Live Attenuated Varicella Vaccine: The KMcC Strain in Healthy Children

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VOLUME

71 #{149}MARCH 1983 #{149}NUMBER 3

Pediatrics

Live

Attenuated

Varicefla

Vaccine:

The

KMcC

Strain

in Healthy

Children

Allan

M. Arbeter,

MD, Stuart

E. Starr,

MD,

Robert

E. Weibel,

MD,

Beverly

J. Neft,

PhD,

and

Stanley

A. Plotkin,

MD

From the Department of Pediatrics, The Children s Hospital of Philadelphia and The

University of Pennsylvania School of Medicine, Philadelphia; and Virus and Cell

Biology Research, Merck Institute for Therapeutic Research, West Point, Pennsylvania

ABSTRACT. The KMcC strain of live, attenuated

van-cella-zoster virus vaccine was studied in healthy children

as a preliminary step towand vanicella vaccine studies with this strain in children with leukemia. Forty-three children were immunized: 26 with the 40th passage vac-cme and 17 with the 50th passage. Studies included surveillance for clinical reactivity, oropharyngeal excre-tion of vaccine virus, viruria, and viremia. Antibody re-sponses were assayed by fluorescent antibody to mem-brane antigens and immune adherence hemagglutination. Cell-mediated immune responses were assayed by lym-phocyte proliferation to vanicella-zoster virus specific an-tigens. There was 100% seroconversion to the KMcC passage 40 and 50 vaccines (by fluorescent antibody to membrane antigen assay). Every child studied developed in vitro lymphocyte proliferation to vanicella-zoster virus antigens. Papular skin lesions, probably vaccine related, occurred in 31% of the 40th passage vaccinees but in only

6% of the 50th passage vaccinees. The 50th passage KMcC strain vaccine is sufficiently immunogenic and safe to initiate clinical studies with leukemia patients. Pediatrics

1983;71:307-312; vaccination, chickenpox, varicella.

strain, designated KMcC, had been developed and studied in the United States since 1968. The recent

clinical studies of varicella vaccines were sparked by the increasing evidence for severe clinical disease in immunocompromised individuals.4 Recent

epi-demiologic studies by Preblud5 and Fleisher et al6 indicated that among normal individuals varicella morbidity and mortality are higher than previously assumed. Neonatal varicella7 and Reye’s syndrome8

are recent additions to the list of complications of

natural varicella.

In this report we describe the results of studies conducted with the KMcC strain of VZV vaccine.

The studies were performed in healthy children in

order to gain sufficient experience to select a

vac-cine virus passage level so that further studies in immunocompromised

patients

could be conducted. These studies with the KMcC strain complement

our previously reported clinical trials with the OKA strain of VZV in healthy children.2

A live, attenuated vanicella-zoster virus (VZV)

vaccine designated the OKA strain has been studied in clinical trials since the early 1970s in Japan’ and

since 1979 in the United States.2 Another VZV

Received for publication April 14, 1982; accepted June 16, 1982. Reprint requests to (A.M.A.) Division of Infectious Diseases, The Children’s Hospital of Philadelphia, 34th St and Civic

Center Blvd, Philadelphia, PA 19104.

METHODS

Vaccine

The development and early clinical trials with several tissue culture passage levels of the KMcC strain were recently reported by Neff et al. The virus was recovered from vesicle fluid of an other-wise healthy child with typical varicella. The strain was originally identified as vanicella-zoster virus by plaque reduction neutralization tests. In vitro and

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as required by the Bureau of Biologics. Clinical trials were conducted with tissue culture passage

levels 10, 30, 40, 45, 50, and 60 by Neff and

col-leagues.3 In addition, the 40th and 50th passages

were independently studied by us as described in

this report.

The vaccine was prepared as a frozen aqueous suspension of 1.2 mL per vial. The dose of vaccine

was 0.5 mL and was administered subcutaneously.

The vaccine was always transported in dry ice and stored at -70#{176}C. Vaccine was thawed within ten

minutes before administration, and unused portions

of a thawed vial were discarded. Each dose of KMcC passage 40 and KMcC passage 50 vaccines con-tamed 1,150 and 6,500 plaque forming units, respec-tively, as determined by the manufacturer (Merck Laboratories, mc, West Point, PA).

Vaccinees

Healthy varicella-susceptible children were re-cruited from the private practices of pediatrics in

the Philadelphia area. Susceptibility was confirmed

by the absence of VZV antibody in serum specimens

obtained on the day of immunization. A parent of

each child confirmed the absence of known recent exposure to natural varicella, written consent was

required before immunization. At KMcC passage

40, there were 26 vaccinees (16 male and ten female)

with mean age of 5.0 years (range 18 months to 16

years). At KMcC passage 50, there were 17 vacci-nees (six male and 1 1 female) with mean age of 6.4

years (range 18 months to 14 years).

