Download Correlations among measles virus

et al.
Clinical and Experimental Immunology
ORIGINAL ARTICLE
doi:10.1111/j.1365-2249.2005.02931.x
Correlations among measles virus-specific antibody,
lymphoproliferation and Th1/Th2 cytokine responses following
measles–mumps–rubella-II (MMR-II) vaccination
N. Dhiman,* I. G. Ovsyannikova,*
J. E. Ryan,* R. M. Jacobson,*†
R. A. Vierkant,‡ V. S. Pankratz,‡
S. J. Jacobsen‡ and G. A. Poland*§
*Mayo Vaccine Research Group, †Department of
Pediatric and Adolescent Medicine, ‡Department
of Health Sciences, and §Program in Translational
Immunovirology and Biodefense, Mayo Clinic
College of Medicine, Rochester, MN, USA
Accepted for publication 3 August 2005
Correspondence: Gregory A. Poland MD, Mayo
Vaccine Research Group, Program in Translational Immunovirology and Biodefense, Mayo
Clinic and Foundation, 611 C Guggenheim
Building, 200 First Street, SW, Rochester, MN
55905, USA.
E-mail: [email protected]
Summary
Immunity to measles is conferred by the interplay of humoral and cellular
immune responses, the latter being critical in maintaining long-term recall
response. Therefore, it is important to evaluate measles-specific humoral and
cellular immunity in populations several years after vaccination and understand the correlations among these measures of immunity. We examined
measles-specific antibodies, lymphoproliferation and the Th1/Th2 signature
cytokines, interferon (IFN)-γγ and interleukin (IL)-4, in a population-based
cohort of healthy children from Olmsted County, Minnesota after two doses
of measles–mumps–rubella-II (MMR-II) vaccine. We detected positive measures of measles-specific cellular and humoral immunity in the majority of
our study population. However, a small proportion of subjects demonstrated
an immune response skewed towards the Th2 type, characterized by the presence of either IL-4 and/or measles-specific antibodies and a lack of IFN-γγ
production. Further, we observed a significant positive correlation between
lymphoproliferation and secretion of IFN-γγ (r = 0·20, P = 0·0002) and IL-4
(r = 0·15, P = 0·005). Measles antibody levels were correlated with lymphoproliferation (r = 0·12, P = 0·03), but lacked correlation to either cytokine type.
In conclusion, we demonstrated the presence of both long-term cellular and
humoral responses after MMR-II vaccination in a significant proportion of
study subjects. Further, a positive correlation between lymphoproliferation
and IL-4 and IFN-γγ suggests that immunity to measles may be maintained by
both Th1 and Th2 cells. We speculate that the Th2 biased response observed
in a subset of our subjects may be insufficient to provide long-term immunity
against measles. Further examination of the determinants of Th1 versus Th2
skewing of the immune response and long-term follow-up is needed.
Keywords: antibody, cellular immunity, cytokines, lymphoproliferation,
measles
Introduction
Measles leads to nearly 1 million deaths each year, due primarily to failure to vaccinate and due in part to vaccine
failure [1–6]. Antibody responses to measles infection or
vaccination have been studied extensively and are used as
correlates of protection against measles. Vaccine failure has
been defined in terms of antibody response alone, and the
markers of measles virus-specific cellular immunity after
vaccination have been poorly characterized. Although the
role and relative contribution of cellular immunity in
imparting protection against measles in response to vaccina498
tion is unknown, CD4 T cell responses are known to
influence indirectly the antibody responses [7–10]. Measles
infection is known to cause an acute and profound state of
immune suppression in few individuals, characterized by
polarization of effector CD4 T cells to produce T helper type
2 (Th2) cytokines [7–9,11]. Similarly, measles vaccination
can also cause immune suppression and predominant IL-4
production in some cases [10,12]. This skewing of the
cytokine response towards predominant Th2-type cytokines
results in a deficient or low Th1 cellular immune response
and increases susceptibility to co-infections with various
other pathogens [13,14]. The preferential expansion of the
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 142: 498–504
Humoral and cellular immunity to MMR-II
CD4 T cell pool into Th2 cells is characterized by an increase
in IgE, IgG1 and IgG4, diminished production of the Th1
signature cytokine interferon (IFN)-γ and heightened production of interleukin (IL)-4 (a cytokine critical for establishing the Th2 type response) [8,15]. Further, cellular
immunity is known to play a critical role in controlling
measles virus replication. Children with cellular immune
deficiencies are known to develop severe complications
associated with measles virus infection and recover poorly
compared to their healthy counterparts [16,17]. Because cellular immunity plays a critical role in the maintenance of
recall responses and control of measles virus infection, it is
important to evaluate the presence of measles-specific cellular immunity in addition to antibody levels in populations
several years after vaccination and understand the possible
correlations among these measures of immunity.
