Evaluation of Recombinant Vaccinia Virus—Measles Vaccines in Infant Rhesus Macaques with Preexisting Measles Antibody

Home , Modified vaccinia Ankara, Vaccinia

Virology 276, 202–213 (2000) doi:10.1006/viro.2000.0564, available online at http://www.idealibrary.com on

Yong-de Zhu,*,1 Paul Rota,† Linda Wyatt,‡ Azaibi Tamin,† Shmuel Rozenblatt,‡,2 Nicholas Lerche,* Bernard Moss,‡ William Bellini,† and Michael McChesney*,§,3

*The California Regional Primate Research Center and §Department of Pathology, School of Medicine, University of California, Davis, California 95616; †Measles Virus Section, Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; and ‡Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received June 20, 2000; returned to author for revision July 28, 2000; accepted August 1, 2000

Immunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication- defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants. © 2000 Academic Press

INTRODUCTION and effective, they are neutralized in vivo by maternal antibody (Albrecht et al., 1977). To avoid the interference Measles virus (MV) is a negative-strand RNA virus in of maternal antibody, vaccine is administered at the age the Morbillivirus genus of the family Paramyxoviridae, of 9 months in developing countries and at 12–15 months and it is the causative agent of measles (Griffin and of age in the developed nations. The existence of a Bellini, 1996). Measles is a highly contagious childhood window of susceptibility to infection, between the time of disease characterized by fever and respiratory and sys- decay of maternal antibody below a protective level and temic infection. Measles in the infant is frequently asso- the time of vaccination, contributes to a worldwide child- ciated with serious complications and a high mortality hood measles mortality of 1,000,000 annually, mainly in rate. Measles cases have dropped dramatically world- the developing countries (World Health Organization, wide since the introduction of live, attenuated vaccines 1996). Recently, several reports demonstrated that in- in the 1960s, especially in the developed countries. fants even less than 4 months old can contract measles There were only 100 reported cases in the U.S. in 1999 (Oshitani et al., 1998; Wairagkar et al., 1998). The use of (CDC, 2000), and the interruption of indigenous MV trans- high-titered or inactivated measles vaccines was found mission in the U.S. has been maintained since late 1993 to be unacceptable as a means to overcome the inter- (CDC, 1996; Watson et al., 1998). Although the currently ference of maternal antibody (Aaby et al., 1988, 1993; used live, attenuated measles vaccines are very safe Fulginiti et al., 1967; Halsey, 1993; Kumar et al., 1998; Whittle et al., 1988). The two envelope glycoproteins of MV, the hemagglu- 1 Present address: Seattle Biomedical Research Institute, Seattle, tinin (H) and fusion (F) proteins, are important compo- WA. nents of a measles vaccine. During MV infection, the H 2 Present address: Tel Aviv University, Tel Aviv, Israel. and F proteins elicit both neutralizing antibodies and 3 To whom reprint requests should be addressed at California Re- gional Primate Research Center, University of California-Davis, Davis cellular immunity. Neutralizing antibodies prevent the CA 95616-8542. Fax: (530) 752-2880. E-mail: mbmcchesney@ucdavis. spread of extracellular virus and the cellular immune edu. response plays a critical role in the recovery from infec-

FIG. 1. Experimental design: vaccination of infant macaques with vaccinia recombinants expressing MV H and F proteins in the presence or absence of MIG or maternal antibody. tion (Burnet, 1968; Good and Zak, 1956). CTL epitopes RESULTS have been mapped to H and F in a human population (Jaye et al., 1998a,b). Thus, the expression of MV H and Experimental design and humoral immune responses F in a recombinant vector like vaccinia virus might be after vaccination able to circumvent the effect of maternal antibody and to As shown in Fig. 1, infant monkeys were divided into immunize the newborn host to H and F antigens. groups that received 0, 3.5, or 7 ml of MIG 48 h prior to Unlike rodents, Asian macaques contract measles vaccination. At 48 h, the geometric mean neutralizing naturally or experimentally, and the clinical signs of antibody titer (GMT) in the blood was 160 in Group 4, measles are similar to those in humans; thus, the which received 3.5 ml MIG (range 80–320), and the GMT monkey is a practical model for the study of measles was 452 in Group 5, which received 7 ml MIG (range pathogenesis and for the development of new measles 320–2560, Table 1). The GMTs achieved after adminis- vaccines (Griffin and Bellini, 1996; Hall et al., 1971; tration of MIG were similar to the GMT of 226 (range Kobune et al., 1996; McChesney et al., 1997; van Bin- 80–2560) measured in eight monkeys with maternally nendijk et al., 1994). Several studies have shown that derived antibody (Table 2). At the same 48 h time point recombinant vaccinia viruses expressing the MV H (day of vaccination), sera from six monkeys that received and F proteins can protect against MV infection in both 3.5 or 7 ml MIG were also measured by the conventional mice and monkeys (Drillien et al., 1988; van Binnendijk plaque reduction neutralization method, and a GMT of et al., 1997; Wild et al., 1992). The highly attenuated 2466 was calculated (range 1393–3734). These titers are modified vaccinia Ankara (MVA) has been used as a in the range for human newborns with maternal antibody recombinant expression vector and is being devel- (range 32–32,768; Sato et al., 1979). oped as candidate vaccine for humans (Moss et al., The infant monkeys were vaccinated by the intrader- 1996). The aim of the present study was to compare mal (id) route with either WR H&F or WR ␤ gal or by the the immunogenicity and protective efficacy of two vac- intranasal (i.n.) and intramuscular (im) routes with MVA cinia viruses, the WR strain and MVA, which express H&F or MVA control. Humoral immune responses to the measles H and F proteins, in infant monkeys in the vaccination were evaluated by virus neutralization and by presence of neutralizing antibody. MV neutralizing an- radio-immunoprecipitation (RIP) for antibodies to the H tibodies and CTL responses were measured after vac- and the nucleoprotein (N). Table 1 shows the titers of cination in the presence or absence of MV immune neutralizing antibody to MV after vaccination with WR ␤ globulin (MIG) or natural maternal antibody. Differing gal or WR H&F in the presence or absence of MIG. Very levels of protection were observed after a respiratory- low levels of MV neutralizing antibody (titers of 10–20) mucosal challenge with wild-type MV. were detected in the two infants of Group 1 vaccinated 204 ZHU ET AL.

