[PDF] Recombinant Bacille Calmette-Guérin Expressing the Measles Virus

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1445
Recombinant Bacille Calmette-GueÂrin Expressing the Measles Virus Nucleoprotein Protects Infant Rhesus Macaques from Measles Virus Pneumonia

Yong-de Zhu, Glenn Fennelly, Christopher Miller,California Regional Primate Research Center, Department of Pathology,

Microbiology, and Immunology, School of Veterinary Medicine, and

Ross Tarara, Inger Saxe, Barry Bloom,

Department of Pathology, School of Medicine, University of California, and Michael McChesney Davis; Department of Pediatrics and Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York Measles virus infection continues to be a major cause of infant mortality. There is a need for a measles vaccine that can be administered at birth in the presence of maternal neutralizing antibody. Infant rhesus monkeys were immunized with recombinant bacille Calmette-Gue rin expressing the full-length measles virus nucleoprotein (BCG-N) and subsequently challenged with measles virus. Nucleoprotein-speci®c lymphocyte proliferative responses were detected in the absence of anti-N antibody after vaccination. Vaccination with BCG-N did not prevent systemic measles virus infec-

tion; however, there was a signi®cant reduction of lung in¯ammation after challenge. Virus titers

in lymph nodes were signi®cantly lower, and the duration of nasopharyngeal viral shedding was shorter in some vaccinated monkeys after challenge. These results suggest that measles virus± speci®c T cells were primed by BCG-N vaccination and that they prevented virus-induced lung

pathology.Measles is a highly contagious infectious disease that is these high-titer vaccines were associated with increased mortal-

characterized by fever, respiratory tract and systemic infection, ity, especially in female children, caused not by measles virus and immunosuppression. The number of measles cases has but by other infections [8, 9]. The present vaccination strategy, been reduced dramatically by the widespread use of live attenu- the use of existing standard-titer vaccines at 9±12 months of

ated measles vaccines, butoe1 million children continue to dieage, leaves the millions of infants who lose protective maternal

from measles each year worldwide [1]. Virus-induced local measles virus±neutralizing antibody before this age at high tissue damage as well as virus-mediated suppression of the risk for measles. immune system commonly lead to bacterial superinfections. The present goal is to develop a new vaccine that is safe for Measles virus pneumonia or secondary bacterial pneumonia is infants, that is not neutralized by maternal antibody, and that the primary cause of deaths in infants from measles [2, 3]. induces immune responses that prevent serious disease. It is

One-third of measles cases occur in infants'9 months of agenot unreasonable to design a vaccine that, even if ineffective

in developing nations, and the fatality rate in this group is veryat eliciting protective antibody, can induce T cell responses

high. Live attenuated measles virus vaccines are ineffective in that may lead to the rapid clearance of an initial measles virus the ®rst 6±9 months of life because of neutralization by mater- infection and to the reduction of serious disease and death. nal antibody [4, 5].

Bacille Calmette-Gue

Ârin (BCG) is a live attenuated bovine

In an attempt to overcome maternally acquired immunity in tubercle bacillus widely used to immunize against tuberculosis, the early months of life, high-titer measles vaccines (oe104.7

and it can be given at or any time after birth. In 1994, almostpfu) were administered to infants at 4 and 6 months of age

90% of children'1 year of age throughout the world werein several developing countries. These infants showed goodimmunizedwithBCG[1].BCGhasbeendevelopedasarecom-serologic responses and protection against measles [6, 7], butbinant vaccine vector [10], and it effectively delivers antigen

to the respiratory mucosa [11]. BCG was given orally to infantsin the early part of the century, but the intradermal route was

Received 16 May 1997; revised 25 July 1997.subsequently adopted as more convenient for accurate dosing.