In the current study, we examined the correlation among
measles virus-specific antibodies, lymphoproliferation and
Th1/Th2 cytokine responses following two doses of measles–
mumps–rubella-II (MMR-II) vaccine. The purpose of this
study was to identify markers of immune response to each
effector arm of immunity and to assess if correlations exist
among these measures of immunity.
Materials and methods
Human subjects
The details of our study sample recruitment have been
described previously [18]. Briefly, we recruited a populationbased, stratified random sample of 346 healthy children and
young adults (12–18 years of age) from all socio-economic
strata residing in Rochester, Minnesota. Study subjects had
documentation in their medical records of receiving two
doses of the MMR-II vaccine containing the Edmonston
strain of measles virus (tissue culture infective dose
TCID50 ≥ 1000), the Jeryl Lynn B-strain of mump virus
(≥ 20 000 TCID50) and the Wistar RA 27/3-strain of rubella
virus (≥ 1000 TCID50) on or after the age of 12 months. There
had been no circulating wild-type measles virus in this geographical area of residence during the subjects’ lifetimes. We
determined the level of measles-specific antibodies, lymphoproliferation and cytokine production after ex vivo measles stimulation to characterize overall immunity to measles
in our study population. We included 339 subjects in the final
analysis for whom we had all four measures of immunity. The
study was approved by the Mayo Clinic’s Review Board. We
obtained written, informed consent or assent (from younger
subjects) from subjects and/or parents/guardians from all the
subjects at the time of enrolment in the study.
Antibody assays
Quantitative levels of measles-specific IgG antibody titres for
all serum specimens were determined by the Enzygnost
(Dade Behring, Marburg, Germany) anti-masern-virus/IgG
enzyme immunoassay (sensitivity = 99·6%; specificity =
100%), as described previously [18]. Briefly, sera were
separated from whole blood by centrifugation. Sera were
aliquoted and stored at −80°C until the time of the assay.
Serum samples were added in duplicate to a microtitre plate,
which contained two parallel wells coated with a test whole
measles virus antigen or control antigen derived from noninfected cells. Optical densities were determined at 450 nm
and corrected at 650 nm on a microplate reader (Molecular
Devices Corporation, Sunnyvale, CA, USA). The difference
in mean absorbance between the viral test antigen and the
control antigen for each sample was calculated and multiplied by a correction factor (determined by dividing a kitspecific nominal value by the mean of the reference standard) to give the corrected change in absorbance (∆A).
Measles-specific IgG antibody levels (mIU/ml) were
calculated from the antilog of the following formula:
log10 = α*∆Aβ (where α and β are lot-dependent constants).
Positive antibody response was defined as a change in absorbance (∆A) > 0·2. The coefficient of variation for this assay in
our laboratory was 3·8%.
