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Center for Immunization Research, Bloomberg School of Public Health, Johns Hopkins University, Baltimore
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Division of Infectious Diseases, Saint Louis University Health Sciences Center, St. Louis, Missouri
Wyeth Vaccines Research, Pearl River, New York
Division of Pediatric Infectious Disease, Vanderbilt University Medical Center, Nashville, Tennessee
Background.
Recombination technology can be used to create live attenuated respiratory syncytial virus (RSV) vaccines that contain combinations of known attenuating mutations.
Methods.
Two live attenuated, recombinantly derived RSV vaccine candidates, rA2cp248/404SH and rA2cp248/404/1030SH, were evaluated in 31 adults and in 95 children 6 months old. rA2cp248/404/1030SH was subsequently evaluated in 44 infants 12 months old. These vaccine candidates share 4 attenuating genetic elements and differ only in a missense mutation (1030) in the polymerase gene.
Results.
Both vaccines were highly attenuated in adults and RSV-seropositive children and were well tolerated and immunogenic in RSV-seronegative children. Compared with that of rA2cp248/404SH, replication of rA2cp248/404/1030SH was restricted in RSV-seronegative children (mean peak titer, 104.3 vs. 102.5 plaque-forming units /mL), indicating that the 1030 mutation had a potent attenuating effect. Although rA2cp248/404/1030SH was well tolerated in infants, only 44% of infants who received two 105.3-pfu doses of vaccine had detectable antibody responses. However, replication after administration of the second dose was highly restricted, indicating that protective immunity was induced. At least 4 of 5 attenuating genetic elements were retained in recovered vaccine viruses.
Conclusions.
rA2cp248/404/1030SH is the first RSV vaccine candidate to be sufficiently attenuated in young infants. Additional studies are needed to determine whether rA2cp248/404/1030SH can induce protective immunity against wild-type RSV.
Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness (LRI) in infants and children [1], but a vaccine is not available because of several obstacles, including the need to vaccinate early in life [24] and the history of immune-mediated enhancement of naturally occurring RSV disease among RSV-naive recipients of a formalin-inactivated RSV vaccine [57]. Enhanced RSV disease has never been observed after natural infection or administration of candidate live attenuated RSV vaccines [2, 810]. Live virus vaccines administered intranasally also afford better mucosal immunity than do inactivated virus vaccines administered parenterally [11, 12]. For these reasons, live attenuated vaccines are being developed for RSV-naive populations.
Live attenuated RSV vaccines have been in development for several decades. A cold-passaged (cp), nontemperature sensitive (ts) derivative of RSV, cpRSV, caused mild respiratory illness in young children [13]. Chemical mutagenesis of cpRSV produced several ts mutants [1416] that were evaluated in clinical trials during the 1990s. cpts248/955 was insufficiently attenuated in RSV-seronegative children and older infants, precluding further evaluation in younger infants [9]. However, cpts248/404 was highly attenuated in RSV-seronegative children and was the first RSV vaccine to be administered to 12-month-old infants. Unfortunately, cpts248/404 caused nasal congestion in these infants, an unacceptable adverse effect in this population. The nasal congestion was temporally associated with vaccine virus shedding, and the mean peak titers shed were 104.0 and 104.9 pfu/mL at the 2 dose levels tested [2]. Although cpts248/404 was insufficiently attenuated in this target population, this study provided important information regarding (1) the level of attenuation necessary for infants, (2) the ability of live attenuated RSV to replicate and induce antibody responses in infants who have maternally derived RSV antibody, and (3) preliminary evidence of protection against illness associated with wild-type (wt) RSV infection [2].