Colony-bred male and female newborn rhesus macaques(Macaca mulatta)

Intranasal immunization of mice with recombinant BCG elic-were housed in accordance with the American Association for Accreditation

ited long-lasting secretory IgA and serum IgG responses to of Laboratory Animal Care. The investigators adhered to the guidelines of the Committee on Care and Use of Laboratory Animals, National Resources foreign antigens, presumably because of BCG persistence in

Council.

the nasal-associated lymphoid tissue [11]. Consequently, both Grant support: NIH (RR-00169, AI-35167, AI-01238, and AI-01387). the intranasal and intradermal routes of inoculation were stud- Reprints or correspondence: Dr. Michael McChesney, California Regional

Primate Research Center, University of California-Davis, Davis, CA 95616-ied in the present experiment.

8542.
Immunization with BCG or vaccinia virus expressing the The Journal of Infectious Diseases 1997;176:1445±53 full-length measles virus N protein was shown to accelerate q1997 by The University of Chicago. All rights reserved.

0022±1899/97/7606±0005$02.00

clearance of rodent-adapted measles virus from the brains and

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1446Zhu et al.JID 1997;176 (December)

virus, were housed in accordance with the American Associationto protect against fatal encephalitis in rodents [12, 13]. The

for Accreditation of Laboratory Animal Care. Three groups of 4 immunization strategy here rests on the following assumptions. newborn rhesus monkeys were inoculated with BCG control, BCG First, BCG-N can be given shortly after birth and will not be

11.7, and BCG 6.1C, respectively. In each group, 2 infants were

neutralized by maternal antibody. N protein is the most abun- inoculated by the intradermal (id) route and the other 2 by the dant viral protein in measles virus±infected cells and it is not intranasal (inl) route with a dose of 1110 6 cfu/animal. After 3 a target for neutralizing antibody [14]. Second, cytotoxic T cell months, the infants were given a second dose by the same routes. (CTL) and T cell proliferative responses induced by measles Two months after the ®rst inoculation, 0.1 mL of tuberculin (Tu- virus N protein have been demonstrated in mice and humans bersol 250; Connaught Laboratories, Swiftwater, PA) was inocu- [15±17]. Finally, measles virus N protein±speci®c memory T lated id on the abdomen. The tuberculin skin test was considered

cells engendered by vaccination with BCG-N may induce rapidpositive if the induration wasoe2 mm at 48 h. Before challenge,

a skin test to measles virus N protein was performed with 0.1 mg helper T cell function for the generation of protective levels

of N protein [26] inoculated id in 0.1 mL of PBS.of antibody to the measles virus hemagglutinin (H) and fusion

Calmette observed BCG bacillemia in infants 4 h after they (F) proteins via intermolecular help, as observed in a murine received 10 12 cfu of live BCG given by mouth. This suggested in¯uenza model [18]. that BCG are rapidly taken up by M cells in the intestinal epithe- Nonhuman primates can be infected with measles virus, and lium [27]. To determine whether bacillemia occurred following they show clinical signs and pathology similar to the human BCG administration in the present study, blood samples were disease [19±23]. The aim of the present study was to investi- drawn between 4 and 8 h after inoculation and directly plated onto gate immune responses to measles virus N protein expressed

Middlebrook 7H10 agar with kanamycin.

by BCG in infant rhesus monkeys and to determine whether Five months after the second inoculation, the monkeys were vaccination with BCG-N can prevent systemic infection or challenged by inl inoculation of 10 4.2 TCID 50
of the Davis 87

disease after measles virus challenge.isolate of measles virus grown in rhesus peripheral blood mononu-

clear cells (PBMC) [19]. The animals were monitored daily for clinical signs. Two BCG 6.1C±vaccinated monkeys and 2 control monkeys were sacri®ced on day 14 after challenge to assess pathol-

