JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2064-20700095-1137/92/082064-07$02.00/0Copyright © 1992, American Society for Microbiology

Evaluation of a Live Attenuated, Cold-Adapted ParainfluenzaVirus Type 3 Vaccine in Children

ROBERT B. BELSHE,1* RUTH A. KARRON,2 FRANCES K. NEWMAN,1 EDWIN L. ANDERSON,1SHARON L. NUGENT,1 MARK STEINHOFF,2 MARY LOU CLEMENTS,2 MODENA H. WILSON,2

SUSAN L. HALL,3 EVELINE L. TIERNEY,3 AND BRIAN R. MURPHY3Center for Vaccine Development, Division ofInfectious Diseases, Departments ofInternal Medicine and

Pediatrics, St. Louis University School ofMedicine, and St. Louis Veterans Administration Medical Center,St. Louis, Missouri 631041; Center for Immunization Research, Johns Hopkins University, Baltimore,

Maryland 212052; and Laboratory of Infectious Diseases, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 208923

Received 27 January 1992/Accepted 20 May 1992

Cold passage 18 (CP18) parainfluenza virus type 3 (PIV-3) vaccine was evaluated in a double-blind,randomized, placebo-controlled study of 95 infants and young children. None of 19 seropositive older children41 to 124 months old became infected when 106 50% tissue culture infective doses (TCID50) ofvaccine virus wasadministered intranasally. Two of nine and seven of twenty-four young seropositive children given lo- or 106TCIDso of CP18 PIV-3, respectively, became infected. Each of four seronegative young children becameinfected, as indicated by virus shedding and antibody response, when given 106 TCID50 of CP18 PIV-3intranasally. Illness was not observed in seropositive children. Two of the four seronegative children developeda mild illness characterized by rhinorrhea and wheezing on auscultation; none had fever. In one case, vaccinevirus spread from a vaccinee to a sibling control but did not cause illness. The vaccine is attenuated relative towild-type PIV-3, but additional attenuation will be required to achieve a satisfactory PIV-3 vaccine.

Parainfluenza virus infections cause significant morbidityin children under the age of 3 years and are a major cause ofserious respiratory illness resulting in the hospitalization ofinfants and children (22). Among the parainfluenza viruses,type 3 (PIV-3) is the leading cause of serious respiratoryillness. Previous studies demonstrated that parenteral injec-tion of killed PIV-3 or respiratory syncytial virus (RSV)vaccine failed to protect against subsequent natural infectionwith these viruses (12, 14). Inactivated vaccines for RSV andmeasles virus not only failed to protect against naturalinfection but caused potentiation of disease in the vaccineeswhen they were naturally infected with these agents (11, 14).Live attenuated vaccines against measles and mumps aresafe and efficacious in children 15 months old (5, 17). SincePIV-3 causes serious disease within the first 6 months of life,immunization has to be initiated in the presence of maternalantibodies. Parenteral administration of live RSV vaccine ininfants with maternal antibody was unsuccessful, in partbecause vaccine virus was neutralized by maternal antibod-ies (4). As a result of these studies and the success ofintranasally administered cold-adapted (CA) reassortant in-fluenza virus vaccines, the direction for PIV-3 vaccinedevelopment has turned toward live attenuated vaccinesgiven intranasally (3, 7, 23). A series of candidate PIV-3vaccines was generated by cold adapting a human PIV-3strain, the JS strain, to replication at 20°C (1, 9, 10). Threecandidate vaccines were previously evaluated in tissue cul-ture and small animals; these included biologically clonedviruses selected after cold passages 12, 18, and 45 and aredesignated by cold passage level (CP12, CP18, and CP45) (9,10). In addition to exhibiting enhanced growth at 20°C(cold-adapted phenotype) compared with wild-type (WT)PIV-3, the cold passage viruses manifested various degrees

* Corresponding author.

of temperature sensitivity during replication in cell culture(TS phenotype).

