Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus.
ABSTRACT Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8(+) T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8+ T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K(b) and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection.
Article: Measles: old vaccines, new vaccines.[Show abstract] [Hide abstract]
ABSTRACT: Isolation of measles virus in tissue culture by Enders and colleagues in the 1960s led to the development of the first measles vaccines. An inactivated vaccine provided only short-term protection and induced poor T cell responses and antibody that did not undergo affinity maturation. The response to this vaccine primed for atypical measles, a more severe form of measles, and was withdrawn. A live attenuated virus vaccine has been highly successful in protection from measles and in elimination of endemic measles virus transmission with the use of two doses. This vaccine is administered by injection between 9 and 15 months of age. Measles control would be facilitated if infants could be immunized at a younger age, if the vaccine were thermostable, and if delivery did not require a needle and syringe. To these ends, new vaccines are under development using macaques as an animal model and various combinations of the H, F, and N viral proteins. Promising studies have been reported using DNA vaccines, subunit vaccines, and virus-vectored vaccines.Current topics in microbiology and immunology 02/2009; 330:191-212. · 4.86 Impact Factor
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ABSTRACT: Measles virus is a highly prevalent neurotropic virus capable of causing persistent infections within the central nervous system. We measured IgG class antibodies to measles in 820 individuals including 138 with recent onset psychosis, 378 with persistent schizophrenia, and 304 non-psychiatric controls. Levels of antibodies among the groups were compared by bivariate and by multivariate analyses and correlated with clinical and demographic variables. The level of measles antibodies in individuals with a recent onset of psychosis was greater than the level of antibodies in individuals with persistent schizophrenia or individuals without a history of a psychiatric disorder (p<.00001). The level of measles antibodies in the individuals with persistent schizophrenia was greater than the level of measles antibodies in the controls (p<.001). Recent onset of psychosis was associated with having elevated levels of measles antibodies, defined as the 90th percentile of the levels of the controls, with an odds ratio of 8.0 (95% CI 4.6, 14.0); persistent schizophrenia was associated with having this level with an odds ratio of 2.3 (95% CI 1.4, 3.7). Within the psychiatric groups, measles antibody levels were associated with age, race, and current treatment with the antipsychotic medication, olanzapine. The reasons for elevated levels of measles antibodies in the psychiatric groups are not known with certainty and should be studied in prospective investigations.Schizophrenia Research 06/2010; 119(1-3):89-94. · 4.59 Impact Factor
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ABSTRACT: Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.Journal of Virology 04/2010; 84(8):3798-807. · 5.08 Impact Factor
Protective immunity provided by HLA-A2 epitopes for fusion and
hemagglutinin proteins of measles virus
SangKon Oha,b,⁎, Brian Stegmana, C. David Pendletona, Martin O. Otac, Chien-Hsiung Panc,
Diane E. Griffinc, Donald S. Burkeb, Jay A. Berzofskya,⁎
aVaccine Branch, National Cancer Institute, National Institutes of Health, Bldg. 10-Rm 6B-09, NIH, Bethesda, MD 20892-1578, USA
bDepartment of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
cW. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
Received 21 February 2006; returned to author for revision 23 March 2006; accepted 28 April 2006
Available online 15 June 2006
Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8+T-cell-mediated immune responses that may play a
central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and
fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H
protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a
T2-binding assay, and four from H and two from F could induce peptide-specific CD8+T cell responses in HLA-A2 transgenic mice. Further
experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed
and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both
A2Kband HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F.
Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study
will contribute to the development of new vaccines against MV infection.
Published by Elsevier Inc.
