen had antibodies for all three typesof poliomyelitis virus at the time ofdelivery.17 In a survey made in 1958,83 per cent of 100 pregnant women,most of them recently vaccinated, hadantibodies to all three types of virus.26

This increasing incidence of maternalantibodies is reflected in the presentstudy where over 90 per cent of theinfants had antibodies for one or moretypes three months following birth. Asimilar study in Canada by Wilson,et al.,19 reports that less than 50 percent of their infants had maternal anti-body to poliomyelitis before vaccina-tion; however, over one-third of themwere five months of age or older, whichmay explain the better serological re-sponse to multiple vaccine found intheir study in comparison with thepresent data.The concern for possible inhibition of

poliomyelitis antibody production in in-fants does not apply equally to theresponse to the diphtheria, tetanus, andpertussis components, since few of themothers had detectable antibodies tothese diseases as reflected by the pre-vaccination testing of their infants inthe present study. In the 1956 reportmentioned above only 5 of 95 mothershad pertussis agglutinins at the timeof delivery, and in the 1958 survey,15 of 100 were positive.

In view of the data presented it wouldseem that there should be serious con-sideration of delaying poliomyelitis vac-cination of infants until maternal anti-bodies are more likely to be gone orat least reduced to a low level. The

child is less frequently exposed earlyin infancy, and furthermore he shouldbe protected during this interim by thesame passively acquired antibodieswhich suppress the response to vaccine.Such a delay in administration wouldnot necessarily preclude the combi-nation of poliomyelitis vaccine with"triple" vaccine since perhaps it, too,could be delayed without serious con-sequences.

Summary

Forty-eight infants from two to fourmonths of age were injected three timesat intervals of one month with 0.5 mlof a multiple vaccine against diphtheria,tetanus, pertussis, and poliomyelitis.Thirty-one of the children received anadditional or booster dose from 6 to12 months later.

Serologic tests on prevaccinationblood specimens revealed that very fewof the infants had detectable antibodiesto diphtheria, tetanus, or pertussis, butthe majority of them had poliomyelitisantibodies at this time.The response of the majority of the

infants to the poliomyelitis componentsof the vaccine was definitely poorerthan to the diphtheria, tetanus, andpertussis components.The lower serologic response to the

poliomyelitis antigens was clearly asso-ciated with the presence of maternallyacquired passive antibodies in the in-fant's serum at the time of vaccination.The few children who had prevaccina-tion antibodies to diphtheria, tetanus,and pertussis also had poorer responsethan did those who were negative be-fore vaccination.The suppressive effect of maternal

antibodies affected not only the pri-mary, but also the booster response tothe several antigenic components; teta-nus antibody production seemed to beleast influenced. The significance of thefindiags is discussed.

OCTOBER, 1960 1 537

ACKNOWLEDGMENT-The authors gratefullyacknowledge the technical assistance of JackSchieble and Margaret Cook.

REFERENCES

1. Kendrick, P. L., and Brown, G. C. Immune Re-sponse in Guinea Pigs and Monkeys to the IndividualComponents of a Combined Diphtheria-Pertussis-Tetanus Antigen Plus Poliomyelitis Vaccine. A.J.P.H.47:473-483, 1957.

2. Schuchardt, L. F.; Munoz, J.; and Verwey, W. F.Studies on a Combined Diphtheria-Pertussis-Tetanus-Poliomyelitis Vaccine. Ibid. 50 :321-328, 1960.

3. Batson, R.; Christie, A.; Mezur, B.; and Barrick,J. H. Response of the Young Infant to PoliomyelitisVaccine Given Separately and Combined with OtherAntigens. Pediatrics 21:1-6, 1958.

4. Barrett, C. D., Jr.; Timm, E. A.; Molner, J. G.;Wilner, B. I.; Anderson, C. P.; Carnes, H. E.;and McLean, I. W. Multiple Antigen for Immuniza-tion Against Poliomyelitis, Diphtheria, Pertussis, andTetanus. 1. Response of Infants and Young Childrento Primary Immunization: Preliminary Report.J.A.M.A. 167:1103-1106 (June 28), 1958.

5. Barrett, C. D.; Timm, E. A.; Molner, J. G.; Wilner,B. I.; Fahey, M. F.; and McLean, I. W. MultipleAntigen for Immunization Against Poliomyelitis,Diphtheria, Pertussis, and Tetanus. II. Response ofInfants and Young Children to Primary Immunizationand Eighteen-Month Booster. A.J.P.H. 49:644-655,1959.

6. Committee on Evaluation of Multiple Antigens,American Public Health Association. J. Ipsen, Chair-man. Letter to Editor re: Combined Poliomyelitis-Diphtheria-Pertussis and Tetanus Prophylactics. Ibid.49:678, 1959.