Studies with two CA mutants of PIV-3 (CP12 and CP18)were conducted with adults (7). Although volunteers wereselected for low levels of nasal wash antibodies to PIV-3,few were infected with vaccine virus (7). Also, a study ofCP12 PIV-3 was undertaken with seronegative chimpanzeesto assess this vaccine candidate in a fully susceptible sero-negative host (7). The CP12 vaccine was nonreactogenic inchimpanzees and was restricted in growth in the upper andlower respiratory tracts by >100-fold compared with WTPIV-3. Although the CP12 vaccine virus exhibited limitedreplication in chimpanzees, it induced resistance to chal-lenge with WT PIV-3. Some of the viruses recovered fromthe chimpanzees inoculated with the CP12 PIV-3 vaccinecandidate had lost the TS property. Since the CP18 PIV-3vaccine is more attenuated in hamsters than is the CP12virus, and the CP45 PIV-3 vaccine was noninfectious forhamsters, CP18 was chosen for evaluation in young childrenfor safety and immunogenicity as a vaccine candidate withproperties intermediate between those of CP12 and CP45.

MATERIALS AND METHODS

Vaccine. Isolation of the parental WT virus (JS strain),cold adaptation procedures, selection of biologic clones, andcharacterization of the clones have been previously de-scribed (1, 9, 10). The CP18 mutant (clone 1146) of humanPIV-3 JS WT virus is a CA and TS mutant which replicateswell at 20°C and is restricted in growth at 39°C. CP18 PIV-3was adapted to growth in DBS-FRhL-2 cells by passage ofthe CP18 virus three times at 31°C in DBS-FRhL-2 cells,further biological cloning of the virus with three terminaldilution passages in DBS-FRHL-2 cells, and expansion ofthe virus suspension with one additional passage in DBS-FRhL-2 cells. The vaccine suspension was prepared in the

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same cell line by Flow Laboratories, McLean, Va. Twoadditional passages of CP18 in DBS-FRhL-2 cells yieldedseed virus for vaccine; vaccine was produced in flasks ofDBS-FRhL-2 cells. The lot designation for this vaccinesuspension was HPI3-2.

Study population. Two groups of children and infants, 3 to10 years and 6 to 36 months of age, were recruited for the JSPIV-3 CP18 study. Individuals selected for participation inthe study were considered to be normal, healthy infants andchildren without evidence of acute or chronic illness. Vol-unteers from households with siblings under the age of 6months or with pregnant women or immunodeficient personsresiding in the household were excluded from the study.

Clinical design. The clinical trials with CP18 PIV-3 wereconducted in a placebo-controlled, double-blind fashion.Nasopharyngeal swab samples or nasal washes were col-lected from each volunteer immediately prior to vaccinationas previously described (18). Volunteers were inoculatedintranasally with 0.5 ml of either CP18 PIV-3 vaccine dilutedin Leibovitz medium (L-15) or a placebo (L-15 alone). TheCP18 PIV-3 vaccine was diluted in L-15 so that the finalquantity of virus given to each participant was 105 or 106 50%tissue culture infective doses (TCID50) per dose. In the SaintLouis University study, each child was visited at home by anurse daily for 11 days following vaccination to assess theclinical response to the vaccine and to collect nasopharyn-geal swab specimens for virus isolation. Subjects in theJohns Hopkins University study were observed for 1 to 2 heach day for 12 consecutive days (3 days preceding and 9days following vaccination) at the Center for ImmunizationResearch; nasal washes were collected daily as previouslydescribed (18). In addition, parents were instructed to obtainthe rectal temperature of each study subject at least onceeach evening and to retake the temperature within 15 min ifthe reading was above 38.1°C. Clinical definitions of illnesswere as follows: (i) fever, rectal temperature of >101'F (ca.38°C) or oral temperature of >100°F; (ii) upper respiratoryillness, rhinorrhea or pharyngitis for 2 or more consecutivedays; and (iii) lower respiratory illness, wheezing or pneu-monia. Otitis media was diagnosed by a pediatrician and wasdefined as loss of normal tympanic membrane landmarks anddecreased mobility. An illness was attributed to infectionwith the CP18 PIV-3 vaccine virus when the vaccine viruswas recovered from nasal secretions and/or a fourfold in-crease in the titer of hemagglutination inhibition (HAI)antibody to PIV-3 occurred.Laboratory analysis. Specimens collected at Saint Louis