Keywords: Measles; Vaccines; CD8; Cytotoxic T lymphocytes; Epitopes
The live-attenuated measles vaccine has been used for over 30
years and has virtually eliminated measles virus (MV) in many
industrialized countries. However, measles control remains a
challenge in developing countries due to the low seroconversion
rates in young infants. Under the age of 9 months, the live-
attenuated vaccine is poorly immunogenic because of both the
immaturity of the immune system and the presence of maternal
antibody (Gans et al., 1998; Halsey et al., 1985). The decay rates
of maternal antibody are not the same in all individuals, resulting
in a variable window for the susceptibility to MV before routine
immunization. Early vaccination with a high titer vaccine at the
age of 4–6 months was attempted but discontinued because of
high mortality rates among the female recipients (Garenne et al.,
1991; Holt et al., 1993).
To circumvent the limitations of current live-attenuated
vaccines, new vaccine strategies are being tested. These
strategies include recombinant vaccinia expressing MVantigens
(Drillien et al., 1988; Wild et al., 1992, 1993), immune
stimulating complex (ISCOM)-based vaccines using subunits or
proteins, peptide-conjugated vaccines (Stittelaar et al., 2000;
van Binnendijk et al., 1997), plasmid DNA vaccines (Polack et
al., 2000; Stittelaar et al., 2002), and alphavirus replicon particle
vaccines (Pan et al., in press). Although these strategies have
varied in their ability to induce protective B and T cell
responses, current vaccine strategies use specific antigens, viral
Virology 352 (2006) 390–399
⁎Corresponding author. S. Oh is to be contacted at Baylor Institute for
Immunology Research, Baylor University Medical Center, 3434 Live Oak,
Dallas, TX 75204, USA. Fax: +1 214 820 4813. J.A. Berzofsky, fax: +1 301 402
E-mail addresses: email@example.com (S. Oh),
firstname.lastname@example.org (J.A. Berzofsky).
0042-6822/$ - see front matter. Published by Elsevier Inc.
proteins, and immunogenic peptides. This may resolve the
problems caused by maternal antibodies and induce more potent
immune responses against MV infection.
Although antibody-mediated immune responses are important
on neutralizing antibodies to hemagglutinin (H) and fusion (F)
et al., 2000; Permar et al., 2003; van Binnendijk et al., 1990; van
Els and Nanan, 2002) indicate that cell-mediated immunity plays
an important role in the protection against MV infection. More
importantly, Tcells can be primed in infants by MV vaccine even
in the presence of passively transferred antibody (Gans et al.,
1999; Siegrist et al., 1998). Therefore, vaccines eliciting cell-
mediated immunity as well as humoral immunity are likely to be
the best choice for the eradication of MV. However, cellular
fully studied, partiallybecause of(Griffin etal.,1994; Roseetal.,
1984) limited knowledge of MV epitopes presented by MHC
al., 1995; van Binnendijk et al., 1992; van Els and Nanan, 2002).
Therefore, characterization of human CD8+T cell epitopes
from MV proteins is important for new vaccine development and
will help to evaluate cellular responses induced after vaccination
or MV infection. For this purpose, we have initiated a
comprehensive plan to identify HLA-A2 epitopes of MV
proteins. Previous studies, using prediction and peptide elution,
and L proteins and 3 in N (Herberts et al., 2001; Nanan et al.,
H and F proteins. Using HLA-A2 transgenic mice, we report
identification of 3 new HLA-A2 epitopes in H and one in F. All
four peptides were endogenously processed and presented by
HLA-A2 and induced peptide-specific CD8+T-cell-mediated
protectiveimmunityagainst challenge withrecombinant vaccinia
virus expressing H or F protein.
HLA-A201 epitope prediction and binding affinity
Forty peptides, twenty 9-mers and twenty 10-mers, were
predicted from the H and F proteins of five MV strains,
Edmonston (Alkhatib and Briedis, 1986; Richardson et al.,
1986), AIK-C (Mori et al., 1993), Halle (Buckland et al., 1987;
Gerald et al., 1986), IP-3-Ca (Schmid et al., 1992), and
Yamagata (Komase et al., 1990a, 1990b), using the algorithm
developed by Parker et al. (1994). Both Edmonston and AIK-C
are vaccine strains (Hirayama, 1983). All peptides predicted in
this study are commonly expressed in the five strains of MV
(data not shown).
