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Background.A 30-second aerosol measles vaccination successfully primes children 12 months of age and older but is poorly immunogenic when given to 9-month-old children. We examined the immune responses when increasing the duration to aerosol exposure in 9-month-olds.
Methods.One hundred and thirteen healthy 9-month-old children from Mexico City were enrolled; 58 received aerosol EZ measles vaccine for 2.5 minutes and 55 subcutaneously. Measles-specific neutralizing antibodies and cellular responses were measured before and at 3 and 6 months postimmunization.
Results.Adaptive immunity was induced in 97% after aerosol and 98% after subcutaneous administration. Seroconversion rates and GMCs were 95% and 373 mIU/mL (95% confidence interval [CI], 441–843) following aerosol vaccination and 91% and 306 mIU/mL (95% CI, 367–597) after subcutaneous administration at 3 months. The percentage of children with a measles-specific stimulation index ≥3 was 45% and 60% in the aerosol versus 55% and 59% in the subcutaneous group at 3 and 6 months, respectively. CD8 memory cell frequencies were higher in the aerosol group at 3 months compared with the subcutaneous group. Adverse reactions were comparable in both groups.
Conclusions.Increasing exposure time to aerosol measles vaccine elicits immune responses that are comparable to those seen when an equivalent dose is administered by the subcutaneous route in 9-month-old infants.
The fourth millennium goal of the United Nations is to reduce childhood mortality. To achieve this goal, one of the main efforts has been to conduct global measles immunization campaigns . As a consequence, measles mortality has decreased 78%, from an estimated 733000 measles deaths in 2000 to 164000 in 2008 . Despite this reduction in mortality, measles continues to account for 278358 registered cases each year worldwide .
Infants have the highest risk for fatal measles infection and may be difficult to immunize due to immunological immaturity, which may be further complicated by passively acquired maternal antibodies to measles [4–8] that inhibit vaccine take and diminish the primary antibody responses . Aerosol measles vaccine may overcome the obstacle posed by maternal antibodies by entering via the mucosal route [10, 11]. The many other potential advantages of aerosolized measles vaccine have been described elsewhere  and include the delivery of vaccine to large numbers of children during mass campaigns using a needle-less device .
Seroconversion rates following aerosol measles vaccination range from 33% to 100% in numerous clinical trials, and this method has been successfully used in mass campaigns in Mexico [10, 12, 14–18]. Aerosol vaccination with the Edmonston-Zagreb (EZ) strain was found to be highly immunogenic in school-age children when administered as a second dose in studies in South Africa and Mexico [15, 16], and it was immunogenic in 12-month-old Mexican children as a primary immunization . In contrast, when 9-month-old Mexican children were immunized using the same aerosol delivery method and providing the same vaccine dose (30-s exposure) used successfully in 12-month-olds, the seroconversion rate was only 33% . We hypothesized that lengthening aerosol exposure time, would increase the inhaled dose and improve immune responses in this age group.
While serum-neutralizing antibodies have been associated with protection against infection, measles-specific cellular immunity is also known to be an essential component of the host response that is crucial for virus clearance. Measles-specific cellular immunity can be detected in 6- and 9-month-old infants after subcutaneous immunization, even in the presence of passive antibodies [4, 19, 20] and may be better sustained than measles immunoglobulin G (IgG) antibodies in some vaccinated children . We have demonstrated induction of cellular and humoral immunity to measles following aerosol administration of measles vaccine in 12- and 9-month-old children [17, 18]. Thus measles-specific T-cell responses may be a sensitive marker of immunological priming following vaccination even in young infants when antibody responses are low or undetectable. Unfortunately, few studies have looked at the durability of the cellular response after measles vaccination. While all memory T cells are identified by CD62L and CCR7 surface markers, differences in marker expression distinguish central memory T cells (TCM CD62hi CCR7+) that persist in secondary lymphoid organs from effector memory cells (TEM CD62Llow CCR7−)  that persist in an activated state in nonlymphoid tissues. Memory B cells can be detected through their surface markers CD19+ and CD27+ .
The aim of the present study was to assess T- and B-cell immunity following immunization with aerosol measles vaccine using the EZ strain at a higher dose than previous studies (via increased exposure time) in 9-month-old infants. Immune responses elicited by aerosol measles vaccine were compared with those of 9-month-old infants given measles vaccine subcutaneously. A second dose of measles vaccine (as MMR) was administered subcutaneously at 12 months of age in keeping with current recommendations, and immune responses examined 3 months thereafter to discern differences in priming according to the route of primary exposure.