University for virus isolation were inoculated onto primaryrhesus monkey kidney (RMK) tissue culture cells in dupli-cate and incubated at 32°C for 14 days. The RMK cellcultures were observed for a cytopathic effect and tested forhemadsorption with guinea pig erythrocytes on days 5, 9,and 14 following inoculation. Positive specimens were iden-tified as PIV-3 by the standard HAI test and frozen at -70°Cfor further characterization. Nasal wash specimens collectedfrom participants at Johns Hopkins University were inocu-lated onto LLC-MK2 monolayers in quadruplicate, andspecimens showing hemadsorption were positively identifiedas PIV-3 by indirect immunofluorescence (Bartels Micros-can; Baxter Healthcare Corp., Bellevue, Wash.).Blood was collected from each of the children prior to

vaccination and 28 days after vaccination to assess antibodyresponses. An HAI assay utilizing homologous antigen wasused to determine antibody titers in serum (7).PIV-3 isolates recovered from vaccinees were character-

ized to determine their TS and CA properties. The virus

titers in most of the original nasopharyngeal swab sampleswere low (<3.5 PFU/ml), and therefore the characterizationof the phenotype of the isolates was performed by using thetissue culture harvest from the first primary RMK passage(1). Plaque assays were performed at 32 and 39°C on L-132cell culture monolayers to determine whether or not eachisolate retained the TS phenotype. The parental JS WT virusexhibited a less-than-threefold (0.5 log1o) reduction in effi-cacy of plaque formation at 39 versus 32°C; in contrast, theCP18 vaccine virus exhibited a >5.0 log1o reduction inplaque formation at 39 versus 32°C. Viruses recovered fromvolunteers were considered TS if the plaque titer at 39°C was>100-fold less than the plaque titer at 32°C. To assess theCA phenotype, isolates were grown in RMK cells at 20°C for14 days or at 32°C for 7 days and the amount of virusproduced was determined by plaque assays at 32°C in L-132cells. The JS WT virus produces 102.0 PFU or less at 20°Cbut >107.0 PFU at 32°C; in contrast, the CP18 virus produces>105.0 PFU at both 20 and 320C. Viruses exhibiting differ-ences in growth of < 100-fold at 20 and 32°C were consideredto have the CA phenotype.

Viral genome sequence analysis. Virion RNA was purifiedas previously described (15) from representative PIV-3 iso-lates from vaccinees (designated viruses 1 through 5) or asibling control (virus "sibling of no. 5") passaged once onRMK and twice on LLC-MK2 cell monolayers. RNAs fromJS WT and other PIV-3 WT strains were purified followingpassage once on LLC-MK2 cells. The noncoding leaderregion of the fusion (F) gene was sequenced from purifiedviral genomic RNA by using a modification of the Sangermethod (21).

RESULTS

Clinical response. Ninety-five children participated in thisstudy. Fifty-six children were vaccinated with live, attenu-ated, CA CP18 PIV-3 vaccine. An additional 39 volunteersreceived a placebo. Four groups of children were studied.Nineteen seropositive children over 3 years of age re-

ceived 106 TCID50 of the CP18 vaccine virus intranasally(Table 1). Parainfiuenza virus was not isolated from anychild, and an antibody rise was not detected in this oldergroup of vaccinees. None of the children developed upper orlower respiratory disease. One child became febrile andsubsequently had a varicelliform rash.Nine seropositive infants and children 6 to 35 months old

received 105 TCID50 of CP18 PIV-3 intranasally (Table 1).None of the vaccinees in this group shed PIV-3; however,two children had a fourfold or greater increase in antibodytiter to PIV-3. Respiratory illness did not occur; however,two children had fever. One of these two children had otitismedia, but neither was infected with vaccine virus; neitherhad a rise in the titer of antibody to PIV-3.Twenty-four seropositive infants and young children (aged