7. Gianmmona, S., and Watson, E. (Unpublished data.)8. Kencirick, P. L.; Lawson, G. McL.; and Miller, J.

"Hemophilus Pertussis," in Diagnostic Proceduresand Reagents (3rd ed.). New York, N. Y.: Ameri-can Public Health Association, 1950, p. 146.

9. Kendrick, P. L. Rapid Agglutination Technic Ap-plied to B. pertussis. A.J.P.H. 23:1310-1312, 1933.

10. Melnick, J. L., and Opton, E. M. Assay of Polio-myelitis Neutralizing Antibody in Disposable PlasticPanels. Bull. World Health Organ. 14:129-146, 1956.

11. Talmage, D. W. Allergy and Immunology. Ann.Rev. Med. 8 :239-256, 1957.

12. di Sant' Agnese, P. A. Combined ImmunizationAgainst Diphtheria, Tetanus and Pertussis in New-born Infants. II. Duration of Antibody Levels, Anti-

body Titers After Booster Dose. Effect of PassiveImmunity to Diphtheria on Active Immunization withDiphtheria Toxoid. Pediatrics 3:181-194, 1949.

13. Edsall, G. Active and Passive Immunity of theInfant. Ann. New York Acad. Sc. 66:32-43, 1956.

14. da Silva, M. M.; Prem, K. A.; Johnson, E. A.;McKelvey, J. L.; and Syverton, J. T. A Responseof Pregnant Women and Their Infants to Poliomye-litis Vaccine. J.A.M.A. 168:1-5 (Sept. 6), 1958.

15. Perkins, F. T.; Yetts, R.; and Gaisford, W. Sero-logical Response of Infants to Poliomyelitis Vaccine.Brit. M. J. 1:68-71 (July), 1958.

16. Report of Symposium on Immunization of Childhood.Brit. M. J. 1:1342-1346 (May 23), 1959.

17. Brown, G. C., and Carroll, C. J. Antibody Re.sponse of Pregnant Women to Poliomyelitis Vaccineand Passive Transfer to Infants. J. Immunol. 81:389-395, 1958.

18. Russell, K. Antibody Responses to British Polio-myclitis Vaccine of Children with Naturally Occur-ring Antibodies. Brit. M. J. 1:622-624 (Mar. 15),1958.

19. Wilson, R. J.; Moss, G. W. O.; Potter, F. C.; andMacLeod, D. R. E. Diphtheria and Tetanus ToxoidsCombined with Pertussis and Poliomyelitis Vaccines.Clinical Trial of a Quadruple Antigen. Canad.M. A. J. 81:450-453 (Sept. 15), 1959.

20. Perkins, F. T.; Yetts, R.; and Gaisford, W. AComparison of the Responses of 100 Infants to Pri-mary Poliomyelitis Immunization with Two and ThreeDoses of Vaccine. Brit. M. J. 1:1083-1086 (Apr. 25),1959.

21. Brown, G. C.; Rabson, A. S.; and Craig, D. E.Poliomyelitis Vaccine Studies. Pub. Health Rep. 71:604-611 (June), 1956.

22. Brown, G. C.; Rabson, A. S.; and Schieble, J. H.The Effect of Gamma Globulin on Subclinical In-fection in Familial Associates of Poliomyelitis Cases.II. Serological Studies and Virus Isolations fromPharyngeal Secretions. J. Immunol. 74:71-80, 1955.

23. Brown, G. C., and Smith, D. C. Serologic Responseof Infants and Preschool Children to PoliomyelitisVaccine. J.A.M.A. 161:399-403 (June 2), 1956.

24. Lipton, M. M., and Steigman, A. J. Neonatal Im-munity. I. Disparity in Maternal-Infant Polio VirusAntibody. Proc. Soc. Exper. Biol. & Med. 96:348-352, 1957.

25. Strean, G. J.; Gelfand, M. M.; Pavilanis, V.; andSternberg, J. Maternal-Fetal Relationships: PlacentalTransmission of Poliomyelitis Antibodies in Newborn.Canad. M. A. J. 77:315-323 (Aug. 15), 1957.

26. Brown, G. C., and Kendrick, P. L. (Unpublisheddata.)

The authors are associated with the department of epidemiology and viruslaboratory, School of Public Health, University of Michigan, Ann Arbor, Mich.

This paper was presented before a Joint Session of the Epidemiology, Labora-tory, and Maternal and Child Health Sections of the American Public HealthAssociation at the Eighty-Seventh Annual Meeting in Atlantic City, N. J.,October 20, 1959, and at the Symposium on a Calendar of Immunization forChildren, Paris, France, October 19-21, 1959.

This study was aided by grants from the National Foundation and RackhamResearch.

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