Materials and Methods

ogy and virus titers in the lungs.Construction of recombinant BCGvectors expressing the N geneIsolation of measles virus from PBMC, tissues, and nasopharyn-of measles virus.The Connaught strain of BCG was used togeal aspirates.Heparinized venous blood was obtained at 7, 14,obtain BCG recombinants expressing the N protein of measlesand 28 days after challenge. Nasopharyngeal aspirates were col-virus. The full-length Edmonston strain measles virus N gene waslected at 7, 10, 14, and 28 days after challenge, and lymph nodesubcloned intoEscherichia coli±mycobacterial expression vectorsbiopsies were performed on day 14 after challenge. QuantitationpNS261 and pMV361, based on the plasmids pMV206 andofvirusloadinPBMC andlymphnodemononuclearcells(LNMC)pMV306, respectively [10]. Both expression vectors include theby end-point dilution coculture with Raji cells was as previouslyaphgene encoding kanamycin resistance, anE. coliorigin of repli-described [28]. One milliliter of nasopharyngeal aspirate was cul-cation, and a synthetic multiple cloning site. By use of thetured with 1110

6

Raji cells in T25 ¯asks in 10 mL of RPMImulticopy (average, 5) extrachromosomal vector, pNS261, theN1640 medium (Irvine Scienti®c, Santa Ana, CA) supplementedgene of measles virus was fused downstream of the 3*end of awith 10% fetal bovine serum (FCS; Intergen, Purchase, NY) andportion of the coding sequence of the in¯uenza virus nonstructural1% glutamine, penicillin, and streptomycin (Sigma, St. Louis). The

protein as previously described [12].cultures were passaged twice per week. All cultures were moni-

To ensure the stability of vector expression in the absence oftored for cytopathic effect and scored positive by an indirect immu-

kanamycin selection in vivo, a single-copy integrating vector,no¯uorescence assay by use of the KK2 monoclonal antibody to

pMV361, whichinserts into the mycobacterialchromosomal DNA,measles virus N protein [29]. The virus titer was expressed as the

was used to design a recombinant (r) BCG construct that stablyTCID 50
per 10 6 cells and was determined by the method of Reed

expresses the full-length measles virus N protein. The strains ofand Muench [30]. Lung tissues were collected at necropsy, minced

rBCG(pNS261::N)andrBCG(pMV361::N)weredesignatedBCGthrough a sieve, and cultured with Raji cells. The virus titer was

6.1C and BCG 11.7, respectively. For a negative control, BCGexpressed as the TCID

50
per gram of lung tissue.

was transformed with a pMV206-based plasmid containing theAnti±measles virus antibody levels.Monkey sera or plasma

vector sequence without the measles virusNgene. Plasmid DNAsampleswerecollectedattimepointsbeforevaccinationandbefore

was electroporated into BCG as described [24]. Lysates prepared and after challenge. Anti±N protein IgM and IgG antibody titers from recombinant BCG containing the plasmid were separated by were measured by an EIA as described previously [26, 28, 31].

8% SDS-PAGE, electroblotted onto nitrocellulose transfer mem-

Measurement of measles virus±speci®c lymphocyte prolifera- branes, and immunolabeled with a monoclonal antibody directed tion and CTL responses.To measure measles virus±speci®c cel- against N [25]. lular immune responses, PBMC were isolated from heparinized Immunization of rhesus monkeys and challenge with measles blood at time points between vaccination and measles virus chal-

virus.Colony-bred male and female newborn rhesus macaqueslenge as described [28]. For the lymphocyte proliferation assay, 1

(Macaca mulatta),seronegative for simian type D retroviruses,110 5

PBMC per well were incubated in RPMI 1640 (Irvine

Scienti®c) supplemented with 10% FCS (Intergen) and 1% gluta-simian T cell lymphotropic virus, and simian immunode®ciency

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1447JID 1997;176 (December) BCG Measles Vaccine

Figure 1.Expression of measles virus Nmine, penicillin, and streptomycin (Sigma) in 96-well round-bot-

protein in recombinant BCG. Lysates of 21 tom plates (Falcon, Lincoln Park, NJ) with 10mg/mL recombinant 10 8 cfu of either BCG 11.7 (lane 1), con- N protein orSpodoptera frugiperda(Sf 9) cell lysate as an antigen taining single copy of N gene integrated into control [26] for 7 days ending with an 18-h pulse of [ 3

H]thymidine

mycobacterial chromosomal DNA, or BCG at 1mCi/well. The plates were harvested onto ®berglass ®lters