TABLE 1 Neutralizing Antibody Titers to Measles Virus in Monkeys Immunized with Recombinant Vaccinia (WR) in the Presence or Absence of MIG

Reciprocal neutralizing antibody titer in immunized monkeys

Group 2 Group 1 (WR ␤gal ϩ Group 3 Group 4 Group 5 (WR ␤gal) MIG 7 ml) (WR H&F ϩ PBS or NMS) (WR H&F ϩ 3.5 ml MIG) (WR H&F ϩ 7 ml MIG) wksa 30998b 31031 30999 31005 30305 30308 30989 31017 30310 30991 30990 31025 30997 31024 30327 30328 30330 30332

0 20 10 320 160 10 20 10 10 20 160 160 160 320 80 320 320 320 320 220Ͻ10 160 80 80 80 80 40 10 160 80 80 80 40 160 160 160 160 810Ͻ10 40 20 160 320 1280 160 40 20 20 160 40 20 80 10 20 10 10 Ͻ10 Ͻ10 20 10 160 640 1280 160 80 10 20 20 10 20 40 20 10 20 20 Ͻ10 Ͻ10 Ͻ10 Ͻ10 640 1280 1280 320 40 Ͻ10 10 20 10 20 10 20 20 40 22c 1280 640 1280 320 5120 1280 2560 1280 2560 640 320 1280 40 1280 640 1280 10 1280

a MIG, PBS, or NMS (normal monkey serum) was inoculated at 48 h prior to immunization at Week 0. Monkeys were boosted at 8 weeks and challenged at Week 20. b Monkey number. c 2 weeks postchallenge. with WR ␤ gal in the absence of MIG. In another two waned over the vaccination period even after boosting. control infants in Group 2, the subcutaneous (sc) inocu- This was also demonstrated by RIP (Fig. 2, Monkeys lation of 7 ml MIG induced titers of 320 and 160 that 30991 and 30997). MV neutralizing antibodies were not waned to an undetectable level at 20 weeks, the time of detected or barely detected in these animals at 20 challenge. In contrast, these infant monkeys developed a weeks, and there was no difference in antibody re- strong antibody response to vaccinia virus (data not sponse to vaccination between the monkeys receiving shown). In Group 3 monkeys vaccinated with WR H&F 3.5 or 7 ml MIG. Also, infants that had maternal antibody without MIG, MV neutralizing antibody developed after at the time of vaccination, either low to medium (Group 6) vaccination, and the monkeys had a high titer of antibody or high titer (Group 7), had waning neutralizing titers in at 20 weeks except for Monkey 30310, with a titer of 40. the same ranges as those monkeys that received 3.5 or Only two of five monkeys in this group had a rise in 7 ml MIG (Table 2). antibody titer after the boost at 8 weeks. Antibody to the Immunoprecipitation experiments confirmed the re- H protein but not to the N protein, which was absent from sults of the neutralization assays (Fig. 2). In the monkeys the vaccine, was detected by RIP (Fig. 2, Monkey 30989). vaccinated with WR H&F in the absence of MIG, antibody However, in monkeys vaccinated in the presence of 3.5 to H was easily detected by RIP on the day of challenge. or 7 ml MIG, Groups 4 and 5, neutralizing antibody titers In contrast, the amount of H that could be precipitated by

TABLE 2 Neutralizing Antibody Titers to Measles Virus in Monkeys Immunized with Recombinant Vaccinia (WR) in the Presence of Maternal Antibody