Efforts were next made to develop a live RSV vaccine that was slightly more attenuated than cpts248/404 in RSV-naive infants and children. The mutations present in cpRSV and 6 cpts derivatives were identified by means of cDNA technology [17], permitting the generation of recombinant RSV (rRSV) vaccine candidates that contained new combinations of attenuating cp and ts point mutations [1821] and deletions () of nonessential genes (e.g., the SH, NS1, NS2, and M2-2 genes) [22, 23]. On the basis of preclinical studies, 1 deletion mutation (SH) and 1 ts mutation (1030) were selected for addition to cpts248/404, to generate rRSVs that might be more attenuated in humans. SH attenuated wt RSV in mice [22] and chimpanzees. Although the levels of attenuation of 248/404SH and cpts248/404 were similar in chimpanzees [23], SH was chosen because studies in infants might show an additional attenuating effect, given that the young infant is a more permissive host for RSV than the chimpanzee [2]. The 1030 mutation was added to create rA2cp248/404/1030SH, because rA2cp248/404/1030 was more ts in vitro and more attenuated in mice than was cpts248/404 [24].
rA2cp248/404SH contains 4 independent attenuating genetic elements: cp, which is based on 5 missense mutations in the N and L proteins and the F glycoprotein that together confer the non-ts attenuation phenotype of cpRSV and that are considered to be a single attenuating genetic element [25]; ts248, a missense mutation in the L protein [16, 19]; ts404, a nucleotide substitution in the gene-start transcription signal of the M2 gene [20]; and SH, complete deletion of the SH gene [20, 23, 25]. rA2cp248/404/1030SH contains 5 independent attenuating genetic elements: those present in rA2cp248/404SH and ts1030, another missense mutation in the L protein [24]. Here, we report the evaluation of the safety, immunogenicity, and phenotypic stability of rA2cp248/404SH and rA2cp248/404/1030SH in adult and pediatric populations.
PARTICIPANTS, MATERIALS, AND METHODS
Vaccines.
Construction, rescue, and biological cloning of infectious rA2 RSV strains were conducted at the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD); Wyeth-Ayerst Research (Gosport, United Kingdom); and Wyeth Vaccines (Pearl River, NY) [18, 24, 25]. rA2cp248/404SH was rescued and biologically cloned by means of 3 successive plaque-to-plaque purifications and was amplified twice, all in HEp-2 cells. Virus seed was passaged once in Vero cells and was purified by means of 3 successive plaque-to-plaque isolations in Vero cells. The final virus clone was amplified by means of 1 passage in Vero cells and 1 passage in Vero cell microcarrier culture at 30°C.
rA2cp248/404/1030SH was rescued in HEp-2 cells and biologically cloned by means of 2 additional terminal dilutions. The final virus clone was amplified by means of 2 passages in Vero cells followed by 1 passage in Vero cell microcarrier spinner culture. This material was used to prepare the vaccine in microcarrier culture at 30°C.
Viral suspensions for clinical trials were produced in Vero cells and were found to be free of adventitious agents by Wyeth Vaccines. The titers of rA2cp248/404SH and rA2cp248/404/1030SH were 107.0 pfu/mL and 107.5 pfu/mL, respectively. To achieve the necessary titers, vaccines were diluted as described elsewhere [2]. Diluent was also used as placebo.
Study design.
Both rRSV vaccines were evaluated in open-label trials in adults and in randomized, double-blind, placebo-controlled trials in RSV-seropositive and RSV-seronegative children (table 1). rA2cp248/404/1030SH was also evaluated in infants. The studies were conducted between April and November over several years. One RSV-seronegative placebo recipient and 2 recipients of 105.3 pfu of rA2cp248/404/1030SH were naturally infected with community-acquired wt RSV, as determined by sequence analysis of recovered virus. Their data were excluded from the analysis of safety and immunogenicity (tables 1 and 2) but were included in comparisons of phenotypic stability (table 3).
Eligible individuals were healthy and had no contact with immunosuppressed individuals or infants <6 months old [2, 9]. Each 0.5-mL dose of vaccine or placebo was intranasally administered. After inoculation, physical examinations were performed, and nasal wash specimens were obtained for viral culture (table 1) [2, 9]. Fever, respiratory illnesses, and otitis media (OM) were defined as described elsewhere [9]. We also monitored nasal congestion in infants <6 months old [2]. In our analysis, we included nasal congestion that interfered with eating or sleeping or that resulted in obligatory mouth breathing.