6 to 36 months) were subsequently inoculated with 106TCID50 of CP18 vaccine virus. Four children shed PIV-3(vaccine virus [vide infra]) in their respiratory secretions,and five had a fourfold or greater rise in antibody to PIV-3;a total of seven children were infected by PIV-3, as indicatedby either shedding of PIV-3 and/or development of anantibody response. Among the seven children infected withvaccine virus, two had fever but both shed other viruses(enterovirus or PIV-2) and one had rhinorrhea and shed arhinovirus and cytomegalovirus in respiratory secretions.Two children developed a lower respiratory illness charac-terized by wheezing. Wheezing occurred for 1 day in one

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2066 BELSHE ET AL.

ts volunteer (day 9 postinoculation) and for 4 days in anotherz o N r t volunteer (days 6 through 9 postinoculation). Neither child

who wheezed was infected by the vaccine virus, as indicatedby absence of an antibody response and failure to detect

.0 r, >vaccine virus in respiratory secretions. Other viruses were3.0 , oe m tSI', not isolated from the two children who wheezed.z Four seronegative infants and children, 7 to 30 months old,

were vaccinated with 106 TCID50 of CP18 vaccine. An HAIQ~ |'gC. C Q{ antibody assay using guinea pig erythrocytes purchased fromX_.o o ;> a commercial supplier and of undetermined age after harvestX0> xo o E 3 m W x N x =had indicated that these children were seropositive prior to

vaccination; subsequent tests using fresh guinea pig erythro-cytes revealed that these individuals were seronegative. Virus

_u.=OOo o x m S | was shed by each of the seronegative vaccinees, and eachm 0.r.tc ;t-~ tr; t;<vaccinee developed an antibody rise. Fever did not occur;

e? o_ E Mhowever, two children developed mild upper respiratory2t |illness (rhinorrhea on days 3 through 11 and 6 through 11) and

these individuals had mild wheezing, heard only on ausculta-A4 & 8°M ~ N 0tion on 4 days (days 7, 8, 10, and 11 postvaccination) and 3

days (days 5, 6, and 11 postvaccination), respectively. Thechild who wheezed for 4 days had a history of a previouswheezing episode that was associated with upper respiratory

orr> illness, and this episode had not required treatment withX=.o;>o~Lbronchodilators. During the vaccine study, this child was

treated with an antibiotic for otitis media and a bronchodilatorbeginning on day 8 of the study; otitis and wheezing resolved

=S°;>.mZ within 3 days. This child later developed recurrent episodes of"r 00roo w wk r wheezing. Long-term follow up revealed that this child had

subsequent respiratory and other common childhood ill-8A0OOznesses. These illnesses occurred 22 days postvaccination

rA;hX tib ° i(febrile upper respiratory illness with wheezing; virus not

U4mcNXNNmisolated), 9 months postvaccination (febrile upper respiratoryUZ.;,g Q illness and wheezing; virus not isolated), 13 months postvac-> cination (febrile upper respiratory illness with no wheezing;

,|oN|..; rhinovirus and adenovirus isolated), and 17 months postvac-o .r Cu.c i cination (impetigo of the external ear; virus not isolated from

wheezing on auscultation and did not require medication.Of the 39 placebo recipients, 4 were febrile and 4 had

.-3Q<a°Ot Nt O . J symptoms of upper respiratory illness. None of the 39> 6 ^ cL developed wheezing or other manifestations of lower respi-0~~~~~~~~~oz;Cu ratory illness. PIV-3 was prevalent in the community at both

study sites, St. Louis and Baltimore, during the study. Threei0OOplacebo recipients shed PIV-3, and three had an antibody

e.°.8Q rise; overall, five placebo recipients were infected withU o'?CuZOt m WO O £ .¢ = PIV-3 during the study.