Reciprocal neutralizing antibody titers in monkeys immunized with WR H&F

Group 6 Group 7 (With low/medium titer maternal Ab) (With high titer maternal Ab) wksa 30985 b 30992 31004 31018 31026 30995 31000 31027

0 160 80 160 80 160 320 2560 320 2 40 80 40 40 80 160 320 160 8 2080201040104040 10 20 40 20 Ͻ10 10 10 20 10 20 10 40 10 Ͻ10 10 10 Ͻ10 10 22c 1280 640 1280 20 640 320 40 640

a MIG or PBS was inoculated at 48 h prior to immunization at Week 0. Monkeys were boosted at 8 weeks and challenged at Week 20. b Monkey number. c 2 weeks postchallenge. PREEXISTING ANTIBODY AND MEASLES VACCINATION 205

FIG. 2. Immunoprecipitation of MV H and N proteins by sera from vaccinated monkeys. Six monkeys’ sera are shown by vaccine group: Monkey 30998 in Group 1 vaccinated with WR ␤gal, Monkey 30989/Group 3/WR H&F, Monkeys 30991 and 30997/Group 4/WR H&F ϩ 3.5 ml MIG, Monkey 31000/Group 7, and Monkey 31026/Group 6/WR H&F ϩ maternal antibody. Four lanes are shown for each monkey corresponding to prevaccination (and before administration of MIG), 2 weeks postboost, day of challenge, and 4 weeks postchallenge. The last two lanes at right show precipitation of H and N with monoclonal antibodies specific for each protein. Note that a nonspecific band migrates with the N protein. sera from monkeys that were vaccinated in the presence neutralizing antibody at 20 weeks. In contrast, monkeys of maternal antibody or MIG decreased over time and vaccinated with MVA H&F in the absence of MIG (Group little antibody to H was detectable on the day of chal- 10) developed MV-specific antibody after vaccination, lenge. Since N was not present in the vaccine, antibody and the antibody titers increased after boosting in two of to N in the postchallenge sera provided an indicator for four animals. All animals had a neutralizing antibody titer replication of the challenge virus. In monkeys vaccinated Ն1:80 at 20 weeks (Table 3), although these titers were with WR H&F in the absence of MIG or maternal anti- lower in general than those of monkeys vaccinated with body, relatively little N was precipitated by the postchal- WR H&F. A decline in neutralizing antibody titers was lenge serum sample. Sera from monkeys vaccinated in demonstrated in monkeys that received 7 ml MIG before the presence of MIG or maternal antibody precipitated MVA H&F vaccination in Group 11 with no response to large amounts of N due to replication of the challenge the booster vaccination. virus (Fig. 2). In contrast to MV neutralizing antibody responses, Table 3 shows the titers of neutralizing antibody to MV antibody responses to vaccinia virus were detected in all after vaccination with recombinant MVA in the presence animals that received MIG or PBS, whether by vaccina- of 0 or 7 ml MIG. In the MVA control monkeys (Groups 8 tion with WR or MVA (Table 4). Antibody titers to vaccinia and 9), antibody titers were similar to those of WR ␤-gal- virus increased after boosting with MVA H&F, but these vaccinated monkeys. No neutralizing antibody response titers returned to the levels before boosting, which were to MV was induced, a decay of transferred measles similar to those of monkeys vaccinated with WR H&F. No antibody was observed, and there was little detectable antibody response to MV was detected in Monkey 30330

TABLE 3 Neutralizing Antibody Titers to Measles Virus in Monkeys Immunized with Recombinant Vaccinia (MVA) in the Presence or Absence of MIG

Reciprocal neutralizing antibody titers in immunized monkeys

Group 8 Group 9 Group 10 Group 11 (MVA) (MVA ϩ 7 ml MIG) (MVA H&F) (MVA H&F ϩ 7 ml MIG) wksa 30270b 30277 30267 30269 30273 30280 30281 30282 30319 30326 30297 30298

0 160 10 320 640 20 20 Ͻ10 Ͻ10 640 2560 320 320 2 160 10 80 320 10 10 20 Ͻ10 320 640 160 80 8 401080801080808080804080 10 10 10 10 20 320 320 160 80 10 20 40 80 20 10 Ͻ10 Ͻ10 10 160 80 80 80 10 20 10 40 22c 640 2560 320 320 640 1280 1280 5120 1280 1280 5120 5120

a MIG or PBS was inoculated at 48 h prior to immunization at Week 0. Monkeys were boosted at 8 weeks and challenged at Week 20. b Monkey number. c 2 weeks postchallenge. 206 ZHU ET AL.