Of those forty peptides, six from H and five from F were
selected based on the binding scores generated by the
algorithm. The binding affinity of each peptide to HLA-A2
molecules was first assessed by the T2-binding assay and
the data are summarized in Table 1. Nine (5 for H and 4 for
F) out of eleven peptides bound to HLA-A2 molecules, but
three of them, H9-117, F9-205 and F10-452, bound more
weakly (FI50> 50 μM) than the other six. H10-516 and F9-
57 showed the highest binding affinity to the HLA-A2
molecule. The other four peptides, H9-477, H9-567, H10-
250, and F9-63, bound relatively strongly to HLA-A2
molecule. However, the affinity of these six peptides,
FI50 = 5–12 μM, was lower than the affinity of a well-
characterized HLA-A2 epitope of the influenza virus matrix
protein (FMP: GILGFVFTL), FI50= 0.5 μM.
Immunogenicity of the peptides in HLA-A201-transgenic mice
To test whether those peptides could induce peptide-specific
CD8+T cells, we immunized animals with either the peptide-
adjuvant mixture or DC pulsed with the peptides. In both
experiments, animals were boosted once, and then CD8+Tcells
from the immunized mice were restimulated with syngeneic
splenocytes pulsed with the immunized peptides for 1 week.
Data in Fig. 1 show the lytic activity of CD8+Tcells against
target cells pulsed with individual peptides from H. Four
peptides, H9-477, H9-567, H10-250, and H10-516, induced
measurable levels of peptide-specific CD8+T cell responses.
These CD8+Tcells lysed target cells pulsed with 10 or 0.1 μM
of the corresponding peptides. In terms of the magnitude of lytic
activity, CD8+CTL induced with H9-567 and H10-516 were
slightly higher than CD8+CTL induced with other peptides
when target cells were pulsed with a 10 μM concentration of the
peptides. In this assay, Jurkat-A2Kbcells, expressing α1 and α2
of HLA-A2 and α3 of Kb, but no murine MHC molecules, were
used as target cells. Therefore, the CTL are specific for those
peptides and are restricted by HLA-A2, not murine MHC
Two out of four peptides from F protein could also induce
peptide-specific CD8+T cell responses when animals were
HLA-A201 epitopes for both hemagglutinin and fusionprotein andtheir binding
affinity to HLA-A201 molecules
A. 9-mers from hemagglutinin
B. 10-mers from hemagglutinin
C. 9-mers from fusion protein
D. 10-mers from fusion protein
aConcentration, μM of peptide that shows geometric fluorescence index is
0.5. FI = (mean fluorescence with peptide − mean fluorescence without
peptide) / mean fluorescence without peptide. Background fluorescence without
887.2 was subtracted for each individual value.
bBinding scores were predicted by the algorithm developed by
Bioinformatics and Molecular analysis section at National Institute of Health
(Parker KC 1994 JI 152:163).
391S. Oh et al. / Virology 352 (2006) 390–399
immunized with the peptide-adjuvant mixtures (Fig. 2). F9-57
and F9-63 showed higher binding affinity to HLA-A2
molecules than the other two peptides from F, indicating that
binding affinity of peptide to the MHC molecules could impact
peptide-specific CD8+T cell responses. Consistently, F9-57
induced a higher frequency of peptide-specific CD8 CTL than
F9-63. It is also interesting that CD8+T cell responses induced
with the peptides from F were slightly weaker, albeit very
reproducible, than the responses induced with the peptides from
H in our repeated experiments even though there was no
dramatic difference in their binding affinities.
To confirm these data, we immunized animals with peptide-
pulsed DC, and we measured the lytic activities of the peptide-
specific CD8+CTL (Fig. 3A). Consistent with the data in Fig. 1,
H9-567 induced a higher frequency of peptide-specific CD8+T
cells than three other peptides from the same protein. In addition
to the increased frequency of CD8+T cells, CTL induced with
either 0.1 or 10 μM H9-567 produced a similar magnitude of
lytic activity against target cells pulsed with 10 μM peptide.