Surveillance.
To determine whether immunization with a live attenuated RSV vaccine was associated with enhanced disease, RSV-seronegative children and infants who received either vaccine or placebo were monitored for wt RSV infection during the subsequent RSV season [2, 9].
Virus isolation, quantitation, and phenotypic characterization.
Virus was isolated from snap-frozen nasal wash specimens and identified as RSV, as described elsewhere [9]. Infectivity was quantitated by plaque assay [9]. Specimens that were negative by culture were assigned a titer of 100.3 pfu/mL.
To determine the level of temperature sensitivity of virus present in nasal wash, specimens from recipients of rA2cp248/404SH were tested for efficiency of plaque formation (EOP) at 32°C, 36°C, 37°C, 38°C, and 39°C, and specimens from recipients of rA2cp248/404/1030SH were tested for EOP at 32°C, 35°C, 36°C, 37°C, and 38°C. On the basis of comparison with titers observed at 32°C, the EOP of rA2cp248/404SH was 100-fold reduced at 38°C, and the EOP of rA2cp248/404/1030/SH was 100-fold reduced at 35°C.
Genetic characterization.
Sequence analysis was performed on 9 viruses recovered from 5 recipients of rA2cp248/404/1030/SH. These viruses plaqued efficiently at 36°C and, in one case, at 37°C. Sequencing was performed directly on 1 specimen and otherwise on uncloned, passaged preparations, as described elsewhere (table 4).
Immunologic assays.
Serum specimens were tested for titers of antibodies to RSV by 60% plaque reduction neutralization assay [26] and for IgG and IgA antibodies to RSV F and G glycoproteins by ELISA [9]. All titers are expressed as mean reciprocal log2 values.
Data analysis.
Participants were considered to be infected if at least 1 of the following criteria was met: vaccine virus was isolated, a 4-fold increase in neutralizing antibody titer occurred, or 4-fold increases in 2 of the ELISA titers (serum IgG to RSV F glycoprotein, serum IgG to RSV G glycoprotein, serum IgA to RSV F glycoprotein, and serum IgA to RSV G glycoprotein) occurred (table 1). Mean peak titers of vaccine virus shed (log10 pfu/mL) were calculated for infected vaccine recipients. To calculate means, neutralizing antibody and ELISA reciprocal titers were log2 transformed. The Mann-Whitney U test was used to compare titers. Rates of illness were compared by Fisher's exact test (2-tailed). P < .05 was considered to be statistically significant.
RESULTS
Response of adults and RSV-seropositive children.
In adults and RSV-seropositive children, rA2cp248/404/SH and rA2cp248/404/1030SH were well tolerated and highly restricted in replication (table 1). One seropositive recipient of rA2cp248/404/1030SH experienced pneumonia on study day 5; this child did not have evidence of infection with vaccine virus, but enterovirus was detected on study days 4 and 5 (table 1). After vaccination, 4-fold increases in antibody titers rarely occurred (table 2), suggesting that these vaccines are minimally infectious or immunogenic in nonRSV-naive populations.
Response of RSV-seronegative children.
In RSV-seronegative children, rA2cp248/404SH and rA2cp248/404/1030SH were infectious and immunogenic. The frequency of illnesses was similar in vaccine recipients and placebo recipients (table 1). OM was observed slightly more often in RSV-seronegative vaccine recipients than placebo recipients, but it occurred sporadically throughout the study period and was not consistently associated with vaccine virus shedding. LRIs were not observed in vaccine recipients.