TABLE 4 Antibody Titers to Vaccinia Virus in Monkeys Immunized with Recombinant Vaccinia (WR or MVA) in the Presence or Absence of MIG

Reciprocal ELISA titers in immunized monkeys

Group 3 Group 5 Group 10 Group 11 (WR H&F) (WR H&F ϩ 7ml MIG) (MVA H&F) (MVA H&F ϩ 7 ml MIG) wksa 30305b 30308 30310 30327 30328 30330 30332 30273 30280 30281 30282 30297 30298 30319 30326

0 200 Ͻ200 Ͻ200 Ͻ200 200 200 200 Ͻ200 Ͻ200 Ͻ200 Ͻ200 200 200 200 200 8 6400 12800 3200 6400 6400 1600 12800 1600 1600 1600 800 3200 1600 800 6400 10 6400 6400 6400 3200 6400 3200 6400 51200 12800 25600 6400 25600 12800 3200 25600 20 3200 6400 6400 3200 6400 3200 6400 6400 3200 6400 1600 3200 1600 1600 12800

a MIG, PBS, or NMS (normal monkey serum) was inoculated at 48 h prior to immunization at Week 0. Monkeys were boosted at 8 weeks and challenged at Week 20. b Monkey number. at 2 weeks after challenge (Table 1). All other animals study of MV-specific CTL activity. No CTL response to developed strong antibody responses to MV after chal- MV was detected before vaccination, and there were no lenge (Tables 1–3). detectable CTL responses to MV throughout the entire vaccination period in monkeys vaccinated with WR ␤ gal CTL responses to MV after vaccination in the or the MVA control (Groups 1, 2, 8, and 9, Figs. 3 and 4). presence or absence of MIG In Group 3 animals vaccinated with WR H&F without Peripheral blood mononuclear cells (PBMC) were col- MIG, three of five animals had CTL responses to MV at lected from all monkeys at Weeks 0, 8, 10, and 20 for the 8 weeks postvaccination, and five of five had MV-specific

FIG. 3. MV-specific CTL responses in monkeys immunized with WR vaccinia recombinants. The time point is Week 10, 2 weeks after the second immunization. PREEXISTING ANTIBODY AND MEASLES VACCINATION 207

lenge (Table 5). Skin rashes correlated positively with viral loads in PBMC on Days 7 or 14 after challenge and negatively with CTL activity prior to challenge (see the statistical analysis below). PBMC on Days 7, 14, and 28 after MV challenge were cocultured with Raji cells to quantitate MV viremia. All four WR ␤ gal-vaccinated monkeys (with and without MIG, Groups 1 and 2) had a peak of viremia on Day 7, then the viral load declined on Day 14, and MV was not detected on Day 28 (Fig. 5A). This viremia kinetics is similar to that of naive animals inoculated with measles virus (Zhu et al., 1997). No viremia was detected in the five animals of Group 3 vaccinated with WR H&F without MIG (Fig. 5B); in Group 4, one of four monkeys vaccinated with WR H&F with 3.5 ml MIG had no detectable virus in PBMC postchallenge (pc), and the other three infants had low and transient viremia on Day 7 (Fig. 5B). In monkeys vaccinated with WR H&F in the presence of 7 ml MIG (Group 5), there was no detectable viremia pc FIG. 4. MV-specific CTL responses in monkeys immunized with MVA in Monkey 30330, which had a strong CTL response after vaccinia recombinants. The time point is Week 10, 2 weeks after the vaccination; the other three monkeys had high viral loads second immunization. which were similar to those of WR ␤ gal-vaccinated monkeys (Fig. 5B). Thus a dose response to MIG was suggested by the kinetics and peak of viral loads after CTL activity at 2 weeks postboost and at 20 weeks (Fig. challenge. The eight monkeys in Groups 6 and 7 with 3, Table 5). In the monkeys that received 3.5 ml MIG maternal antibody at the time of vaccination had viral before vaccination with WR H&F (Group 4), three of five loads that were similar to the monkeys vaccinated in the developed CTL activity after vaccination and boosting, presence of 3.5 or 7 ml MIG; five of eight had high viral and in the monkeys receiving 7 ml MIG before vaccina- loads and only one monkey had no detectable viremia tion in Group 5, two of four developed CTL responses to after MV challenge (Table 5). MV (Fig. 3, Table 5). Three of eight monkeys with mater- In the MVA control monkeys of Groups 8 and 9, the nal antibody at the time of vaccination developed CTL kinetics of viremia pc were similar to those of the WR responses after vaccination or boosting (Groups 6 and 7, ␤-gal monkeys (Fig. 6A). Vaccination with MVA H&F, in Table 5). Monkeys vaccinated with MVA H&F had results the absence of MIG (Group 10), resulted in one of four similar to those monkeys vaccinated with WR H&F. If the animals with no detectable viremia; the other three mon- animals were not given MIG before vaccination (Group keys had transient viremia on Day 7 pc, which was 2 10, Fig. 4), four of four had CTL responses after vaccina- logs lower than that of the MVA controls (Fig. 6B). One tion and boosting, but only one of four monkeys in Group 10 of four monkeys receiving 7 ml MIG before vaccination 11 had detectable CTL activity to MV after receiving MIG with MVA H&F (Group 11) had transient viremia on Day 7 (Fig. 4, Table 5). pc, and the other three monkeys had high viral loads Clinical signs and viral loads after MV-Davis which were similar to those of the MVA controls (Fig. 6B). challenge Summary analysis of data (Table 5) All the monkeys were inoculated i.n. with MV-Davis at 20 weeks after the first vaccination. All of the monkeys Animals with preexisting antibody at the time of vac- vaccinated with WR ␤ gal or the MVA control developed cination were significantly less likely to develop a de- skin rashes between Days 8 and 12 after MV challenge tectable CTL response (P ϭ 0.004) and neutralizing an- (Groups 1, 2, 8, and 9 and Table 5). However, monkeys tibody (P ϭ 0.0001) postimmunization, than animals hav- that were vaccinated with WR H&F or MVA H&F after ing no preexisting antibody. The postchallenge peak viral receiving 0 or 3.5 ml MIG (Groups 3, 4, and 10) remained load was significantly reduced in animals having no healthy and no skin rashes were observed. Two of four preexisting antibody at the time of vaccination compared monkeys developed skin rashes after MV challenge in to those with low titers (P ϭ 0.0135) or high titers (P ϭ Groups 5 and 11, vaccinated with WR H&F or MVA H&F 0.0002) of preexisting antibody. Although there was an after receiving 7 ml MIG (Table 5). Three of eight mon- obvious overall trend toward increased peak viral load keys in Groups 6 and 7 that had maternal antibody at the with increasing levels of preexisting antibody at the time time of vaccination developed skin rashes after MV chal- of vaccination, the difference in viral load between 208 ZHU ET AL.