Preparation of peripheral blood mononuclear cells
(PBMC)
PBMC were separated from heparinized venous blood by
Ficoll-Hypaque (Sigma, St Louis, MO, USA) density gradient centrifugation and washed in RPMI-1640 medium
(Celox Laboratories, Inc., St Paul, MN, USA) supplemented
with 100 µg/ml streptomycin (Sigma), 100 U/ml penicillin
(Sigma) and 8% heat-inactivated fetal calf serum (FCS)
(Hyclone, Logan, UT, USA). Cells were then counted, resuspended in RPMI freezing media containing 10% dimethyl
sulphoxide (Sigma), 20% FCS, frozen at −80°C overnight
and stored in liquid nitrogen until cultured.
Lymphoproliferation assay
Lymphoproliferation to measles vaccine virus was assessed
using an in vitro [3H]-thymidine incorporation assay as
described previously [18]. Briefly, PBMC (2 × 105cells/well)
were incubated for 72 h in RPMI-1640 medium, supplemented with 5% autologous sera, in the presence of live
attenuated measles vaccine virus [75 plaque-forming units
(pfu)/well]. Plain RPMI media served as the unstimulated
control, and phytohaemagglutinin (PHA, 5 µg/ml) was
used as a positive control for lymphoproliferation. The
PBMC were then pulsed with [3H]-thymidine for 18 h, harvested on glass fibre filters and counted for the amount of
incorporated radioactivity. Results were then expressed as
stimulation indices (SI), which are defined as the ratio of
the median counts per minute (cpm) of antigen-stimulated
wells to the median cpm of unstimulated control wells.
Stimulation indices of three or higher were considered to be
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 142: 498–504
499
N. Dhiman et al.
an indicator of a positive lymphoproliferation response
[19].
Measurement of IFN-γγ and IL-4 cytokines
The details of the IFN-γ and IL-4 cytokine assays are
described in detail elsewhere [20]. Briefly, for IFN-γ determination, thawed PBMC were cultured at a concentration of
2 × 105 in RPMI 1640 media containing 10% normal human
serum (NHS), 100 U/ml penicillin and 100 µg/ml streptomycin with or without measles (MV) virus (Edmonston B
vaccine strain of MV cultured in African green monkey kidney cells) at a multiplicity of infection (moi) of 0·5 for 6 days.
For IL-4 determination, we cultured thawed PBMC at a concentration of 4 × 105 in RPMI-1640 media, supplemented
with 10% NHS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured in the presence of 2 µg/ml of
monoclonal IL-4 receptor antibody (R&D Systems, Minneapolis, MN, USA) with measles virus at a moi of 0·1 for
6 days as previously described [21,22]. These doses were
chosen carefully to operate in approximately 70% measlesvirus stimulation dose for immune activation (and not too
high or too low a dose) to avoid skewing the immune
response to either direction due to overloading or underpresentation of the antigen. Stimulation with PHA, 5 µg/ml,
was used as a positive control. Vero cell lysate was used as
unstimulated control. Cell culture supernatants were collected and assayed for IFN-γ and IL-4 using a standard
enzyme-linked immunosorbent assay (ELISA) protocol
(OptiEIA Human IFN-γ and IL-4) (PharMingen, San Diego,
CA, USA). The IFN-γ and IL-4 concentration of each test
sample was calculated by reference to the standard curve.
Median background levels of IFN-γ and IL-4 cytokine production in unstimulated wells were subtracted from the
median measles-induced responses to produce ‘corrected’
secretion values. Any value above the unstimulated control
was considered positive. Negative corrected values indicate
that the unstimulated secretion levels were, on average,
higher than the stimulated secretion levels.
Statistical analysis
Data were summarized descriptively using frequencies and
percentages for all categorical variables and medians and
interquartile ranges (IQR) for all continuous variables. Associations of immune response with demographic and clinical
variables were assessed using analysis of variance methods.
Due to data skewness, all P-values were calculated using
rank-transformed values.
An intraclass correlation coefficient (ICC) and corresponding 95% confidence interval was calculated to assess
the magnitude of variance across the four different immune
response measures relative to error variance [23]. Ranktransformed values were used to calculate the ICC due to
data skewness in the immunity variables. Spearman’s
500
Table 1. Demographic variables of the study population.