Although both rA2cp248/404SH and rA2cp248/404/1030SH readily infected RSV-seronegative children, the level of viral replication differed significantly. The mean peak titer shed by recipients of the 105.0-pfu dose of rA2cp248/404SH was 50-fold greater than that shed by recipients of the 105.3-pfu dose of rA2cp248/404/1030SH (104.3 vs. 102.5 pfu/mL, respectively; P = .009) (table 1), and this difference was observed throughout the study period (figure 1). The dose of rA2cp248/404/1030SH influenced the frequency of infection (69% for the 104.3-pfu dose vs. 100% for the 105.3-pfu dose) but not the mean peak titer shed (table 1).
Despite differences in replication, both vaccines were immunogenic in RSV-seronegative children. Eighty-eight percent of recipients of the 105.0-pfu dose of rA2cp248/404SH had 4-fold increases in neutralizing antibody and RSV F and G antibody titers (table 2); similarly, 100% of recipients of the 105.3-pfu dose of rA2cp248/404/1030SH developed antibody responses, with mean postvaccination titers similar to those achieved with rA2cp248/404SH (table 2). The dose of rA2cp248/404/1030SH also influenced the frequency and magnitude of the antibody response: only 4 of 12 RSV-seronegative children who received the 104.3-pfu dose developed a neutralizing antibody response, compared with 7 of 8 who received the 105.3-pfu dose (P = .03), and the postvaccination mean reciprocal log2 titers were 5.2 and 7.2, respectively. This difference can be explained by the increased rate of infection with vaccine virus among the recipients of the 105.3-pfu dose and suggests that increases in dose may be one strategy for enhancement of the immune response to highly attenuated RSV vaccines.
Response of infants.
rA2cp248/404SH was not evaluated further because its level of replication in RSV-seronegative children was similar to that of cpts248/404, which caused congestion in infants [2]. In contrast, replication of rA2cp248/404/1030SH was highly restricted in RSV-seronegative children, making it suitable for evaluation in infants. Two doses of either 104.3 or 105.3 pfu of rA2cp248/404/1030SH or of placebo were administered to infants. Of the 44 infants enrolled, 2 were withdrawn before the second 104.3-pfu dose of vaccine was administered, and 3 were withdrawn before the second dose of placebo was administered.
Mild illnesses occurred frequently and at similar rates in infants who received vaccine or placebo (table 1). Rates of illness were highest in recipients of the first 104.3-pfu dose (69%) (table 1), but illnesses occurred at similar rates in infected and uninfected vaccine recipients (70% vs. 67%, data not shown) and were not temporally associated with viral replication. LRI was observed in 2 infants who received vaccine: rhinovirus was recovered from 1 infant with bronchiolitis (study days 414), and parainfluenza virus type 3 was recovered from 1 infant with pneumonia (study days 512). Neither infant shed vaccine virus.
As was observed for the RSV-seronegative children, replication of rA2cp248/404/1030SH was highly restricted in infantsmean peak titers after the first dose of vaccine was administered were 102.4 pfu/mL for recipients of the 104.3-pfu dose and 103.5 pfu/mL for recipients of the 105.3-pfu dose (table 1). Increasing the dose increased the percentage of infected infants, from 63% to 94%; similar increases were observed for the RSV-seronegative children (table 1).
Surveillance.
RSV-seronegative children and infants enrolled in the present vaccine studies also participated in surveillance for RSV disease during the winters after vaccination. Enhanced disease was not observed when children and infants initially infected with vaccine virus were naturally infected with wt RSV.
Phenotypic and genetic analysis.
rA2cp248/404SH and rA2cp248/404/1030SH are ts viruses, with 100-fold reductions in EOP at 38°C and 35°C, respectively. In 42 nasal wash specimens from 8 recipients of rA2cp248/404/SH, plaques were not detected at 39°C (data not shown), indicating stability of the ts phenotype. One hundred forty-one nasal wash specimens from 45 recipients of rA2cp248/404/1030SH contained virus that formed plaques at 32°C (table 3). Of these specimens, 48 contained virus that formed plaques at 35°C, 27 contained virus that formed plaques at 36°C, and 1 contained virus that formed plaques at 37°C (table 3). In contrast, 9 specimens from 3 children naturally infected with wt RSV contained virus that formed plaques at 35°C, 36°C, 37°C, and 38°C (table 3).