Healthy children ages 9 months ± 4 weeks, identified from birth records of the Hospital General de Mexico in Mexico City were recruited by phone. Parents provided written informed consent. Children were randomly assigned to receive aerosol or subcutaneous measles vaccine using a list of random numbers generated using Epistats. Exclusion criteria included history of previous measles vaccination, family history of allergy to egg proteins, gestation <36 weeks, birth weight <2500 g, malnutrition, and acute or chronic illness. Enrollment took place between April 2008 and April 2009. No measles cases were identified in Mexico City during this time interval or the previous year. The study was approved by the IRB of the School of Medicine UNAM and the Hospital General de México.
Live, attenuated EZ measles virus vaccine, with a potency of 103.85 TCID50/0.5 mL, produced by the Serum Institute of India, was used. Lyophilized vaccine was provided in 10-dose vials to which 5 mL of diluent was added and used immediately. The subcutaneous dose (103.85 TCID50/0.5 mL) was injected in the area over the left deltoid. The aerosol administration technique has been described elsewhere [10, 13, 15, 16]. Briefly, a 12W compressor (Pulmo Aide 3655, DeVilbiss) pumps air into a nebulizer (Medex 4107) containing 10 doses inside an opaque cup with crushed ice. Aerosolized vaccine is administered through a plastic tube attached to a plastic cone held over the nose and mouth. The aerosolized dose (103.85 TCID50 in 0.5 mL exposed over 2.5 min) is used to calculate the estimated retained dose as follows: 0.5 mL contains 7079 infectious viral particles from which ~20% are inhaled [18, 24], providing an estimated retained dose of 1416 infectious viral particles.
All infants were given measles, mumps, rubella (MMR) vaccine (Serum Institute of India, LTD) by subcutaneous injection at 12 months of age.
Postvaccination phone evaluations for adverse reactions (fever, rhinitis, cough, conjunctivitis, diarrhea, arthralgias, others) were performed every other day by the investigators for 14 days and during the day 30 consult. Children were seen immediately if signs and/or symptoms were a concern.
Blood samples (2–5 mL) were collected before and at 3 and 6 months after immunization (6-month samples also correspond to 3 months post-MMR).
Fresh peripheral blood mononuclear cells (PBMC) were added to 96-well microtiter plates at 3 × 105 per well in RPMI 1640 (Gibco), and 10% normal human sera (Sigma). Measles antigen (Enders strain), prepared from infected Vero cell lysates, or uninfected cell control, was added at dilutions of 1:8, 1:16, and 1:32 to triplicate wells. T-cell proliferation was measured by adding tritiated thymidine (2.5 μCi per well) after 5 days for 6 to 18 hours. Phytohemagglutinin (Difco) was used as the positive control. Proliferation results were expressed as the stimulation index (SI), the ratio of mean counts per minute (cpm) measles antigen-stimulated wells over mean cpm of control wells. Past studies have shown that SI ≥ 3 is a positive response [4, 17–19, 21].
The cellular immune response was also measured by flow cytometry as follows; 3 × 105 PBMC per well were incubated in RPMI 1640 (Gibco) and 10% normal human sera (Sigma) and stimulated with measles antigen or Vero cell control for 5 days. The cells were stained with anti–CD8-PE Cy7, anti–CD4-PE Cy5, anti–CD19-PE, anti–CD45RA-PE, anti–CD27-FITC, anti–CD62L-APC/AF750, and anti–CCR7-APC monoclonal antibodies (Pharmingen, Becton Dickinson), 6 colors per well were measured and compensation was performed with nonstimulated cells with simple staining for each color; 10000 cells per patient were acquired in a FacsCanto II cytometer (Becton Dickinson) and analyzed with FacsDiva software (Becton Dickinson). Mean cell frequencies per group before and 3 and 6 months after vaccination and between vaccine groups were compared.
IFN-γ production by T cells in response to measles antigen stimulation was evaluated. Blood samples were collected before and 3 and 6 months after immunization. PBMCs were separated and stimulated with measles antigen. Supernatants from PBMCs were collected on day 5, stored at −70°C, and tested for IFN-γ by ELISA (Biosource International). Peak IFN-γ concentration was used for data analysis.