Variable
n = 339
Age, years (median, IQR*)
Gender (n, %)
Female
Male
Race (n, %)
Caucasians
Others
Age, months at first MMR-II immunization
(median, IQR)
Age, years at second MMR-II immunization
(median, IQR)
Time, years since last MMR-II immunization
to blood draw (median, IQR)
15 (13,17)
159, 47%
180, 53%
316, 93%
23, 7%
15·6 (15·1, 17·5)
12·1 (11·5, 12·6)
4·8 (2·8, 6·1)
*IQR, interquartile range; MMR-II, measles–mumps–rubella-II.
correlation coefficients were then used to assess all pairwise
associations between the immunity measures. All statistical
tests were two-sided, and all analyses were carried out using
SAS software system (SAS Institute, Inc., Cary, NC, USA).
Results
Demographic variables
Our study population had a median age of 15 years at the
time of recruitment with near equal gender representation
(53% males). The majority of subjects were Caucasians
(93%) and the median age at the first and second immunization was 15·6 months and 12·1 years, respectively. Measles
virus-specific immune responses were measured after a
median time period of 4·8 years after the last immunization
(Table 1).
Immunological variables
The median value for measles-specific IgG was 1553 mIU/
ml, and the median SI for the lymphoproliferation was 3·8
(Table 2). We observed an increase in the antibody levels to
measles with increasing age at the first MMR-II vaccination
(P = 0·05). Similarly, an increase in age at the second MMRII vaccination showed an increasing trend in antibody levels
to measles (P = 0·04) (data not shown). No association was
found between the lymphoproliferative response and any of
the demographic variables (data not shown). Median values
for measles-specific IFN-γ and IL-4 cytokines were 40·7 pg/
ml and 9·7 pg/ml, respectively (Table 2). The IFN-γ and IL-4
responses to measles vaccination were found to be independent of gender, age or time lapsed since last immunization in
our study cohort (data not shown).
The proportion of individuals that had a positive response
for measles specific antibody, lymphoproliferation, IFN-γ
and IL-4 as per our cut-off criteria was 0·86, 0·58, 0·83 and
0·85, respectively (Table 2). Overall, 37·5% of our study
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 142: 498–504
Humoral and cellular immunity to MMR-II
Table 2. Characterization of measles-specific cellular and humoral positive responses in study subjects.
Variable (n = 339)
IgG (mIU/ml)
Stimulation index (SI)
IFN-γ (pg/ml)
IL-4 (pg/ml)
Median (IQR)
n, % Subjects with
positive response*
1553 (737, 2637)
3·8 (1·9, 6·6)
40·7 (8·1, 176·7)
9·7 (2·8, 24·3)
290, 86%
195, 58%
281, 83%
288, 85%
*Positive antibody response = change in absorbance (∆A) > 0·2.
Measles-specific IgG antibody levels (mIU/ml) are calculated from the
antilog of the following formula: log10 = α*∆Aβ (where α and β are lotdependent constants). *Positive stimulation index (SI) response ≥ 3.
*Positive cytokine response is any value above background. Median
background levels of interferon (IFN)-γ and interleukin (IL)-4 cytokine
production in unstimulated wells were subtracted from the median
measles-induced responses to produce ‘corrected’ secretion values. The
uncorrected median (IQR) for IFN-γ (pg/ml) was 40·7 (3·7, 196·3) and
−2·9 (−9·9, 6·85) for measles virus (MV) stimulated and unstimulated
control, respectively. The uncorrected median (IQR) for IL-4 (pg/ml)
was 15·5 (5·0, 30·8) and 3·46 (−1·42, 10·48) for MV stimulated and
unstimulated control, respectively.
subjects had a positive response for all four measures of
immunity, 98·8% for at least one measure of immunity,
while only 1·2% lacked positivity to any of the measured
variables (Table 3). From the 98·8% of subjects that measured positive for at least one variable, 7·4% of the subjects
demonstrated undetectable levels of IFN-γ production and
negative measles-specific lymphoproliferation (Table 3).