MtE M 2 2 1 t i > Virus shedding. The viral shedding pattern exhibited by theX~Wt~n, 3 3four seropositive and four seronegative children who shed

CL i~ r , PIV-3 is illustrated in Fig. 1. The earliest time at which viral0 ON "t "d,~ ".O )X 6 d shedding was detected in a seropositive child was day 2, butoz t 3 m = two of the seronegative children shed virus on day 1. TheXCuQ:° Q o peak titer of virus shedding was observed on day 6 amon.8,: 0 seronegative children, and the mean peak titer was 103

U U o o ooH C; , PFU/ml of the nasal swab specimen among the seronegativeU5 0 so m xoXs z; 0 <Cu h children but only 10 PFU/ml of the nasal swab specimenCu;> 0X 3 ° Qamong the seropositive children. One of four seropositive

"0 _ ; M8children and three of four seronegative children shed vaccinet31|QQ 2 > | 0 ,! ; ¢ virus through day 11 postvaccination. The mean durations of5.t .0exg5 shedding were 8 days among the seropositive children and 11

zmr0'i days among the seronegative children. (Table 1).U)QCui o Viruses isolated from vaccinees and control children were

Env:v: CA characterized for the TS and CA phenotypes. Characteriza-oX..8 | B ° 3otion of all isolates from a representative vaccinee is shown in

E0 .0:;o 5iTable 2. The initial isolate was both TS and CA; however,0 ,ifromdays 3 through 7 during peakvirus shedding, the virus

shed was TS' (i.e., the virus had lost the TS phenotype). The

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ATTENUATED PARAINFLUENZA 3 VACCINE IN CHILDREN 2067

4

E

0F-S

0 1 2 3 4 5 6 7 8 9 10 11 12

DAYS POST VACCINATIONFIG. 1. Virus shedding pattern in nasal swab specimens of

seropositive (0; HAI antibody titer, >1:8) and seronegative (-;I-A antibody titer, <1:8) children vaccinated intranasally with JSCP18 PIV-3 vaccine. Points between 0.7 and 1 log1o PFU/mlrepresent samples which were culture positive in tissue culturetubes but plaque assay negative. The minimum detectable by plaqueassay was 101.0 PFU/ml, and the minimum detectable by tubeculture was 100°7 PFU/ml. Points below 0.7 log1o PFU/ml representsamples which were negative by virus culture and plaque assay.

late isolates obtained on days 8 and 9 again exhibited the TSphenotype. Most of the isolates retained the CA phenotype.A summary of the phenotypic characteristics of all of the

isolates from vaccinees and control children is shown inTable 3. The CA property was more stable genetically thanthe TS property. Of 54 viruses recovered from the eightvaccinees, 45 retained the CA property. In contrast, only 14viruses from vaccinees retained the TS property. Twelveisolates retained both the TS and CA properties. Loss of theTS and/or the CA phenotype by vaccine virus was notnecessarily associated with illness (Table 3). Volunteers 7and 8 were seronegative, shed virus which had lost both theTS and the CA properties, did not become ill, and developeda rise in antibody titer. None of the isolates from controlchildren manifested the CA or TS property. However, twoof these isolates were from control children whose siblings

received vaccine virus, suggesting that the CP18 vaccinevirus spread from a vaccinee to a contact control child.

Since PIV-3 was prevalent in the community at the time ofthese vaccine studies, it was possible that viruses isolatedfrom vaccinees or contacts of vaccinees were derived fromeither CP18 PIV-3 vaccine virus or naturally circulating WTPIV-3. Therefore, viral genome sequence analysis was usedto determine the origin of the PIV-3 isolated from testsubjects. Because biological properties such as reactivitywith monoclonal antibodies can be inconclusive, sequenceanalysis provides a powerful tool that has recently beenexploited in differentiating mumps virus vaccine strains fromWT viruses (24). The region targeted for sequence analysiswas the untranslated leader region of the F gene of viralRNA, since sequence analysis of virion RNA obtained fromseven naturally occurring strains has shown that this regionhas a high degree of sequence diversity compared with thecoding region (21). Sequence analysis was performed onviruses recovered from five vaccinees and one sibling of avaccinee. In addition, the JS WT virus and one communityisolate obtained at the time of this study in St. Louis weresequenced and compared with sequence data from otherisolates previously published (Fig. 2) (8).Comparison of the isolates with seven WT strains (Fig. 2)