6.1C (lane 2), containing multicopy extra-

(Cell Harvester; Inotech, Lansing, MI) and counted by liquid scin- chromosomal vector, wherein N protein is tillation (1540 Microbeta; Wallac, Turku, Finland). The stimula- expressed as fusion protein with in¯uenza vi- tion index was calculated as the ratio of counts per minute in rus nonstructural protein, were electroblotted

stimulated wells divided by counts in antigen control wells. Anonto nitrocellulose transfermembranes and

immunoblotted with N protein±speci®c

monoclonal antibodies. Principal band of fu-To measure measles virus CTL responses, PBMC were restimu-

sion protein in BCG 6.1C lysates migrated lated in vitro, followed by a 4-h chromium release assay as pre- at slightly higher molecular mass than did viously described [28]. The lysis of mock-infected and measles authentic N protein (60 kDa). Levels of N virus±infected autologous B cell lines was measured and the per- protein expression from single-copy vector centage of speci®c lysis was calculated as follows: 1001[(experi- were 50% of those seen with multicopy vec- mental release0spontaneous release)/(maximal release0sponta- tor. neous release)]. Speci®c lysis was considered positive if the lysis of infected targets wasoe2-fold above the lysis of mock-infected targets and if it was at least 10%. Bacillemia was not detected at 4 and 8 h following id or inl Histopathology.To assess the degree of pathology in the inoculation of infant monkeys. No clinical signs or lesions lungs, 2 BCG 6.1C±vaccinated and 2 BCG control monkeys were were observed following inl immunization of monkeys. Local euthanized with sodium pentobarbital on day 14 after challenge,

suppurative skin abscesses and regional lymphadenitis wereand complete necropsies were performed as previously described[32]. Representative tissues were immediately ®xed by infusion

observed in half of the id-inoculated animals. BCG was isolated

of 10% buffered formalin for histology. The ®xed tissues werefrom the lesions of these vaccinated monkeys between 2 weeks

embedded in paraf®n and stained with hematoxylin-eosin. The and 6 months after inoculation. Swabs from draining lymph pulmonary pathology in each animal was evaluated microscopi- node or injection site ulcers were streaked onto Middlebrook cally for the degree and severity of cell necrosis and the in¯amma-

7H10 agar with and without kanamycin. The number of viable

tory cell in®ltration of the alveolus, interstitium, and airway. Lung coloniesthatgrew intheabsenceof kanamycinwascomparable tissue sections were scored as normal, mild, moderate, or severe to the number of colonies that grew on agar in the presence of histopathology. The tissue sections were examined by three pathol- kanamycin. Kanamycin-resistant colonies were subcultured in ogists in a blind fashion. Middlebrook 7H9 liquid medium containing kanamycin for 1 Statistical analysis.The statistical difference in virus titers of week. Lysates fromoe90% of kanamycin-resistant colonies PBMC and LNMC between BCG control and BCG 6.1C± or BCG

subcultured in liquid medium were tested positive for N protein11.7±vaccinated monkeys on days 7 and 14 after challenge wastested by using attest carried out on the coded titers [33].

expression by Western immunoblotting. Thus, both plasmids appeared to be stable in vivo in the absence of kanamycin selection.ResultsSensitization to the BCG vector was tested at 2 months after

the ®rst inoculation by the skin test response to tuberculin withGrowth kinetics of BCG strains in vitro and plasmid stability

in vivo.Recombinant proteins, especially those of eukaryotic the following results: 4 of 4 BCG 11.7±inoculated infants and

2 of 4 BCG 6.1C±inoculated infants had strong responsesorigin, may interfere with the growth of BCG. To comparelevels of protein expression in vitro between the multi- and (average, 8±10 mm induration) indicative of delayed hypersen-

sitivity to BCG. One of 4 BCG controls had a weakly positivesingle-copy BCG vectors, equivalent numbers of colonies ofmid±log phase BCG 11.7 and BCG 6.1C were evaluated by response. We cannot explain why the tuberculin response was

low in BCG control monkeys. Tuberculin skin tests were notWestern immunoblotting. Levels of N protein expression fromthe single copy vector were 50% of those seen with the done after boosting. Skin test responses to the measles virus