Correlation between immunological variables
Table 4 shows the correlation among the pairs of measures of
measles-specific antibody, lymphoproliferation, IFN-γ and
IL-4 as represented by a Spearman’s correlation matrix.
The antibody levels were correlated to lymphoproliferation
(r = 0·12, P = 0·03), but lacked any correlation with the
measures of IFN-γ and IL-4 secretion. Alternatively, we
found a significant correlation between lymphoproliferation
and IFN-γ (r = 0·20, P = 0·0002) as well as lymphoproliferation and IL-4 (r = 0·15, P = 0·005) secretion. Further,
measles-specific IFN-γ had a significant positive correlation
Table 3. Characterization of measles-specific humoral and cellular immunity in study subjects.
Immune measure
Variable positive
Frequency*
Percentage*
% Cumulative
Cellular and humoral
IgG, SI, IFN-γ, IL-4
IgG, SI, IFN-γ
IgG, SI, IL-4
IgG, IFN-γ, IL-4
SI, IFN-γ, IL-4
IgG, SI
IFN-γ, IL-4
IgG, IFN-γ
SI, IL-4
127
20
21
82
17
4
17
15
3
37·5
5·9
6·2
24·2
5·0
1·2
5·0
4·4
0·9
90·2
Th1-biased
SI, IFN-γ
SI
IFN-γ
2
1
1
0·6
0·3
0·3
1·2
Th2-biased
IgG, IL-4
IL-4
IgG
17
4
4
5·0
1·2
1·2
7·4
None
–
4
1·2
1·2
*Frequency and percentage for individual immune measure are based on the cut-off criteria mentioned in the text.
Table 4. Correlation between measles-specific antibody, lymphoproliferation, interferon (IFN)-γ and interleukin (IL)-4 responses in study subjects as
represented by a Spearman’s correlation matrix.
Variable
IgG level (mIU/ml)
Stimulation index (SI)
IFN-γ (pg/ml)
IL-4 (pg/ml)
IgG level
(mIU/ml)
Stimulation
index (SI)
IFN-γ (pg/ml)
IL-4 (pg/ml)
1·00
–
–
–
0·12 (0·03)
1·00
–
–
0·03 (0·56)
0·20* (0·0002)
1·00
–
0·04 (0·45)
0·15* (0·005)
0·29* (< 0·0001)
1·00
The values in parentheses represent the corresponding P-values. *P-value ≤ 0·005 are shown in bold type.
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 142: 498–504
501
N. Dhiman et al.
with measles-specific IL-4 secretion (r = 0·29, P < 0·0001)
(Table 4). The overall value of the intraclass correlation for all
four measures of immunity for measles was 0·14 (95%
CI = 0·09–0·19). However, as these correlations are weak in
magnitude (r < 0·3), they should be interpreted with caution.
Discussion
In the current study, we demonstrate the presence of longterm cellular and/or humoral immunity after vaccination
with two doses of the currently recommended MMR-II vaccine in the majority of our study population by measuring
measles-specific lymphoproliferation, antibodies and Th1/
Th2 signature cytokines, IFN-γ and IL-4. However, a small
fraction of individuals (7·4%) demonstrated a Th2-biased
immune response characterized by the presence of antibodies and IL-4 and undetectable IFN-γ and lymphoproliferative response.
Although protective efficacy of measles vaccine is defined
in terms of antibodies alone, several ‘experiments of nature’
demonstrate that cellular immunity is critical and sufficient
to recover from measles infection. Children with agammaglobulinaemia have a normal course of clinical measles and
recover uneventfully from measles infection, while those
with defective cellular immunity (e.g. leukaemia, HIV)
develop progressive illnesses and have high mortality from
measles infection [14,24]. Previous studies demonstrate that
measles infection can result in an acute and profound state
of immune suppression and skew the immune response
towards the Th2 phenotype, characterized by high production of IL-4 and increased immunoglobulin levels [7–11].