showed that the isolates are highly related to the JS WTvirus. For this analysis, the parent WT strain (JS WT), whichhad been cold adapted to generate the CP18 vaccine virus,was considered the reference strain. Extensive homologybetween the JS WT strain and the five viruses isolated fromthe vaccinees was shown. Virus isolated from the sibling ofone of the vaccinees was also vaccine virus-like. In contrast,WT PIV-3 strains isolated between 1957 and 1987 variedfrom the JS sequence by 8 to 28 nucleotides. These resultsdemonstrate that the vaccinees were infected by vaccinevirus and that the vaccine virus spread to at least one controlchild whose sibling was vaccinated with CP18.

DISCUSSION

The CP18 vaccine was not infectious for older seropositivechildren (3 to 10 years old), and the response of this groupwas similar to that observed during previous studies with

TABLE 2. Characterization of TS and CA properties of PIV-3 isolateda from a representative seronegative child (vaccinee 6)

Day postvaccination virus Plaque titer at 39oC/32OCl TS phenotype Growth at 20'C/32'C' CA phenotypeisolated (log10 PFU/ml) interpretationc (log10 PFU/ml) interpretation'

1 < 1.0/5.5 TS 6.3/6.3 CA2 <1.0/6.7 TS 3.6/7.1 Not CA3 6.7/6.9 Not TS NT4 6.7/7.1 Not TS 4.5/6.3 CA5 5.6/6.5 Not TS 7.0/7.3 CA6 5.1/6.1 Not TS 6.2/7.2 CA7 5.1/6.0 Not TS 6.2/7.2 CA8 1.8/5.5 TS 6.0/6.9 CA9 < 1.0/6.5 TS 5.3/6.3 CA

Control virusesJS WT 5.8/6.0 Not TS 1.5/7.2 Not CACP18 inoculum <1.0/5.5 TS 5.9/7.2 CA

a First-passage isolates were characterized.b Plaque titers after 72 h on L-132 tissue culture cells.c TS if 100-fold or greater reduction in titer at 39°C compared with 32°C.d Growth at 20'C for 14 days in RMK tissue culture cells and 7 days at 32°C in RMK was determined by plaque assay in L-132 tissue culture cells at 32'C. NT,

not tested.' CA if <100-fold reduction in growth at 20C compared with 32°C.

3

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2068 BELSHE ET AL. J. CLIN. MICROBIOL.

TABLE 3. Correlation of clinical observations, antibody responses, and the TS and CA properties of PIV-3 isolates from childrenintranasally inoculated with either CP18 vaccine or a placebo

Reciprocal HAI antibody titer No. retaining indicated phenotype(s)Vaccinee or No. of Clinicalcontrol no. Prevaccination 28 Days isolates tested CA TS Both Neither finding(s)0

postvaccination

Vaccinees1 32 512 3 3 3 3 0 A2 16 64 7 7 2 2 0 C3 8 8 1 0 0 0 1 E4 32 16 5 5 0 0 0 C5 <8 64 10 8 5 4 1 B6 <8 64 gb 7 4 3 0 D7 <8 256 10 8 0 0 2 C8 <8 256 9 7 0 0 2 C

Total for all vaccinees 54 45 14 12 6

Controlsc9 256 512 1 0 0 0 1 C10 <8 32 2 0 0 0 2 C11 64 128 1 0 0 0 1 C

a A, fever to 101'F (ca. 38'C), rhinorrhea, coughing, shedding of PIV-3 (days 3, 6, and 7) and PIV-2 (days 4, 5, 8, and 10); B, no fever, wheezing (days 7, 8,10, and 11), rhinorrhea (days 3 to 11), coughing (days 3, 5, and 6 to 11), shedding of PIV-3 on 10 days; C, no symptoms or signs of illness; D, no fever, wheezing(days 5, 6, and 11), rhinorrhea (days 6 to 11), coughing (days 8 and 10), shedding of PIV-3 on 9 days; E, rhinorrhea (days 10 and 11), shedding of rhinovirus andcytomegalovirus on day 8, shedding of PIV-3 on 1 day.

b One virus was not tested for the CA property.c These control children were siblings of vaccine study children; one (no. 10) was shedding PIV-3 on day 0 of the study, and the vaccinee sibling of no. 10 did

not shed virus. Control 9 and vaccinee 2 are siblings, and cross-infection occurred (see text). Control 11 and vaccinee 3 are siblings, and cross-infection may ormay not have occurred.