TABLE 5 Summary of Immune Responses Prior to MV Challenge and Viral Load and Skin Rash after Challenge

Experimental Monkey Neutralizing MV viral Skin group Vaccine Ϯ Ab No. antibodya CTLb loadc rash

WR Ϫ ␤gal 30988 Ϫ Ϫ ϩϩϩ ϩ 1 ϩ PBS 31031 Ϫ Ϫ ϩϩϩ ϩ 30999 Ϫ Ϫ ϩϩϩ ϩ 2 ϩ 7mlMIG 31005 Ϫ Ϫ ϩϩϩ ϩ 30305 ϩϩ ϩ Ϫ Ϫ WR H&F 30308 ϩϩ ϩ Ϫ Ϫ 3 ϩ PBS or NMS 30310 ϩϩϪϪ 30989 ϩϩ ϩ Ϫ Ϫ 31017 ϩϩ ϩ Ϫ Ϫ 30990 Ϫ ϩ ϩϩ Ϫ 30991 ϪϩϩϪ 4 ϩ 3.5ml MIG 30997 ϪϩϪϪ 31024 ϪϪϩϪ 31025 ϪϪϩϪ 30327 Ϫ Ϫ ϩϩϩ ϩ 5 ϩ 7ml MIG 30328 Ϫ Ϫ ϩϩ ϩ 30330 ϪϩϪϪ 30332 ϩ ϩ ϩϩ Ϫ 30985 Ϫ Ϫ ϩϩϩ ϩ 6 ϩ low/medium-titer 30992 ϩ Ϫ ϩϩϩ Ϫ maternal Ab 31004 Ϫ ϩ ϩϩϩ Ϫ 31018 ϪϩϪϪ 31026 Ϫ Ϫ ϩϩϩ ϩ 7 ϩ high-titer 30995 Ϫ ϩ ϩϩ Ϫ maternal Ab 31000 ϪϪϩϪ 31027 Ϫ Ϫ ϩϩϩ ϩ MVA 30270 Ϫ Ϫ ϩϩϩ ϩ 8 ϩ PBS 30277 Ϫ Ϫ ϩϩϩ ϩ 30267 Ϫ Ϫ ϩϩϩ ϩ 9 ϩ 7ml MIG 30269 Ϫ Ϫ ϩϩ ϩ 30273 ϩϩϪϪ MVA H&F 30280 ϩϩϩϪ 10 ϩ PBS 30281 ϩϩϩϪ 30282 ϩϩϩϪ 30297 Ϫ Ϫ ϩϩϩ ϩ 11 ϩ 7ml MIG 30298 Ϫ Ϫ ϩϩ Ϫ 30319 Ϫ Ϫ ϩϩϩ ϩ 30326 ϩϩϩϪ