Although DC immunization did not increase the numbers of
CD8+CTL dramatically compared to peptide-adjuvant mix-
tures, we confirmed that all four peptides could induce peptide-
specific CD8+T cells and those CD8+T cells were able to
recognize target cells in an antigen-specific manner. For the
peptides from F protein, we selected F9-57 and F9-63 from the
data in Fig. 2. The data in Fig. 3A indicated that the number of
peptide-specific CD8+CTL induced by F9-57 was slightly
higher than CD8+CTL induced by F9-63 as observed in Fig. 2.
Fig. 1. Immunization with peptides from measles hemagglutinin induces peptide-specific CD8+Tcell responses in A2Kbtransgenic mice. For each peptide, 3–4 mice
were immunized s.c. in the base of the tail with 100 μl of an emulsion containing 1:1 IFA and PBS with antigens and cytokines (50 nmol CTL epitope, 50 nmol HBV
core 128–140 helper epitope, 3 μg of IL-12, and 5 μg of granulocyte macrophage colony stimulating factor (GM-CSF). Mice were boosted s.c. with the emulsion on
3–4 weeks after the primary. Three to four weeks after the boost, pooled-spleen CD8+Tcells were restimulated with a 10 or 0.1 μM concentration of the peptides used
for immunization for 1 week. In a 5-h51Cr release assay, Jurkat-A2Kbcells were used as target cells. Target cells were pulsed with 10 or 0.1 μM peptides. Control cells
were not pulsed with peptides. Data from two repeated experiments were consistent and error bars represent mean ± SE of a triplicate assay.
Fig.2. Immunization with peptides frommeasles fusionprotein induces peptide-
specific CD8+Tcell responses in A2Kbtransgenic mice. For each peptide, 3–4
mice were primed and boosted as described in Fig. 1. Three to four weeks after
the boost, pooled-spleen CD8+T cells were restimulated with a 10 or 0.1 μM
concentration of the immunized peptides for 1 week. For the 5-h51Cr release
assay, Jurkat-A2Kbcells pulsed with the peptides (10 or 0.1 μM) were target
cells. Control cells were not pulsed with peptides. Data are representative of two
repeated experiments showing consistent results and error bars represent
mean ± SE of a triplicate assay.
392 S. Oh et al. / Virology 352 (2006) 390–399
In addition to measuring functional activity of CD8+CTL, the
frequency of peptide-specific CD8+T cells was measured by
counting the number of γ-IFN-producing CD8+T cells when
3B). All peptides tested induced significant numbers of γ-IFN-
producing CD8+T cells. Moreover, H9-567 and F9-57 induced
the other peptides when CTL were stimulated with 10 μM
concentrations of the peptides. The differences in the magnitude
of functional activity could be the result of in vitro expansion of
CD8+CTL before measuring CTL activity (Fig. 3A).
Peptide epitopes of H and F proteins are endogenously
processed and presented on HLA-A2
Four peptides from H and two from F were immunogenic in
HLA-A2 transgenic mice and CD8+CTL induced with these
peptides recognized the peptide-pulsed target cells (Figs. 1–3).