N protein before challenge were negative in all monkeys.multicopy vector (®gure 1). To compare levels of replicationin vitro, equivalent numbers of colonies of both BCG-N strainsAnti±measles virus N protein antibody and CTL responses

after vaccination.All juvenile rhesus macaques at the Cali-were inoculated into Middlebrook 7H9 broth, and the opticaldensity at 600 nm was measured and compared with that of fornia Regional Primate Research Center are vaccinated against

measles virus to prevent epizootic infection [34]. The 12 infantan equivalent inoculum of wild type BCG Connaught. Strik-ingly, the single-copy strain, BCG 11.7, replicated at a rate monkeys in this experiment had weak anti±measles virus N

protein IgG antibody levels before the ®rst BCG inoculationcomparable to wild type, while the strain containing themulticopy vector, BCG 6.1C, had a markedly reduced rate of because of maternal antibody, but no IgM antibody was de-

tected (data not shown). No anti±measles virus N protein IgMreplication.

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1448Zhu et al.JID 1997;176 (December)

ci®c T cell responses were detected before challenge in the

BCG control monkeys.

Clinical signs and histopathology after measles virus chal- lenge.Five months after the second vaccination, the 12 mon- keys were challenged with 10 4.2 TCID 50
of measles virus inl. All monkeys developed skin rash and other mild clinical signs, such as lethargy and anorexia. Unlike the control monkeys, which had a peak of skin rash on day 7, some of the vaccinates had a skin rash peak on day 10 after challenge, and the rash was more macular. To compare lung pathology, 2 BCG control and 2 BCG 6.1C±vaccinated monkeys were euthanized on day

14 after challenge. Evaluation of the lung tissue from these

monkeys showed that the BCG 6.1C±vaccinated monkeys had dramatically reduced interstitial pneumonitis and bronchiolitis compared with the BCG control monkeys (®gure 3). About

50%±75% of lung tissue from the control animals had moder-

ate to severe interstitial involvement, compared with 10%±

20% mild involvement in BCG 6.1C±vaccinated animals. Two

of the 4 necropsied monkeys had received BCG by the inl route, but there were no granulomata in their lungs and there were no differences in lung pathology between inl- and id- vaccinated monkeys in each group. Other pathology included lymphoid hyperplasia in the lymph nodes and in gut and respi- ratory lymphoid tissues of both vaccinated and control mon- keys. In the lymphoid tissues of the vaccinated monkeys, but not in the BCG control monkeys, there was marked hyperplasia in the T cell±rich paracortex, but there was no expansion of

B cell follicles or germinal centers.

Virus load in PBMC, LNMC, nasopharyngeal aspirates, and lung.All monkeys developed systemic infection after chal- lenge with measles virus. Viremia peaked at day 7 after chal- lenge and then declined by day 28 after challenge. On day 14 after challenge, the virus titer in LNMC of the BCG 11.7± vaccinated group was signi®cantly lower than that of the BCG Figure 2.Measles virus±speci®c CTL response before challenge in 29069 ( A ), BCG 6.1C±immunized monkey, and 29108 (B), BCG control group (®gure 4). There were no statistical differences control monkey. Target cells were autologous B cells mock-infected in virus titers in PBMC between either vaccinated groups or (solid symbols) or infected with measles virus (open symbols). the control animals, but there was nearly 1 log 10 reduction in virus from the day 14 PBMC of rBCG 11.7±vaccinated mon- keys (®gure 4). Nasopharyngeal aspirates were taken on days

7, 10, 14, and 28 after challenge for measles virus isolation toor IgG antibodies were detected at 3 months after vaccinationand 2 months after boosting, with the exception of 1 BCG determine the duration of viral shedding from the nasopharynx.