a Neutralizing antibody titer on the day of challenge: (Ϫ) Յ20 reciprocal titer, (ϩ) Ͼ20 and Յ160, (ϩϩ) Ͼ160. b Measles-specific CTL response detected prior to challenge. c 3 6 6 Viral load after MV challenge: (Ϫ) not detected, (ϩ) Ͻ10 TCID50/10 cells on Day 7 after challenge, (ϩϩ) Յ10 TCID50/10 cells on Day 14 and 3 6 6 (ϩϩϩ) Ͼ10 TCID50/10 cells on Day 7 and Ͼ10 TCID50/10 cells on Day 14. groups with low and high titers of preexisting antibody There were four infant monkeys that had neither neu- was not significant at the alpha ϭ 0.05 level (P ϭ 0.0942). tralizing antibody on the day of challenge nor a detect- A significant inverse relationship was found between the able MV-specific CTL response prior to challenge that presence of a prechallenge CTL response and postchal- were still protected from high viral loads or skin rash lenge skin rash, both for all animals, regardless of pre- upon pathogenic virus challenge (Monkeys 31024 and challenge neutralizing antibody status (P ϭ 0.0001) as 31025 in Group 4, Monkey 31000 in Group 7, and Monkey well as for animals without prechallenge neutralizing 30298 in Group 11, Table 5). This suggests that there are antibody titers (P ϭ 0.001). other protective immune responses to MV that we did not PREEXISTING ANTIBODY AND MEASLES VACCINATION 209

strains of vaccinia virus as vaccine vectors revealed similar levels of humoral and cellular immunity in mice (Ramirez et al., 2000; Sutter et al., 1994). However, in this study, peak levels of MV neutralizing antibody were lower in infant monkeys vaccinated with the MVA vector, and MVA was unable to prevent viremia after challenge. It should be noted that the WR strain of vaccinia is moderately pathogenic in rhesus macaques and that the MVA strain cannot replicate fully in mammalian cells. In contrast to the results reported herein, in another recent study, macaques vaccinated with the same recombinant measles MVA in the presence of MV neutralizing antibody did respond to a booster vaccination at 1 month, and these animals were protected from systemic infection 1 year after vaccination (Stittelaar et al., 2000). How- ever, that study modeled the vaccination of 6- to 9-month- old infants with lower levels of maternal antibody than newborns (Sato et al., 1979) and with a more mature immune system. In infant monkeys with preexisting antibody, it is strik- ing that the neutralizing antibody response was inhibited after the booster immunization at 8 weeks when MIG or maternal antibody had declined to low levels. In the

FIG. 5. MV viral loads after challenge in monkeys immunized with

WR vaccinia recombinants. In the top graph, the TCID50 end-point titers of MV are shown for monkeys that were immunized with control WR ␤gal in the absence (closed symbols, Group 1) or presence of MIG (open symbols, Group 2). In the bottom graph, viral titers are shown for monkeys that were immunized with WR H&F in the presence of 7 ml MIG (open symbols, 4 monkeys of Group 5) or 3.5 ml MIG (closed symbols, 5 monkeys of Group 4). The five monkeys of Group 3 that did not receive MIG had no detectable viremia and their data are not shown. The symbol legends indicate individual monkeys in each group. measure and that they are operative in the presence of maternal antibody. These immune responses could in- clude antibody dependent cellular cytotoxicity, delayed type hypersensitivity, or antiviral CTL or neutralizing antibody below the level of detection.

DISCUSSION The major observations obtained from this study are (1) that both WR and MVA vaccinia vectors are safe in newborn rhesus monkeys and that they can immunize newborns to recombinant viral proteins with the induction of both neutralizing antibody and CTL responses, (2) that either maternal or passively transferred MV neutralizing antibody effectively blocks the humoral response to FIG. 6. MV viral loads after challenge in monkeys immunized with