Sera were stored at −70°C. Paired specimens from before and 3 and 6 months after primary vaccination were tested for measles antibodies in parallel with the WHO Measles Reference Serum II, 66/202, using a modified plaque reduction neutralization (PRN) assay . The PRN titer was defined as the serum dilution that reduced the number of plaques by 50%. Titers less than 1:8 were considered negative and assigned a titer of 1:4 for purposes of calculating geometric mean concentrations (GMCs). Seroconversion was defined as a 4-fold rise in antibody titer above levels prior to vaccination. Measles seroprotection against severe disease has been defined as a PRN titer ≥1:120  which is equivalent to 120 mIU/mL based on the performance of the WHO Measles Reference Serum II, 66/202 in this assay.
Total enrollment was planned to provide a sample size with an α of 0.05 and 80% power to detect significant differences in cellular and/or humoral immunity . This calculation projected 53 per group and 75 children were enrolled per group to allow for dropouts. Analysis of immune response data was limited to children from whom blood was obtained for testing both pre- and postimmunization. Immune responses were compared by Student t test, χ2 and Mann–Whitney U tests.
In total, 150 children were enrolled; 113 (58 given aerosol and 55 given subcutaneous vaccine) completed participation. No significant differences in age, sex, and weight were observed between groups. Thirty-seven children were excluded from the final analysis—20 in the subcutaneous group and 17 in the aerosol group for the following reasons: 16 received MMR vaccine during vaccination campaigns before their second blood draw, 2 families changed city of residence, and parents of 19 children withdrew participation, but not because of adverse events.
When 9-month-old infants were given aerosol measles vaccine for 2.5 minutes at 103.85 TCID50/0.5 mL, 45% developed measles-specific T cell proliferative responses at 3 months and 60% at 6 months (3 months after the MMR dose) with no differences when compared with responses of 55% and 59% at 3 months and 6 months, respectively, in the subcutaneous group (P = .2 and 0.66, respectively; Table 1). When the mean SI ± standard error (SE) were compared between infants immunized with aerosolized measles vaccine versus those given measles vaccine subcutaneously, there were no differences at 3 or 6 months after immunization (Table 1). Likewise, measles-specific IFNγ production by T cells 3 and 6 months after aerosol and subcutaneous immunization were not significantly different (Table1).
Cell frequencies in response to measles antigen before and after vaccination were measured by analyzing surface markers specific for memory T and B cells. Increases in mean (±SE) cell frequencies of CD8+ CD62L− CCR7− effector-memory T cells at 3 and 6 months after immunization were observed in both vaccine groups, with a higher proportion in the aerosol group (25.48 ± 2.95) compared with the subcutaneous group (18.56 ± 1.72, P = .04) 3 months after immunization, with no differences between groups at 6 months (Figure 1A). Mean cell frequencies of the CD4+ CD62−CCR7− T cells increased at 3 and 6 months after vaccination with no differences between groups (Figure 1B). Similarly an increase in CD19+ CD27+ memory B cells was observed 3 months after immunization with no significant differences attributed to route of vaccine administration. Unexpectedly, a decrease in CD19+CD27+ memory B cells 6 months after primary immunization (and 3 months after MMR) was observed in both groups (Figure 1C). We did not note any differences in the mean cell frequencies before or after vaccine administration or between routes of administration in CD4+ CD27+ T cells, CD8+ CD27+ T cells, CD4+ CD45RA+ T cells or CD8+ CD45+ T cells (data not shown).
Only 5 children (2 given aerosol and 3 given subcutaneous vaccine) had detectable PRN measles antibodies prevaccination. Seroconversion rates 3 months postvaccination were 95% and 91% in the aerosol and subcutaneous groups, respectively (P = .43), and seropositivity was 100% in both groups 3 months after receiving MMR at 12 months of age (P = 1.0). GMCs were comparable before (4 vs 4 mIU/mL), 3 (373 vs 306 mIU/mL), and 6 months (1528 vs 1214 mIU/mL) after vaccination by aerosol and subcutaneous routes, respectively. Interestingly a booster effect with a 4-fold increase in GMCs over the previous level was detected at 15 months of age, 3 months after MMR administration, irrespective of the initial route of measles vaccine administration (Table 1).