CP18 in adults (7). Few infections occurred, virus sheddingwas not detected, and illness was not associated with thevaccine virus in older children or adults. Evaluation ofhighly attenuated strains of PIV-3 in both adults and olderchildren may not be necessary, and trials with adults may besufficient prior to proceeding to studies with younger sero-positive children.

In contrast to the relatively low rate of infection in the

seropositive children, all four of the seronegative infants orchildren were infected with CP18 PIV-3 vaccine virus. Twoof the four vaccinees were asymptomatic, and none hadfever; these findings suggest that the vaccine was attenuatedrelative to the intercurrent WT PIV-3 infections that oc-curred in the control children. However, our finding that twoof four seronegative vaccinees developed wheezing for 3 to4 days indicates that the vaccine was not sufficiently atten-

JSwtJS cpl8-#1JS cplS-#2sibling of #2JS cpl8-#5JS cpl8-#6JS cplS-#7Wash/47885/57Tex/12677/63Wash/1511/73Wash/5-6543/73T-x/545/80T-x/535/81St .Louis/20628/87

AGGACAAAAG AGGTCAATAC CAACAACTAT TAGCAGTCAC ACTCGCAAGA ATAAGAGAGA

A

CC

TG C AT AT G C AT AT A

AGGGACCAAA AAAGTCAAAT AGGAGAAATC AAAACAAAAG GTACAGAACA

TATTTA

A TTTTTTT

TTC

TT C**G

C**

A CA

GA

A C

* * T

AA GC G

JSwt CCAGAJS cpl8-#lJS cplS-#2sibling of #2JS cpl8-#5JS cplS-#6JS cpl8-#7Wash/47885/57Tex/12677/63 AWash/1511/73Wash/5-6543/73T-x/545/80Tex/535/81St.Louis/20628/87 T G

kACAAC AAAATCAAAA CATCCAACTC ACTCAAAACA AAAATTCCAA AAGAGACCGG CAACACAACA AGCACTGAAC ACAATGCCAA

T

TT

G C C

T T TA

G CT TT

CC

G

GGGG

T TT T

TC

T

202 # ofdifferences

CT fxrom JSwt0

* 0

0000

C 28111210118

16

* not determined

FIG. 2. Viral genome sequence analysis of parental JS WT virus; five isolates of CP18 PIV-3 vaccine from volunteers 1, 2, 5, 6, and 7; virusfrom an uninoculated sibling of volunteer 2; and seven WT strains not related to the vaccine strain. The sequence is written 5' to 3' in thecDNA sense.

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ATTENUATED PARAINFLUENZA 3 VACCINE IN CHILDREN 2069

uated for young seronegative individuals. Additional atten-uation will be required to achieve a satisfactory PIV-3vaccine.

Phenotypic characterization of the viruses recovered fromthe nasopharyngeal swab samples of vaccinees indicatedthat the CA phenotype was more stable genetically than theTS phenotype. Virus shed by vaccinees early during vaccinevirus infection were generally TS. The TS property was lostat the time of peak virus shedding but re-emerged toward theend of the shedding period; this phenomenon of loss of theTS marker during peak virus shedding was observed previ-ously during studies of TS mutants of RSV in chimpanzees(2). Loss of the TS lesion was not necessarily associatedwith PIV-3 disease in children vaccinated with CP18 virus,suggesting that the TS lesion was not solely responsible forattenuation of the vaccine.The role of the TS phenotype in attenuation has been