Serologic Assays

Fluorescent antibody to membrane antigen

(FAMA) assay was performed as previously

de-scribed.2 jj serum specimens were tested at least

twice, and positive and negative control sera were

included on each microtiter plate. The high

repro-ducibiity ofthis technique is noted by low variances

for the standard error of the mean. For 40 repeated

assays of high titer control sera and 67 of low titer

control sera the SEMs were 1.21 and 1.25,

respec-tively.

The immune adherence hemagglutination assay

(IAHA) was performed by Dr J. Neff at Merck

Laboratories.3

VZV.Specific Lymphocyte Proliferation

Lymphocyte proliferation was performed as

pre-viously descnbed.2 Ficoll-Hypaque purified

periph-eral blood mononuclear cells were tested for

respon-siveness to heat-inactivated antigens prepared as

previously described.2 Antigens were prepared from

the KMcC and OKA vaccine strains of VZV and

from the Ellen strain, a high passage laboratory

strain. The antigens were used at final dilutions of 1:100 and 1:200, and the better response was

Se-lected for data analysis. Cell cultures were

har-vested after six days of incubation four hours after

addition of tritiated thymidine. Results are

ex-pressed as the stimulation index, ie, counts per

minute in VZV-antigen-stimulated cultures divided

by counts per minute in control antigen-stimulated

cultures.

Virus Cultures

Specimens for viral culture were obtained from oropharyngeal secretions, urine, and peripheral

blood buffy coat cells to detect vaccine virus

shed-ding and viremia in the children given KMcC pas-sage 40. The handling of viral specimens was the

same as previously described.2 Viral cultures were also obtained from the scrapings of skin lesions. All cultures were kept for 3 weeks. before being dis-carded as negative.

Clinical Reactivity

Acute clinical reactions including temperature

elevation and appearance of skin lesions were re-corded daily by a parent of each vaccinee on a form

provided. In the KMcC passage 40 trial, a parent of

each vaccinee was telephoned or the vaccinee was

visited by a trained study assistant every two to

three days until day 22 postvaccination. When skin lesions were reported by one of the parents, one of

the study physicians visited the vaccinee and

ob-tamed lesion scrapings for viral culture.

RESULTS

Peak body temperature elevations during the first

21 days after immunization, which could not be explained by intercurrent illness, were thought to

be vaccine related (Table 1). Of the 26 children who

received the 40th passage vaccine 14 had no

tern-perature elevation whereas four different children

had temperature elevations in each week after

irn-rnunization of between 37.3 and 38.3#{176}C. One child

had a temperature elevation of 40#{176}C during the

third week; this was probably vaccine related inas-much as it could not be explained by an intercurrent

illness. Episodes of fever occurred frequently in the unvaccinated sibling control group, raising doubt

that all of the temperature elevations in the

vacci-nees were caused by the vaccine. However, the

control group was too small to allow definitive

conclusions. A vaccine-related fever >38.3#{176}C was

noted in two vaccinees (12%) who received the

KMcC passage 50 vaccine. One vaccinee had a fever

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2- to 3-mm papular skin lesions. He remained play-fiil and well during this episode, which lasted one day. Viral cultures of scrapings from his lesions were negative.

The distribution of papular skin lesions is shown

in Table 2. Generalized skin lesions, which were

thought to be vaccine related, occurred in 31% of the KMcC passage 40 vaccinees. Four of the

vacci-nees had between five and 15 lesions. The lesions were papular, sometimes pruritic, occurred between days 14 and 23, and lasted one to three days. None

of the lesions was vesicular. Virus recovery was attempted from the lesions of two of the KMcC passage 40 vaccinees. The cultures were negative.

Firm, tender subcutaneous nodules, 0.5 cm in di-ameter, were observed at the injection site in three of the KMcC passage 40 vaccinees. As the nodules

were neither superficial nor vesicular, viral cultures were not attempted. These nodular lesions occurred

during the second and third postimmunization weeks and lasted between 48 and 72 hours. For the trial with the KMcC passage 50 vaccine, the needle used to fill the syringe was replaced with a new needle before vaccine administration. After this

TABLE 1. Vancella Vaccine: KMcC

Strain-Vaccine-Related Temperature Elevations

Vaccine and Temperature Days After Vaccination (#{176}C)

0-7 8-14 15-23

KMcC passage 40

<37.3 22 22 21

37.3-38.3 4 4 4

>38.3 0 0 1*

Control for passage 40 (not vaccinated

<37.3 2 3 2

37.3-38.3 2 1 2

>38.3 0 0 0

KMcC passage 50

<37.3 17 17 14

37.3-38.3 0 0 1

>38.3 0 0 2t

* One vaccine, maximum temperature 40.0#{176}C.

t Two vaccines, maximum temperature 38.8#{176}Cand 39#{176}C.

change in protocol there were no lesions at the

injection site.