H and F expressed by vaccinia virus in a prime-boost MVA vaccinia recombinants. In the top graph, the TCID50 end-point regimen, (3) that the frequency of CTL responses to MV titers of MV are shown for the monkeys that were immunized with the in infants is reduced by preexisting measles antibody, control MVA in the absence (closed symbols, Group 8) or the presence and (4) that some level of protective immunity, probably a of MIG (open symbols, Group 9). In the bottom graph, viral titers are shown for monkeys that were immunized with MVA H&F in the absence cell-mediated response, prevents the measles skin rash (closed symbols, Group 10) or the presence of MIG (open symbols, despite the inhibitory effect of antibody at the time of Group 11). The symbol legends indicate individual monkeys in each immunization. Direct comparisons of the WR and MVA group. 210 ZHU ET AL. absence of MIG, a boosted neutralizing antibody re- immunoglobulin components of MIG and maternally ac- sponse was observed in some infants. Two of five infants quired antibody are not the same; IgG1 and IgG3 have immunized with WR H&F (Table 1, Group 3) and two of high-affinity receptors in the placenta, and they are pref- four infants immunized with MVA H&F (Table 3, Group 10) erentially transferred to the fetus (McNabb et al., 1976). had fourfold rises in antibody titer after the boost at 8 Eight infant monkeys with low- to high-titer maternal weeks. Other investigations have shown that systemic antibodies (average neutralizing antibody titer of 160) immunity to vaccinia virus inhibits the response to a were vaccinated with WR H&F, and no detectable MV- novel recombinant viral protein delivered with a second- specific humoral responses were induced. CTL re- ary vaccinia exposure (Belyakov et al., 1999; Cooney et sponses were detected in three of these eight monkeys, al., 1991; Rooney et al., 1988). In these reports, vaccinia similar to the number of monkeys vaccinated with WR recombinants were neutralized immediately after inocu- H&F after receiving 3.5 or 7 ml MIG. Thus, at the levels lation, and the effectiveness of the vaccine was reduced, of immune response, viremia after pathogenic virus chal- but this rapid neutralization could be overcome by inoc- lenge and clinical disease, MIG closely mimicked natural ulating the virus by a mucosal route (Belyakov et al., maternal antibody. Further, there was no inhibitory effect 1999). Preexisting vaccinia immunity inhibited both hu- on immune responses to vaccination by a pool of normal moral and cellular responses to recombinant antigens monkey sera that was measles seronegative (2 infant (Belyakov et al., 1999). In the present study, antibody monkeys in Group 3, Table 1). responses to the vaccinia vector were not boosted at 8 In these experiments, infant monkeys (1–2 weeks old) weeks in infants immunized with WR (Groups 3 and 5, were immunocompetent: they developed both humoral Table 4), but there was a large booster response to the and cellular immunity after vaccination, but we do not MVA vector (Groups 10 and 11, Table 4). This difference have data from juvenile or adult monkeys to compare may be due to the abortive infection by MVA or to the fact immune responses quantitatively. Recently, Gans et al. that MVA-immunized infants were inoculated by both a reported that 6-month-old human infants have a defi- parenteral and a mucosal route. ciency in the humoral immune response but not in a Maternally acquired antibodies are important in the cellular immune response to measles vaccination by protection of infants from disease in the first few months comparison with 9- or 12-month-old infants (Gans et al., of life, but the presence of maternal antibodies at the 1998). Similar observations were made for respiratory time of vaccination interferes with responses to several syncytial virus infection of infants (Murphy et al., 1986). infant vaccines, such as for measles, parainfluenza, and But immaturity of the infant immune system, particularly respiratory syncytial viruses (Durbin et al., 1999; Halsey at the humoral level, would not preclude successful im- et al., 1985; Siegrist et al., 1998b). We hypothesized that munization of newborns against viral pathogens like MV. the chimeric expression of measles H and F in a vaccinia In this study, the inhibition of humoral responses by vector could overcome the inhibition by maternal anti- preexisting antibody did not preclude protection from the body, which we proposed was due to viral neutralization. measles skin rash. Indeed, the pool of MIG that we used did not neutralize Although neutralizing antibody is a reliable sign of the WR H&F vector in a vaccinia neutralization assay vaccine-induced immunity, cellular immunity is also es- (data not shown), and it is unlikely that MV H and F sential to recovery from measles (Jaye et al., 1998a). glycoproteins are expressed on the surface of this re- Children with congenital agammaglobulinemia that con- combinant vaccinia virion. Contrary to our hypothesis, tract measles develop protective immunity without de- preexisting MV antibody reduced the humoral and cellu- tectable antibody after infection (Good and Zak, 1956). lar immune responses to measles H and F antigens Recently, it was shown that vaccinated children with no expressed by vaccinia virus in vivo. Several similar ob- detectable antibody response are protected from mea- servations from mice, monkeys, and humans have been sles and it was suggested that these children may have reported for MV (Halsey et al., 1985; Kumar et al., 1998; cellular immunity that provides the protection (Carson et Siegrist et al., 1998a; van Binnendijk et al., 1997) and al., 1995; Samb et al., 1995). Although antibody re- other paramyxoviruses (Durbin et al., 1999; Murphy et al., sponses to H and F were undetectable in the vaccinated 1988). This blockade, more effective against the priming monkeys receiving MIG, by either neutralization assay or of B cells than of T cells, may be due to immune complex by RIP, some of these monkeys developed CTL re- formation at the site of vaccinia replication and the sub- sponses to MV. There are several similar observations sequent degradation of antibody-complexed H and F both in monkeys and in mice (Siegrist et al., 1998b; van proteins by tissue macrophages. Alternatively, MV anti- Binnendijk et al., 1997). In these reports, antibody re- body might downregulate the expression of the H and F sponses were inhibited, but cellular immunity was not or at the cell surface (Fujinami and Oldstone, 1979). only partially inhibited. And those monkeys that devel- The MIG pool that was used in the present experiment oped cellular immunity without humoral immunity after has serum proteins other than immunoglobulins that may vaccination had very low cell-associated viremia after have influenced the outcome of vaccination. And the challenge with wild-type virus (van Binnendijk et al., PREEXISTING ANTIBODY AND MEASLES VACCINATION 211