The genetic characterization of selected viruses is shown in table 4. The 5 cp mutations, the 404 mutation, and the SH mutation were present in all isolates tested, demonstrating the stability of these mutations after replication in RSV-naive children. In 5 instances, single nucleotide substitutions were observed at either the 248 or 1030 codons, with reversion to the wt coding assignment. A sixth isolate had a mixed population of nucleotides at the 1030 codon (table 4). Each isolate had nucleotide substitutions at no more than 1 codon.
In some isolates, genetic changes were not detected, despite alteration in ts phenotype (table 4). In these isolates, it is likely either that mutations occurred at the known attenuating sites, but in a proportion of the uncloned virus population that was insufficient to be detected by sequence analysis, or that suppressor mutations occurred elsewhere in the genome.
DISCUSSION
Recovery of infectious virus from cDNA clones of RSV [17] has profoundly influenced the development of live attenuated RSV vaccines, because it is now possible to develop new vaccine candidates by introducing combinations of attenuating mutations into rRSV by direct manipulation of the DNA intermediate. The effect of each mutation cannot be predicted precisely, because the phenotype associated with an individual mutation is not always additive in the context of other mutations. This was observed in the present study, in which deletion of the SH gene did not further attenuate cpts248/404. However, incremental increases in attenuation can be achieved by including additional mutations, as was observed with the addition of the 1030 mutation to rA2cp248/404SH and with other vaccine candidates under clinical development [28]. The flexibility of this technology suggests that future live RSV vaccine candidates will be developed by means of recombination techniques rather than the classical methods of serial cold passage and chemical mutagenesis [28].
Here, rA2cp248/404SH was evaluated in adults and in RSV-seropositive and -seronegative children. It was minimally infectious in adults and seropositive children, which was expected on the basis of our experience with cpts248/404 [2, 29]. Indeed, previous studies have suggested that minimal infectivity in these individuals is a necessary prerequisite for evaluation in RSV-naive infants and children [9]. However, the mean peak titer of rA2cp248/404SH shed by RSV-seronegative children was 104.3 pfu/mL, which is comparable to that observed in seronegative recipients of cpts248/404 [2]. Thus, in the context of the cpts248/404 mutations, deletion of the SH gene does not further attenuate RSV. Because the level of replication is a useful predictor of attenuation for live respiratory viral vaccines [28], it appeared unlikely that rA2cp248/404SH would be more attenuated in young infants than cpts248/404 [2]. For this reason, rA2cp248/404SH was not evaluated in young infants.
In contrast, replication of rA2cp248/404/1030SH was significantly restricted in RSV-seronegative children, with a mean peak titer of 102.4103.5-pfu/mL in nasal wash specimens. These data indicated that the 1030 mutation conferred substantial attenuation in the context of the cp, 248, 404, and SH mutations and that rA2cp248/404/1030SH was sufficiently attenuated to merit evaluation in young infants. Excess respiratory and febrile illnesses were not observed in infants infected with vaccine virus. Specifically, the clinically significant nasal congestion observed after vaccination with cpts248/404 was not observed in infants who received rA2cp248/404/1030SH. Also, the level of replication of rA2cp248/404/1030SH was comparable in infants and RSV-seronegative children, indicating that replication can occur in the upper respiratory tract even in the presence of maternally derived antibody. This finding is consistent with those of previous studies of live attenuated influenza, parainfluenza, and RSV vaccines [2, 3033] and suggests that this outcome should be expected when these vaccines are evaluated in infants.
rA2cp248/404SH and rA2cp248/404/1030SH induced high titers of RSV antibodies in seronegative children. In contrast, only a minority of infants developed antibody responses to rA2cp248/404/1030SH. We previously showed that a majority of infants developed IgA antibody responses after vaccination with cpts248/404 [2], suggesting that the decreased antigenic load associated with the highly restricted replication of rA2cp248/404/1030SH may have diminished the response in this age group. Of note, IgA antibody responses in infants were directed toward RSV F and G glycoproteins with equal frequency, whereas studies in infants with cpts248/404 showed responses primarily directed toward RSV G glycoprotein [2]. Further studies will be needed to address this inconsistency.