A total of 173 viral cultures were prepared from oropharyngeal secretions, urine, and peripheral blood buffy coat cells from the 26 KMcC passage

40 vaccinees. Oropharyngeal and urine cultures were obtained every three to four days between

days 6 and 22 postimrnunization. Cultures of buffy coat cells from peripheral blood were obtained from

half of the vaccinees between days 6 and 10 and half the vaccinees between days 14 and 22. All cultures were negative.

Serologic Results

The FAMA and IAHA serologic assays were used

in each of the clinical studies (Table 3). All of the

children who received either the 40th or 50th

pas-sage KMcC vaccine seroconverted as determined by the FAMA assay on the 6-week blood specimens.

One vaccinee in the KMcC passage 50 group of

vaccinees did not seroconvert by 6 weeks (by the

IAHA assay). A FAMA titer on the same serum

specimen was 1:32, and the child had a lymphocyte proliferations response with a stimulation index (SI)

of 2.7, which is considered weakly reactive (SI >

2.0).

In the KMcC passage 40 trial, 1/13 patients tested

between seven to ten days after vaccination was

positive for FAMA-VZV antibody. However, by 15 to 21 days 9/10 patients seroconverted with a geo-metric mean titer (GMT) only slightly less than

that of the 6-week specimens. The GMTs of the 6-week postimmunization serum specimens from the

KMcC passage 40 and passage 50 vaccinees were

not significantly different.

Cellular Immune Responses

In vitro lymphocyte proliferation responses

as-sayed with antigens of the KMcC strain of VZV are shown in Fig 1. Included are the in vitro lymphocyte

proliferation results of a KMcC passage 45 clinical

trial conducted independently by Dr Robert E.

TABLE 2. Varicella Vaccine: KMcC Strain-Papular Skin Lesions

Lesions at Injection Site No. of Generalized Lesions

KMcC KMcC

Passage 40 Passage 50

KMcC Passage 40 KMcC Passage 50

<5 5 <5

DaysO-7 0 0 0 0 0 0

Days8-14 2 0 2 1* 0 0

Days 14-21 1 0 2 3ff 0

* One vaccinee with 12 lesions on day 14.

t One vaccinee with 15 lesions on day 15; one vaccinee with five lesions on day 17; one vaccinee with eight lesions on day 16.

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KMc C Vaccine IKMCC Strain Antigen

Vaccine Passage 40 45#{149}

KFk p40 Vaccine

24. 22 20 18 16 14 12 0 TO. z Q 6 -J n 4. I-(if) 2

I

: .

:1

:

i I j .

50

A.

MeantTSD

0 7-14 15-21 6-7 6 6

Days Days Days Weeks Weeks Weeks TIME I #{149}: .1#{149} .: 1#{149} I .5. 1

TABLE 3. Serologic Responses in Normal Seronegative Children Following

Immuniza-tion*

Days After V accination with K McC Passage 40 42 Days After Vaccination with KMcC Passage 50

7-10 15-21 42-49

FAMA

No. tested 13 10 26 17

% seroconversion 7.7t 90 100 100

GMTt 1:4 1:27 1:35 1:26

IAHA

No. tested 4 9 24 17

% seroconversion 0 78 100

GMT ... 1:64 1:79 1:64

C Abbreviations used are: FAMA, fluorescent antibody membrane antigen; JAHA, immune

adherence hemagglutination assay; GMT, geometric mean titer. t One vaccinee.

:1:GMT of seroconverters only.

§Not including one seroconverter by FAMA.

Separate clinical trial conducted by R Weibel MD

Fig 1. Lymphocyte proliferation responses of KMcC

vaccine/KMcC strain antigen.

Weibel. None of the 20 KMcC passage 40 vaccinees

studied had a positive in vitro lymphocyte prolif-eration response on day 0 (SI < 2.0). There was

100% agreement between a <1:2 FAMA titer and a VZV SI < 2.0. Of the eight vaccinees studied be-tween days 7 and 14, six had a reactive lymphocyte proliferation response (SI > 2.0).