Overall, 97% and 98% of infants given aerosol and subcutaneous vaccines, respectively, had a PRN concentration ≥120 mIU/mL, an SI ≥3 or both, 3 months after primary immunization; 100% of infants had either or both responses by 6 months after primary immunization and a booster with MMR at 12 months (Table 1). Furthermore, we analyzed the patterns of immune response and 43% of infants in the aerosol group and 48% in the subcutaneous group had serum PRN levels ≥120 mIU/mL and SI ≥3, while an additional 52% of vaccinees in the aerosol group and 43% in the subcutaneous group developed serum neutralizing antibody responses ≥120 mIU/mL postimmunization but had undetectable measles-specific T-cell responses. In contrast, 2% of children given aerosol vaccine and 7% immunized with subcutaneous vaccine had SI ≥3 when tested for measles-specific T-cell proliferation but no detectable PRN antibody postimmunization, and 3% in the aerosol group and 2% in the subcutaneous group had no antibodies nor detectable cellular responses 3 months after their initial dose of measles vaccine (Table 2). Although the sample sizes were small, there were no obvious differences in measles-specific B- or T-cell responses in 9-month-old infants irrespective of route of vaccine administration, and the significance of small differences in T-cell responses between groups is not known.
Infants given aerosolized measles vaccine while sitting in their mother’s lap tolerated the procedure well; 57% cried while 43% were calm when inhaling the nebulized vaccine. All the infants given measles vaccine subcutaneously cried.
No serious temporally associated events were identified in any of the vaccinated children. No significant differences between groups were observed for fever [21% aerosol group (A) vs 17% subcutaneous (S), P = .53], rash (5%A vs 10%S, P = .22), rhinitis (38%A vs 40%S, P = .86), cough (26%A vs 26%S, P = .57), conjunctivitis (9%A vs 6%S, P = .42), diarrhea (17%A vs 17%S, P = .58), arthralgias (1%A vs 0%S, P = .31) or other adverse reactions (2%A vs 4%S, P = .64) including vomiting in 2 infants in each group and hiccups in one child after subcutaneous immunization. Neither vaccinators nor mothers reported adverse events following exposure to nebulized vaccine.
The humoral immune response in children receiving aerosol measles vaccine has been demonstrated with seroconversion rates ranging from 33% to 100% [10, 12, 14–18]. School-age children who received aerosol measles vaccine as a second dose showed an excellent response in seroconversion and seropositivity rates ; when the same dose was given as a primary immunization to 12-month-old children the response to aerosol vaccine was similar to responses seen in children immunized via the subcutaneous route . However, when this same vaccine strain and dose was subsequently given to 9-month-old children, the immune response was lower than that seen in 9-month-olds given subcutaneous vaccine .
There is limited information on vaccinating 9-month-old children using aerosol EZ measles vaccine and scant data defining the minimum immunizing dose needed for this age group when the aerosol route is used; 2 successful studies in 9-month-old children used aerosol EZ measles vaccine with potencies of 104.7 and 105.2 TCID50/dose [10, 11, 27]. Based on these studies, we hypothesized that technical changes to increase the dose administered could potentially improve immune responses.
Using the manufacturer's specifications (10 doses in 5 mL), we increased exposure time from 30 seconds to 2.5 minutes, providing an estimated retained dose of 1416 viral particles. This dose is higher than that provided by a single 30-second burst used previously (vaccine potency 104.28, estimated retained dose of ~646 viral particles) . Since the current minimum dose for subcutaneous measles vaccination is 1000 TCID50 , our calculated estimate provided a good rationale for testing this approach and supported our hypothesis that a longer aerosol exposure would improve the retained dose, enhance immunogenicity, and result in a response equivalent to that seen after subcutaneous immunization.
Using the surrogate marker for protection for measles (measles neutralizing antibodies ≥120 mIU/mL, detected by PRN assay) , we found that 95% of children in the aerosol group achieved this level of measles antibody, which was not significantly different from the 91% observed in the subcutaneous vaccine group. GMCs increased in both groups and were above the protective concentration 3 months after primary vaccination. Moreover, when MMR was administered at 12 months of age, we observed a 4-fold increase in GMCs in both groups, suggesting that aerosol measles vaccine given before 12 months of age primed the immune response for ananamnestic response when a second dose was administered at 12 months .