Measles virus was detected in the nasopharyngeal aspirates oncontrol infant that had persistent, low-titer N antibody, evenon the day of challenge. A measles virus±speci®c CTL re- days 7 and 10 of all monkeys. On day 14, there was no viral

shedding in 2 BCG 6.1C±vaccinated monkeys and in 3 of 4sponse was detected in 2 of 8 BCG-N±vaccinated monkeys(®gure 2), and there was no CTL response in the BCG control BCG 11.7±vaccinated monkeys (table 2). No measles virus

was detected in the nasopharyngeal aspirates on day 28 aftermonkeys.

Lymphocyte proliferative responses to measles virus N pro-challenge. Lung virus titers were low (10±100 TCID

50
/g) in

both vaccinated and control monkeys necropsied on day 14tein after vaccination.Lymphocyte proliferation to the N pro-

tein was measured after BCG vaccination and boosting. Three after challenge (data not shown).

Measles virus±speci®c IgM and IgG antibody responsesof 4 monkeys in each of the vaccinated groups had low-levelproliferative responses to the N protein on at least one occasion,after challenge.Before challenge, none of the monkeys with

the exception of 1 control monkey had detectable anti±N pro-and the number of responding animals increased after the sec-ond immunization (table 1). No measles virus N protein±spe- tein IgG. This control monkey developed a primary IgM and

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1449JID 1997;176 (December) BCG Measles Vaccine

Table 1.Lymphocyte proliferative responses to measles virus N protein after immunization and boosting with recombinant BCG.

Response after vaccinationResponse after boost*

Route of

Group, animal vaccinationSf9MV-NSISf9MV-NSI

BCG control

29040inl64{437{80.674{1743{30.6

29041id145{19 180{22 1.2197{5103{90.5

29108inl53{1562{11.2865{256877{409 1.0

29111id89{1166{90.7784{186876{301 1.1

BCG 11.7

29066inl65{22 107{33 1.7112{44291{232.6

29069id73{13 124{54 1.7206{521186{548 5.8

29090inl145{19 180{22 1.2107{11274{352.7

29093id53{1562{11.2245{7367{961.5

BCG 6.1C

29096inl65{22 107{33 1.787{16346{150 4.0

29101inl73{13 124{54 1.7337{20484{183 1.4

29105id80{15 169{23 2.1 1684{514 3370{329 2.0

29107id46{9123{75 2.7 1112{184 2475{123 2.2

NOTE. Peripheral blood mononuclear cells (10

5

/well) were cultured for 7 days in presence of 10mg/mL control cell lysate (Sf9) or measles nucleoprotein

(MV-N), and pulsed with [ 3

H]thymidine for 18 h before harvest. Results are expressed as mean cpm{SD of triplicate cultures. inl, intranasal vaccination; id,

* 3 months after ®rst immunization.

IgG response after challenge (tables 3, 4); thus, we reasoned had marked follicular hyperplasia in lymph nodes after chal-

that this reactivity in the EIA was due to either persistence of lenge with wild type measles virus [19], and 4 of 5 vaccinated maternal antibody or nonspeci®c cross-reactivity. After mea- monkeys were protected from systemic infection and clinical sles virus challenge, IgM antibody was detected on day 7, measles [28]. peaked around day 14, and declined to low levels after 12 The immunization strategy used here was to prime a T weeks after challenge in all of the monkeys (table 3). IgG cell response to measles virus antigen in the context of BCG antibody was detectable on day 7 from the BCG 6.1C group infection [12]. The major hypothesis to be tested was and on day 14 in BCG 11.7 vaccinates and control monkeys. whether, on challenge with measles virus, the presence of IgG antibody titers reached a peak at 4 weeks after challenge activated CD8 CTL or CD4 helper T cells, if not protective and were maintained at high levels over a 12-week observation against local viral replication, might protect against systemic period (table 4). infection or extensive virus-induced tissue destruction that, in humans, may predispose to pneumonia or death. As BCG

is used worldwide currently to vaccinate infants against tu-Discussionberculosis, a measles virus N-expressing rBCG that inducesmeasles virus±speci®c T cell responses is a feasible goalAlthough these novel recombinant BCG vectors expressing

for an infant vaccine to protect against measles. This, inthe measles virus N gene did not prevent systemic infection

turn, may attenuate measles virus±induced immunosuppres-afterpathogenic measlesviruschallenge, therewas asigni®cant

sion and tissue destruction.reduction in lung pathology in the vaccinated infant monkeys.