Of eight vaccinees studied between days 15 and

21, seven had reactive lymphocyte proliferation

re-sponse (SI > 2.0) with a mean SI of 4.9. Only one of the 22 vaccinees had not developed a reactive

lym-phocyte proliferation response 6 weeks after

im-munization. The mean SI at 6 weeks was 9.2 for the

KMcC passage 40 vaccinees whereas the mean SI

for the 45th and 50th passage vaccinees was 9.6 and

8.6, respectively. These differences are not

signif-icant. The results of in vitro lymphocyte prolifera-tion assays obtained 6 weeks after immunization

26. 24 22 20 18 16 14 12 z - 10 z 08 .2 I-Co 2

KMCC OKA ELLEN

vzv STRAIN ANTIGENS

Fig 2. Comparison of lymphocyte proliferation

re-sponses of KMcC passage 40 vaccine. Abbreviation used is: VZV, varicella-zoster virus.

with the KMcC passage 40 vaccinees indicated no

difference in stimulation indices when tested against KMcC, Ellen, or OKA strain antigens (Fig 2). Moreover, the responses to the individual

anti-gens were similar in each vaccinee (data not shown).

Vaccine Virus Spread to Contacts

Four sibling control subjects, confirmed to be

varicella susceptible by the absence of anti-varicella antibody, neither seroconverted nor developed a vesicular rash in the 6 weeks of observation. The vaccinated siblings of these control children did not have skin lesions after vaccination.

DISCUSSION

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Takahashi et al’ of the development of the OKA

strain vaccine in Japan. Simultaneously the devel-opment and clinical trials of the KMcC strain of varicella-zoster virus were being carried out in the United States. Studies by Neff et al3 indicated that

100% seroconversion was produced by the KMcC vaccines at passage levels up to 40 but seroconver-sion dropped to 90%, 83%, and 0% for the 45th, 50th,

and 60th passage vaccines, respectively. Further-more, the incidence of a generalized rash was at least 24% with the passages up to and including 40. The trial reported here of the KMcC passage 40 vaccine confirmed the previous findings and added

new information. All of the KMcC passage 40 vac-cinees seroconverted by 6 weeks after immuniza-tion. The incidence of skin lesions was 31%, includ-ing four children who had between five and 15 lesions. The lesions were small, nonvesicular, virus

culture negative, and noncontagious.

The KMcC passage 50 vaccine was studied with the expectation that sufficient data would be

ob-tamed to compare the clinical reaction rates and immune responses with those obtained with the 40th passage KMcC and the OKA vaccines.2 The experience of Neff et al with the 50th passage of

KMcC had been somewhat disappointing in that only 5/6 vaccinees seroconverted and the GMT at 6 weeks after immunization was only 1:11 by IAHA. Furthermore, when the 50th passage vaccine was diluted 1:10 the seroconversion rate dropped to 50%. This decrease in seroconversion rate is noteworthy as there was approximately six times the tissue culture-infective dose of virus in the passage 50 vaccine compared with the passage 40 vaccine. The decreased seroconversion rate, along with a high tissue culture plaque count, suggested increased attenuation.

The experience reported herein with the passage 50 KMcC vaccine indicated that the vaccine was immunogenic, eliciting 100% seroconversion rate (FAMA), and the 6-week postvaccination GMT (FAMA) was not statistically different from that of recipients of the passage 40 vaccine. Although one child developed fever and rash (four lesions), he remained well, his rash was of short duration, and vaccine virus was not recovered from the lesions.

Infectious transmission did not occur with the KMcC strain in the four household contacts of recipients of the passage 40 vaccine. There was no evidence of transmission of the vaccine virus to the 12 sibling control subjects for the KMcC passage 50

study reported by Neff et al.

Protection from natural exogenous infection with

VzV

is probably dependent upon the presence of antibody. Passively administered zoster-immune globulin can prevent or modify natural varicella infection.9 Of five children reported by Neff et al

who received passage 40 or 50 vaccine who had

subsequent close contact with varicella, one of the

passage

50 recipients developed varicella compared with nine cases of varicella in the 12 unvaccinated

control subjects. The one passage 50 vaccinee that subsequently developed varicella had not serocon-verted by IAHA after vaccination.

Antibody was reported by Neff and associates to persist at least 6 months to 1 year after immuniza-tion in 12/12 passage 40 vaccinees although the

IAHA GMT had dropped from 1:36 to 1:25 during

this interval. Follow-up studies of the passage 40 and 50 vaccinees reported in our study are now underway.