Despite the modest antibody responses, young infants, after receipt of the first dose of rA2cp248/404/1030SH, were protected against challenge with the second dose; significant reductions in the proportion of infants shedding virus and the amount of virus shed were observed after the second dose. This protection was likely mediated by RSV-specific immune responses that we were unable to measure but that were effective in preventing infection and in clearing virus from the respiratory tract. Future efforts should be directed toward determination of the correlates of protection in young infants.
Although both rA2cp248/404SH and rA2cp248/404/1030SH are ts viruses, rA2cp248/404/1030SH is more ts than is rA2cp248/404SH. When the ts phenotype of viruses recovered from nasal wash specimens of children who received these vaccines was assessed, there was no evidence of reversion to wt virus. Virus present in specimens from children who received rA2cp248/404SH maintained the ts phenotype of the parent virus (no plaques were detected at 39°C). Some change in the ts phenotype occurred in a fraction of the specimens obtained from recipients of rA2cp248/404/1030SH: 27 of 141 specimens contained virus that formed plaques at 36°C, and 1 specimen contained virus that formed plaques at 37°C. Genetic characterization revealed reversion of either the 248 or the 1030 mutation in several isolates, with preservation of the cp, 404, and SH mutation in all recovered viruses.
It is perhaps not surprising that a subpopulation of viruses that could replicate at 36°C37°C was generated after infection with rA2cp248/404/1030SH, because selective pressure for generation of these viruses would exist in the upper airway. Although the clinical significance of these viruses cannot be determined from the present study, they remained highly ts and would likely be at least as attenuated as cpts248/404, which was attenuated even in young infants [2]. These viruses also retained all of the non-ts attenuating mutations and 2 of the 3 ts attenuating mutations present in the vaccine. Studies of live attenuated influenza and parainfluenza vaccines have shown that ts and non-ts mutations independently contribute to the stability of the attenuation phenotype [3436]; therefore, it is likely that the viruses shed by recipients of rA2cp248/404/1030SH would remain highly attenuated. Also, because the viruses were shed at 103 pfu, they would not likely be transmitted to others, but transmissibility studies are needed to address this issue. The potential transmissibility of these highly attenuated vaccine-derived viruses should be considered in the context of wt RSV, which is highly contagious and infects virtually all children by 2 years of age [37].
These rRSV vaccine candidates are the first to be evaluated in clinical trials. rA2cp248/404SH is not suitable for infants, given its level of replication in RSV-seronegative children. However, rA2cp248/404/1030SH is the first RSV vaccine candidate that appears to be appropriately attenuated for young infants. rA2cp248/404/1030SH shows limited phenotypic instability. It remains to be seen whether attenuating ts point mutations can be stabilized by means of molecular techniques [38] or whether some instability must be expected in highly ts live respiratory virus vaccines. In light of the limited replication after the second dose, multiple doses of rA2cp248/404/1030SH might protect against RSV-associated LRI, despite the limited antibody response in young infants. Field trials should be conducted to address this issue. Strategies to augment the antibody response in infants, such as enhancement of RSV F and G glycoprotein expression by gene shift to promoter proximal positions [39], should also be explored.
Acknowledgments
We thank Elizabeth Schappell, Barbara Burns, Paula Williams-Soro, Zainab Adetoro, Wei Chen, Arlene Kane, Ron Bassler, Lynn Miller, Yen-Huei Lin, Edith Sannella, Alice O'Shea, Sharon Tollefson, and Carlene Musgrave, for expert clinical and technical assistance; Romeo Paredes, for data management; and Martin Blair, for assistance with manuscript preparation.
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