T cell differentiation is in¯uenced by the manner and envi-The BCG-N vaccines elicited weak measles virus N protein±

ronment in which the T cell precursors are stimulated. It hasspeci®c lymphocyte proliferative responses and CTL responses

been demonstrated that low antigen concentration and low-in the absence of speci®c antibody responses. Although the

dose infections tend to induce a Th1-type response, whereasimmunologic basis for the dramatic reduction in lung in¯am-

high-dose antigen induces a Th2 pattern [35]. This may explain mation in the BCG-N±vaccinated monkeys is not clear, the why no anti-N antibody was detected in the infant monkeys detection of low-level T cell proliferative responses after vacci- (0.5 kg body weight) receiving 10 6 cfu of BCG, while anti-Nnation and the remarkable paracortical hyperplasia of the antibody was induced by 2±5110 6 cfu of BCG in mice (1 glymphoid tissues after challenge in the vaccinated monkeys body weight) [12]. Recent studies suggest that immunologic suggests that a protective T cell response was induced by vacci- stimuli that activate macrophages or dendritic cells and trigger nation. In contrast to BCG-N±vaccinated monkeys, monkeys

vaccinated with the Moraten live, attenuated measles vaccine interleukin-12 production tend to initiate Th1 responses, in-

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1450Zhu et al.JID 1997;176 (December)

Figure 3.Histology of formalin-infused

lung tissue from rhesus monkeys 14 days after challenge.A, Lung of monkey that received BCG 6.1C intranasally before challenge. In center of ®eld, bronchiole is shown in cross-section. Note that wall of bronchiole is slightly thickened, with in-

¯ammatory in®ltrate. Surrounding alveolar

structures are essentially normal in appear- ance.B, Lung of BCG control monkey. Ori- entation of this ®eld is same as forA. Wall of bronchiole is thickened, with moderate in¯ammatory in®ltrate, and surrounding al- veolar septa are thickened and alveolar spaces are ®lled with mononuclear in- mm.

cluding help for delayed-type hypersensitivity [36]. BCG repli- After measles virus challenge, virus titers in PBMC and

LNMC were reduced in the vaccinated monkeys relative tocates within macrophages, providing continual and prolonged

antigenic stimulation if the BCG is not destroyed. The BCG- those in controls on day 14 (®gure 4). This reduction was

statistically signi®cant in the LNMC of the vaccine group re-N strains continued to express the N protein after they wererecovered from local injection sites and draining lymph nodes ceiving BCG 11.7, so it seems that BCG 11.7 induced better

protection than did BCG 6.1C. But we cannot explain why IgGseveral weeks to months after inoculation. The positive tuber-culin skin test responses of both vaccinates and controls indi- antibody developed earlier in BCG 6.1C±vaccinated monkeys

than in BCG 11.7±vaccinated monkeys. It is interesting to notecate that delayed-type hypersensitivity was elicited to BCG, soit is possible that a delayed-type hypersensitivity response to that the BCG 11.7, with a single, integrated copy of measles

virus N, replicated in vitro as well as the parental ConnaughtN was also engendered. However, skin test responses to mea-sles virus N protein were not detected after vaccination, so we strain of BCG, while BCG 6.1C, which carries several plasmid

copies of the N gene, was replication-impaired in vitro. Betterspeculate that a delayed-type hypersensitivity response to Nwas weak or absent.replication of BCG 11.7 may have induced better immunity.

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1451JID 1997;176 (December) BCG Measles Vaccine

Table 3.Anti±measles virus N protein IgM antibody titers after challenge. Day

Group,

monkey0 71428 84

BCG control

29040 0 400 6400 6400 0

29041 0 1600 102,400 6400 0

29108* 0 400 1600 NT NT

29111* 0 100 25,600 NT NT

BCG 11.7

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