In addition, there is substantial evidence for a biased Th2
response after measles vaccine administration as documented by decreased responsiveness to mitogens [12] and
predominant IL-4 production [10]. However, we have limited understanding of these measures of immunity to measles in populations several years after vaccination. In the
current cohort, we demonstrate that a majority (90%) of the
subjects had evidence for measles-specific immune markers
for both the humoral and cellular effector arms years after
measles vaccination. However, a small yet significant proportion (7·4%) of subjects did show a preferential bias towards
the Th2 type response. Further, an even smaller fraction
(1%) demonstrated no evidence of any of the four measures
of immunity to measles. Although there is no critical
requirement of vaccine-induced cellular response in imparting protection against infection, the individuals lacking cellular immunity may be at a disadvantage for virus clearance.
No data are available on evaluation of the potential roles of
Th1 and Th2 cells in measles viral clearance in humans.
However, a recent study using the rhesus monkey model of
measles infection demonstrates clearly that CD8 lymphocytes are major mediators of protection, and antibodies have
a limited role in the control of measles replication and clearance [25]. The lack of correlation between measles-specific
502
antibodies and prolonged measles virus shedding in HIVinfected Zambian children also re-emphasizes the importance of cellular immunity in recovery from measles infection [26]. In contrast, antibodies alone can certainly mediate
protection against measles virus infection as infants with
immature cellular immunity can be protected by maternally
acquired antibodies [27], or neutralizing antibodies from
vaccination [28]. Similarly, a formalin-inactivated vaccine
incapable of priming CD8 T cells is protective if antibody
titres are high [29,30]. However, as antibodies wane over
time, cellular immunity may be more important for maintaining long-term immunity and recovery from the disease.
Similar discordance between the contribution of humoral
immunity in viral clearance and protection has been established for other viruses, such as herpes simplex virus and
hepatitis B virus [31,32].
The large sample size from our current study allowed us to
determine correlations between measures of humoral and
cellular immunity after vaccination, compared to solely
looking at antibodies as markers for vaccine-induced
immunity. We demonstrate a significant positive correlation
between lymphoproliferation and two cytokines, IL-4 and
IFN-γ, which play a crucial role in both the generation and
regulation of immune responses. Further, the levels of IL-4
and IFN-γ were correlated positively in the study subjects.
The results suggest that recall immunity to measles is maintained as a fine-tuned and tightly regulated immunological
balance, where both IL-4 and IFN-γ play a key role.
IFN-γ is critical for macrophage activation, T cell proliferation and differentiation and up-regulation of human leucocyte antigens [33–35]. IFN-γ exerts an antiviral effect by
promoting the lysis and clearance of measles-infected cells
and by inhibiting viral gene expression and replication
[25,36,37]. In comparison, a Th2 response is characterized
by IL-4 production and high levels of measles-specific
antibodies after measles infection [8,10,38], which is used
traditionally as the measure of protective immunity against
subsequent infection. Because we found a positive correlation between lymphoproliferation and IL-4 and IFN-γ, our
results suggest that measles immunity is maintained by both
Th1/Th2 cells (or Th0-type T cells that are producers of both
IL-4 and IFN-γ) [39]. Similar Th0 responses to wild-type
measles virus-infected dendritic cells have also been observed [40]. However, measles vaccine-infected dendritic
cells or subjects immunized with the measles vaccine predominantly produce a Th1 cytokine response [40,41]. This
implies that to mount an immune response at various stages
of viral invasion, the host cells maintain a functional pool of
mixed Th1/Th2 cell populations. The Th1 response may help
to clear intracellular viruses from infected host cells, and the
Th2 response may help to clear extracellular viruses, which
can be neutralized by antibodies and inflammatory cytokines. For example, a high intact rate of humoral and cellular
immunity and the presence of both Th1/Th2 cytokines are
observed in influenza vaccinated subjects, which suggests the
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 142: 498–504
Humoral and cellular immunity to MMR-II
presence of Th0 cells [42]. In addition, we did not find any
correlation between antibody levels to measles and measures
of Th1/Th2 cytokines. This agrees with our previous findings
demonstrating that correlations between measles-specific
antibodies and IFN-γ and IL-4-secreting cell frequencies did
not reach statistical significance [43]. Similar disagreement
in quantitative correlation between antibody levels to measles and measures of T cell immunity can also be found in
other studies [44–46]. Further work on the determinants of
Th1 versus Th2 skewing of the immune response is needed.