We next tested whether these peptides were endogenously
processed and presented on HLA-A2 molecules. For this
purpose, animals were immunized with plasmid DNA contain-
ing cDNA of H or F of the Edmonston strain. Data in Fig. 4
show that DNA plasmids expressing H and F induced
measurable levels of peptide-specific CD8+CTL, indicating
that these peptides were endogenously processed and presented
on HLA-A2 molecules followed by the induction of peptide-
specific CD8+Tcells. However, the plasmid DNAs injected did
not induce significant levels of H9-477 or F9-63-specific CD8+
T cell responses. Both H9-477 and F9-63 induced peptide-
specific CD8+Tcell responses when they were administered as
the peptide-adjuvant mixture (Figs. 1 and 2) and peptide-pulsed
DC (Fig. 3), suggesting that these two peptides may not be
processed or presented efficiently on HLA-A2. Although F9-57
showed the highest binding affinity of all peptides tested and
could induce measurable levels of peptide-specific CD8+Tcell
responses when animals were immunized with either peptide-
adjuvant mixture or peptide-pulsed DC, the magnitude of the
F9-57-specific CD8+T cell response induced by plasmid DNA
was marginal. In this study, mice were also immunized with
recombinant vaccinia viruses expressing H or F protein, but no
significant CD8+CTL responses for the H9-477 and F9-63
were observed (data not shown).
Peptide-specific CD8+T cells protect animals challenged with
recombinant vaccinia viruses
The data from Figs. 1–4 indicate that three peptides from H
and one from F protein can be endogenously processed and
presented on HLA-A2 molecules and this results in the
induction of peptide-specific CD8+T cell responses. To test
Fig. 3. Peptide-pulsed dendritic cells (DC) induced peptide-specific CD8+T cell responses. Bone marrow-derived DC were prepared as described in Materials and
methods. On day 7 of the culture, DC were pulsed with 10 μM peptide in serum-free RPMI for 2 h at 37 °C, and mice were immunized s.c. with 1–3 × 105DC/mouse
without washing. Three weeks after priming, mice were boosted with the same peptide-pulsed DC. Three to four weeks after the boost, pooled-spleen CD8+T cells
were restimulated with 10 μM (circles) or 0.1 μM (squares) concentrations of the peptides for 1 week. For the 5-h51Cr release assay, Jurkat-A2Kbcells pulsed with 10
μM peptides were used as target cells. In each experiment, 3–4 mice/group were used and data from two repeated experiments were consistent. Error bars represent
mean ± SE of a triplicate assay. For intracellular IFN-γ staining, cells were stimulated with 10 μM peptides overnight and the data are representative of two repeated
experiments showing consistent results. The number in upper quadrant is % of IFN-γ producing cells in total CD8+T cells.
393 S. Oh et al. / Virology 352 (2006) 390–399
the protective efficacy of those CTL against viral infection,
animals were immunized with each peptide, and then
challenged with recombinant vaccinia virus expressing H or F.
Data in Fig. 5A show that both H9-567 (P = 0.0009) and
H10-250 (P = 0.0034) induced CD8+CTL that result in the
significant protection of animals against recombinant vaccinia
virus expressing H protein. The average vaccinia titer in the
animals immunized with H10-516 (P = 0.0506) was slightly
lower than the titers in the control. Consistent with the data in
Figs. 1 and 4, neither H9-117 nor H9-477 induced any
protection against the virus challenge. Although CD8+CTL
induced with H9-567 and H10-250 are restricted by HLA-A2
(Figs. 1–4), it is possible that CD8+CTL specific for the
mouse H-2Kbor Dbcould also contribute to the protection
against the vaccinia virus challenge. However, the same
experiment was performed in wild-type C57BL/6 animals,
and none of the peptides induced protection against the
recombinant virus challenge (data not shown), suggesting that
the protection observed here was not due to the contribution
CTL specific for the mouse H-2Kb
Furthermore, we performed the same experiment using
HHD-2 mice that express only human HLA-A2 molecules.
Because of the limitation in the number of mice available, we
tested three mice in each group. Nevertheless, the data in Fig.
5B clearly show that all three peptides, H9-567, H10-250, and
H10-516, induced statistically significant levels of protective
immunity against the recombinant vaccinia virus challenge.
The differences in the vaccinia titers between control and
experimental groups were greater in HHD-2 mice than A2Kb
mice, probably due to the increased expression of HLA-A2
molecules in HHD-2 mice (data not shown).
The animals immunized with the peptides from F protein
were challenged with recombinant vaccinia expressing F
(Fig. 6). In A2Kbtransgenic mice, only F9-57 (P = 0.0442)
showed significant protection against the virus challenge.