Previous studies using [3H] thymidine incorporation to detect measles specific T-cell responses gave no information about types of T cells proliferating in response to measles antigens. The present study also describes similar percentages of vaccines with measles specific lymphoproliferative T-cell responses following either measles vaccine subcutaneously or a 2.5-minute exposure to aerosol measles vaccine. We were able to extend these findings by looking for the induction of measles-specific memory cells by incubating PBMCs in the presence of measles antigen for 5 days, on the rationale that memory cell induction takes place several days after initial antigen contact (especially in vitro), and 5 days of incubation would allow clonal expansion of these memory T cells. Afterward, cells were stained with antibodies directed to surface proteins that are known markers of peripheral memory effector T cells . Using this new method, we were able to detect an increase in the proportion of CD8+ memory T cells (CD62L− CCR7−) and in CD4+ memory T cells (CD62L– CCR7–) 3 months after vaccination and demonstrated persistence of these subsets 6 months after the initial dose of measles and 3 months after a dose of MMR. An increase in memory B cells (CD19+ CD27+) in both groups was also observed 3 months after vaccination. Unexpectedly, mean cell frequencies of memory B cells decreased 6 months after primary measles vaccination (ie, 3 months after a dose of MMR vaccine) in both vaccine groups. This phenomenon could be due to a homing of specific memory B cells to lymphoid organs and lower number in peripheral blood or to a higher proportion of memory B cells rapidly differentiating into plasma cells upon contact with measles antigen, but the observation was made after acquiring all the cells, and we unfortunately did not stain for plasma cell surface markers (CD138 SYNDECAN I). This is the first report of the induction of measles-specific peripheral memory CD8+ T, CD4+ T, and B cells in response to the aerosol measles vaccination.
Previous sero-epidemiologic studies suggest that some individuals are protected against measles, even when measles antibody titers are low or undetectable by conventional assays , and some vaccine recipients have a higher or longer lasting cellular response in the absence of a humoral response . Whether these individuals have long-term protection is not known. However, given the importance of cell-mediated immunity in controlling viral infections, children with this pattern of response may be protected against measles infection. CMI responses probably serve as a marker for individual protection, but whether they play any role in preventing infection, transmission, or enhancing herd immunity is not understood and requires further study.
Thus the assays for T-cell immunity with [3H] thymidine and neutralizing antibodies can segregate infants into 4 immune response patterns using a threshold of measles neutralizing antibody of ≥120 mIU/mL along with detectable measles T cell proliferative responses (SI ≥3.0), as illustrated in Table 2. In contrast to the past study in 9-month-old children, where 30 seconds of exposure to aerosolized measles vaccine resulted in 50% of the children failing to develop detectable measles PRN antibodies and with SI <3, a longer exposure time elicited a pattern of immune response that was similar to that seen after subcutaneous administration (Table 2). In the present study, the majority of infants given aerosol measles vaccine developed both T-cell and B-cell responses or humoral response only, while a very low proportion had cellular responses only and a mere 3% in the aerosol group and 2% in the subcutaneous group had no detectable response.
Surprisingly, only 4% of 9-month-old infants in our present study had maternal passive antibodies to measles, even though Mexico experienced a measles epidemic in 1989–1990 . This finding suggests that more children are born to vaccinated younger mothers instead of mothers with immunity from natural measles infection .
Although there was a 5-fold increase in exposure time for aerosolized measles vaccine compared with previous studies, no serious adverse reactions were observed, and the frequency of symptoms occurring after vaccination were equivalent between aerosol and subcutaneous groups.
In summary, this study demonstrated that increasing exposure time is an approach that may improve immunogenicity in 9-month-old infants given their primary measles immunization by the aerosol route; furthermore, this time of exposure was well tolerated. We also detected measles-specific memory CD4+ T, CD8+ T, and B cells in response to the aerosol measles vaccine in 9-month-old children, which has not been previously reported. This method of vaccine administration is potentially adaptable for both mass campaigns and individual dosing with practical advantages for measles vaccination campaigns throughout the world.12
This work was supported by a grant from the Fogarty International Center and the Office of Research on Women's Health (TW006193).
We are indebted to the families of Mexico City who agreed to participate in the study. We are grateful for the generous donation of measles vaccine from the Serum Institute of India, LTD., the nurses Verónica Dominguez Pérez, Sandra Carbajal Cedillo, and Alma Chavez García for their help in the recruitment of patients; to Doris María Arellano Quintanilla for her work with the assessment of the cellular mediated immunity assays, to Dr Ann Arvin for the critical review of the manuscript; and the Liver, Pancreas and Motility unit of the Universidad Nacional Autónoma de México for the use of the FacsCanto II cytometer.