1994). As recently proposed (Seiler et al., 1998), the antibody, were vaccinated with either recombinant WR or present results demonstrate that even though infant vac- MVA (Fig. 1). In the WR groups, all animals were vacci- cines given in the presence of maternal antibody may not nated id with 1 ϫ 10 6 plaque-forming units (pfu), an be able to induce a detectable antibody response or to optimal route and dose for this virus in infant monkeys prevent viral replication, they may stimulate a cellular (M. McChesney, unpublished data). As shown in Fig. 1, response capable of clearing infection and preventing four control monkeys were vaccinated with WR express- severe disease. ing beta galactosidase (WR-␤ gal) in the presence of 7 ml MIG (n ϭ 2) or PBS (n ϭ 2). Fourteen infants were MATERIALS AND METHODS vaccinated with WR H&F in the presence of PBS (n ϭ 3) or NMS (n ϭ 2), 3.5 ml (n ϭ 5) or 7 ml MIG (n ϭ 4). Construction of WR and MVA recombinant vaccinia Another eight infant monkeys that had maternal antibody viruses expressing MV H and F proteins at a low to moderate neutralizing titer (80–160, n ϭ 5) or A recombinant WR strain of vaccinia virus expressing a high titer (320–2560, n ϭ 3) were vaccinated with WR both the H and F proteins of the Edmonston strain of MV H&F. In the MVA groups, all monkeys were vaccinated (WR H&F) was constructed as previously described with 1 ϫ 10 8 pfus by both the im and i.n. routes. Four (Tamin et al., 1994). In this recombinant virus, H was control monkeys were vaccinated with parental MVA in inserted into the TK gene under the control of the P7.5 the presence of 7 ml MIG (n ϭ 2) or PBS (n ϭ 2). Eight promoter and F was inserted into the vaccinia HA gene infants were vaccinated with MVA H&F in the presence and expressed using the M1 promoter. Construction of of 7 ml PBS (n ϭ 4) or 7 ml MIG (n ϭ 4, Fig. 1). All of the MVA H&F, a double-recombinant expressing the H and F animals were boosted with the same recombinant vi- of the Edmonston strain of MV, was described elsewhere ruses and doses at 8 weeks after the first vaccination, (Stittelaar et al., 2000). Briefly, the H and F genes were but MIG was only administered before the first vaccina- placed under control of the modified H5 and P7.5 pro- tion. moters, respectively, and inserted into deletions II and III of the MVA genome, respectively. Both MVA and MVA Measurements of MV neutralizing antibodies and H&F were propagated in chicken embryo fibroblasts as antibodies to H and N proteins by radio- described (Sutter et al., 1994). immunoprecipitation (RIP) Passive transfer of MIG prior to vaccination MV neutralizing antibody was measured by viral antigen reduction in Vero cells in 96-well plates as described MIG is a pool of heated-inactivated sera from monkeys previously (Zhu et al., 1997). Neutralizing antibody titers that contracted measles during an outbreak amongst were calculated as the highest dilution showing 50% juvenile monkeys at the California Regional Primate Re- reduction in optical density of six control wells that con- search Center in 1995. The reciprocal neutralizing anti- tained virus without serum. Selected sera were also body titer of MIG is 5120 by 50% end-point titration. MIG tested for viral neutralization by the plaque reduction (3.5 or 7 ml) was administered to infant rhesus monkeys method (Albrecht et al., 1981). Antibodies to measles H by SC inoculation 48 h prior to vaccination. Control infant and N proteins were detected by RIP as previously de- monkeys were inoculated sc with either 7 ml of phos- scribed (Tamin et al., 1994), with a single exception. The phate-buffered saline (PBS) or pooled, heat-inactivated infected-cell pellet was resuspended in RIP buffer (1% normal monkey sera (NMS) that was negative for mea- sodium deoxycholate, 1% Triton X-100, 0.2% SDS, 150 sles antibody by IgG ELISA (Zhu et al., 1997). mM NaCl, and 50 mM Tris–HCl at pH 7.4). Vaccination with WR and MVA recombinant viruses Measurement of MV-specific CTL Colony-bred male and female newborn rhesus macaques (Macaca mulatta), seronegative for simian type D PBMC were isolated from heparinized blood and cryo- retroviruses, simian T-cell lymphotropic virus, and simian preserved in liquid nitrogen until use. To measure mea- immunodeficiency virus, were housed in accordance sles CTL responses, PBMC were re-stimulated in vitro with the American Association for Accreditation of Lab- with MV antigen, followed by a 4-h chromium release oratory Animal Care. This macaque colony is routinely assay as previously described (Zhu et al., 1997). The lysis vaccinated against measles (Attenuvax, Merck and Co., of mock-infected and measles virus-infected autologous Inc., Westpoint, PA), and ϳ25% of newborn monkeys B cell lines was measured and the percent specific lysis have maternal measles-neutralizing antibody. The inves- was calculated as: 100 ϫ [(experimental release Ϫ spon- tigators adhered to the guidelines of the Committee on taneous release)/(maximal release Ϫ spontaneous re- Care and Use of Laboratory Animals, National Re- lease)]. Specific lysis was considered positive if the lysis sources Council. Infant monkeys (1–2 weeks old, body of infected targets was greater than twofold above the weight ϳ0.5 kg), after assaying for measles-neutralizing lysis of mock-infected targets and if it was Ն10%. 212 ZHU ET AL.

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