The development of specific persistent cellular immune responsiveness following varicella virus vaccine is desirable because of the possible relation-ship between declining cellular immunity and VZV reactivation.’0’ The three KMcC vaccines (pas-sage levels 40, 45, 50) reported here elicited VZV-specific in vitro lymphocyte responsiveness. Fur-thermore, there were no differences in the

magni-tude of the responses. Lymphocytes of the vaccinees were equally stimulated with antigens prepared from the KMcC, OKA, or Ellen strains of VZV. These results suggest that lymphocyte proliferation responses to VZV antigens are not strain specific; however, the results might be different with more purified antigens or use of antigens made from low passage wild strains of VZV.

The kinetics of the antibody and cellular immune responses were studied in the passage 40 vaccinees. Cellular responses were detected between seven

and 17 days and antibody appeared between 14 and

21 days (FAMA). One can speculate that on the

basis of these data, a child exposed to natural infection within one week of vaccination would have a modified disease whereas if exposed more than one week after vaccination clinical illness would probably be prevented. Indeed, a future use of varicella vaccine may be to protect against natural infection by vaccination immediately after expo-sure. This approach was successful in studies with the OKA vaccine in Japan.’

Before large-scale immunizations of healthy chil-dren can be recommended with any varicella vac-cine, certain facts must be accumulated. It will be necessary to include greater length of follow-up in a sufficient number of additional vaccinees to

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SUMMARY

The KMcC strain of varicella virus was used to vaccinate 43 healthy children in order to assess clinical reactivity and immune response. The 50th tissue culture-passaged virus was immunogenic and

sufficiently well tolerated that vaccine studies in leukemic children have been initiated.

ACKNOWLEDGMENTS

This work was supported in part by Merck Sharp and Dohme, mc, West Point, PA, The Canuso Foundation of the Children’s Hospital Cancer Research Center, and The Hassel Foundation.

The authors thank Katalin Abraham, Elinor Proctor, Jeanne Herring, and Elizabeth Greenberg for their tech-nical assistance.

REFERENCES

1. Takahashi M, Asano Y, Kmaiya H, et a!: Prevention and treatment of varicella zoster virus, in Nahmias AJ, Dowdie WR, Schinazi RE (eds): The Human Herpesviruses. New

York, Elsevier, 1980, p 414

2. Arbeter AM, Starr SE, Weibel RE, et a!: Live attenuated varicelia vaccine: Immunization studies with the OKA strain in healthy children. J Pediatr 1982;100:886

3. Neff BJ, Weibel RE, Vallarefore VM, et a!: Clinical and laboratory studies of KMcC strain live attenuated varicella virus. Proc Soc Exp Biol Med 1981;166:339

4. Feldman 5, Hughes WT, Daniel CB: Varicella in children with cancer: Seventy-seven cases. Pediatrics 1975;56:388 5. Preblud SR: Age-specific risks of varicella complications.

Pediatrics 1981;68:14

6. Fleisher G, Henry W, McSorley M, et a!: Life threatening complications of varicella. Am J Die Child 1981;135:896

7. Meyers JD: Congenital varicella in term infants: Risks re-considered. J Infect Dis 1974;129:215

8. Center for Disease Control: Follow up on Reye’s syndrome-United States. MMWR 1980;29:321

9. Arbeter AM, Lange BJ, Abraham KG, et a!: Prevention of varicella in immunosuppressed patients: The use of zoster immune globulin, in Nahmias AJ, Dowdie WR, Schizani RE (eds): The Human Herpesviruses. New York, Elsevier, 1980, p 654

10. Russell AS, Marni RA, Bailey M, et al: Cell-mediated im-munity to varicella-zoster antigen in acute herpes zoster.

Clin Exp Immunol 1972;14:181

11. Ruckdeschel JC, Schimoff SC, Smyth AC, et al: Herpes zoster and impaired cell associated immunity to varicella zoster virus in patients with Hodgkin’s disease. Am J Med

1977;62:77

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Existing instruments (for measuring intellect) represent enormous improve-ments over what was available twenty years ago, but three fundamental defects remain. Just what they measure is not known; how far it is proper to add, subtract, and multiply, divide, and compute ratios with the measures obtained is not known; just what the measures signify concerning intellect is not known. We may refer to these defects in order as ambiguity in content, arbitrariness in

units, and ambiguity in significance.

Submitted by Student

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1983;71;307

Pediatrics

Allan M. Arbeter, Stuart E. Starr, Robert E. Weibel, Beverly J. Neff and Stanley A. Plotkin