investigated for the CP12 vaccine virus and also for otherrespiratory viruses (7, 13, 19, 20). If the TS phenotype weresolely responsible for attenuation, replication in the warmerlower airways would be more restricted than replication inthe cooler upper airways. This has been the case for TSmutants of RSV and influenza A virus (6). In one TS mutantof influenza A virus, the TS phenotype was linked to theattenuation phenotype; TS' virus was shed by a child duringa vaccine study, and this revertant virus was virulent whengiven to adults (19, 20). Importantly, the pattern of sheddingof TS' influenza A virus in which the TS' virus completelydisplaced the TS virus contrasts markedly with the pattern ofshedding of TS' vaccine PIV-3 and RSV, in which thepredominant virus present at the end of the period of virusshedding is TS. The role of the TS phenotype as an attenu-ation factor in the early cold passages of the JS PIV-3 viruseshas recently been examined experimentally (7, 13). WhenCP12 was evaluated in seronegative chimpanzees, it wasequally restricted in replication in the upper and lowerrespiratory tracts; this suggests that non-TS lesions wereresponsible for attenuation of the CP12 virus (7). Twoviruses recovered from children in this CP18 study whichhad lost the TS phenotype and two isolates from chimpan-zees inoculated with the CP12 vaccine which had also lostthe TS phenotype were evaluated in seronegative rhesusmonkeys (13). All four of the non-TS viruses were attenu-ated relative to the parent JS WT virus. The four non-TSvaccine viruses were restricted in replication in the upperand lower respiratory tracts of the rhesus monkeys to adegree similar to that of their parent CP18 and CP12 TSvaccine viruses. Therefore, the attenuation phenotype ofthese low-passage vaccine PIV-3 strains does not appear tobe linked to the TS phenotype.Recovery of PIV-3 from three of the contact control

children who were siblings of vaccinees suggested that eitherWT PIV-3 infection had occurred in volunteers or thevaccine virus was transmissible to household contacts. Bothof these events occurred. In one case, a control child wasfound to be shedding PIV-3 on day 0, i.e., before adminis-tration of vaccine virus to the sibling vaccinee; thus, thisrepresents intercurrent WT PIV-3 infection in the controlchild. One pair of isolates from a vaccinee and a controlsibling revealed sufficient sequence homology to indicatethat vaccine virus was transmitted from the vaccinee to thesibling contact.

Results of this study parallel those of studies conductedwith a CA RSV vaccine given intranasally (16). The RSVvaccine was not infectious in adults, and it exhibited satis-factory attenuation in older children; however, in young

seronegative children mild wheezing or otitis media occurredin some vaccinees. In the present study, only two of the fewseronegative vaccinees developed illness and it was mild.One of the two vaccinees who wheezed was predisposed towheezing in association with upper respiratory illnesses. Forfuture studies, we propose not to vaccinate children with anyprevious episodes of wheezing, even if the wheezing episodewas mild, as in this case. The other child who wheezed didso intermittently, and wheezing was detected as an inciden-tal physical finding. Since the vaccine caused wheezing intwo of four seronegative infants, and the vaccine virusspread from a vaccinee to a control child in at least one case,future studies will involve evaluation of the more attenuatedmutant, CP45, as a vaccine.

Several characteristics of CP45 vaccine PIV-3 suggest thatit may be suitable for use in infants. The virus is morerestricted in replication than CP12 and CP18 viruses inhamsters and chimpanzees, and it is more stable geneticallythan the CP18 virus (9, 10, 15). Careful studies, proceedingstepwise from older to younger children and proceedingfrom seropositive to seronegative children, will allow us todetermine the suitability of CP45 for use as a vaccine toprevent PIV-3 disease.

ACKNOWLEDGMENTSThis work was supported by NIH contracts NO1-AI-05051 and

NO1-AI-15095 and a merit review grant from the Veterans Admin-istration.We gratefully acknowledge the clinical and technical assistance of

Sharon Irby Moore, Lisa Wells, Barbara Bums, Victoria Perkes,Roberta Samorodin, Karen Christina, and Joan Stewart. We thankRobert Chanock for critical review of the manuscript.

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