The major strength of our study is the stratified random
sample of our population. The resulting sample well represents the US Caucasian population with high vaccination
rates. An advantage of an ethnically homogeneous population is decreased confounding variables (ethnicity, previous
viral exposure history, etc.), increasing the confidence in
our findings. Further, the immunity measures were true
responses to vaccination, as these subjects had not encountered wild measles virus in their lifetimes. We understand
that an ideal control to evaluate the specificity of measles
responses would be testing for immune measures to measles
stimulation in naive cells. However, we are confident that the
IFN-γ and IL-4 levels detected in our study are measlesspecific. For example, in our previous studies we were not
able to detect any IL-4 and only very low levels of IFN-γ in
naive cells after strong mitogen stimulation such as PHA
[41]. However, we could detect significant IL-4 and IFN-γ
secretion in response to ex vivo PHA stimulation in PBMC
from these subjects after MMR-II vaccination [41]. This suggests that when we did observe IL-4 and IFN-γ secretion with
measles stimulation in vaccinated individuals, it is represented as measles-specific response. A limitation of our study
may be that we used different stimulation doses for IL-4 and
IFN-γ secretion assays which may influence the differentiation to either Th1 or Th2 response. However, our selected
doses had given us approximately 70% immune activation in
our optimization studies, which is not too high or too low a
dose to skew the immune response to either direction due to
overloading or under-presentation of the antigen. Another
limitation of our study is measurement of whole measles
antibodies instead of neutralizing antibodies. Neutralizing
antibodies would specifically measure protective antibody
titres [28]. We were prompted to measure whole antibodies
due to the correlation of neutralizing antibody titres with
whole antibody enzyme immunoassays [47] and the ease of
performing whole antibody immunoassays on a large sample
size. Further, we measured Th1 and Th2 cytokines in a whole
lymphocyte population instead of isolated CD4 and CD8
populations due to a limitation in cell numbers available.
However, we believe that CD4 cells are the predominant producers of the Th1/Th2 mixed response to measles based on
our previous studies characterizing cytokine secreting cell
types after measles stimulation [22,39].
In conclusion, we demonstrate that the majority of our
study population had detectable measures of measles-
specific cellular and humoral immunity following a median
of 4·8 years after receipt of the second dose of MMR-II vaccine. However, 1·2% lacked a positive response to any measures of measles vaccine induced immunity, and 7·4%
demonstrated a Th2-biased immune response. Our study
provides insights into the mechanisms of measles vaccineinduced immune responses. It may be concluded that, in
most cases, the immune system mounts a balanced Th1/Th2
response to vaccination. However, in a small fraction of individuals, a bias towards either type may be induced. However,
it is speculated that individuals with humoral immunity
alone may be at a disadvantage for decreased viral clearance
and long-term protection due to waning of antibodies over
time. Additional doses of vaccines or improved vaccination
strategies such as peptide-based vaccines may need to be
developed to overcome the lack of cellular immunity in those
individuals who do not respond to the current vaccine. This
is critical for recall immunity and lifelong protection, where
humoral immunity alone may be insufficient to recover from
infection.
Acknowledgements
We thank the parents and children who participated in this
study. We acknowledge the efforts of the research fellows,
nurses and students from the Mayo Vaccine Research Group.
We also thank Carla Tentis for editorial assistance. This work
was partially supported by NIH grants AI 33144 and AI
48793 and Mayo General Clinical Research Center (GCRC)
grant M01-RR00585.
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