This peptide also induced protective immunity in HHD-2
mice (P = 0.031), which express only the human HLA-A2
molecule, and this protection was even greater than in A2Kb
transgenic mice. HHD-2 mice immunized with F9-63 were
not protected to a significant extent.
The type of immune responses to viral infections and the
response conferring protection are dependent on the nature of
the infection. Neutralizing antibody is an important immune
arm for preventing MVinfection. However,cellular immunity is
also critical for controlling MV infection. Moreover, significant
levels of cell-mediated immunity are induced in both naturally
infected and vaccinated individuals (Bautista-Lopez et al.,
Fig. 4. Immunization with DNA plasmids expressing measles hemagglutinin or fusion protein induces peptide-specific CD8+T cell responses. Three to four A2Kb
mice in each group were immunized intramuscularly with 100 μg of the plasmids, SINCP/H, or SINCP/F. At 3 week-intervals, mice were boosted twice. Three to
four weeks after the final immunization, pooled-spleen CD8+T cells were restimulated with 10 μM (circles) and 0.1 μM (squares) peptides for 1 week. For the 5-
h51Cr release assay, Jurkat-A2Kbcells pulsed with peptides, 10 μM were used as target cells. Data are representative of three-repeated experiments and error bars
represent mean ± SE of a triplicate assay.
394S. Oh et al. / Virology 352 (2006) 390–399
2000; Gans et al., 1999; Jaye et al., 1998a, 1998b; Nanan et al.,
1995, 2000; Ovsyannikova et al., 2003; Siegrist et al., 1998; van
Binnendijk et al., 1990; van Els and Nanan, 2002; Ward et al.,
1995). Impaired T cell immunity results in more severe disease
and delayed recovery from measles (Bautista-Lopez et al.,
2000; Gans et al., 1999; Jaye et al., 1998a, 1998b; Nanan et al.,
1995, 2000; Ovsyannikova et al., 2003; Siegrist et al., 1998; van
Binnendijk et al., 1990; van Els and Nanan, 2002; Ward et al.,
1995) and MV-specific long-term memory CD8+Tcells are also
generated after MV infection (Jaye et al., 1998a, 1998b; Nanan
et al., 1995; Ovsyannikova et al., 2003). Therefore, new
vaccines against MV should include components that induce
cellular immunity against MV infection and finding new
epitopes for CD8+T cells will be necessary for development
and evaluation of new MV vaccines.
HLA-A2 is common in most populations, and therefore we
have comprehensively examined HLA-A2 epitopes from the H
and F proteins in this study. We have identified 3 new HLA-
A2-restricted epitopes in H and the first HLA-A2-restricted
epitope in F. Furthermore, we have shown that these CD8+T
cell epitopes were endogenously processed and presented on
HLA-A2 molecules, and they induced protective immunity in
animal models. Because most new vaccine strategies against
MV focus on H and F for inducing neutralizing antibodies and
little is known about human T cell epitopes of both proteins,
new CD8 T cell epitopes for both F and H will be useful in
The expression levels of both H and F are lower than MV
proteins NP and M during viral infections (van Binnendijk et
al., 1992). Both H and F are glycosylated surface viral proteins
that are cotranslationally inserted into the ER. Maturation and
translocation to the cell membrane take place via the Golgi
network, limiting opportunities for processing by the classical
MHC class I pathway. Even without using the classical
pathway, however, peptide could be presented by alternative
pathways, such as proteosomal and TAP-independent pathways
(Watts and Powis, 1999) and by back trafficking of proteins into
the cytosol (Wiertz et al., 1996). For the H protein, 3 out of 6
peptides tested were presented by HLA-A2. Although we did
not show all peptides predicted in this study, higher numbers of
HLA-A2 epitopes showing higher binding scores were
predicted from H compared to F protein even though F is
more abundant than H in MV-infected cells (Herberts et al.,
2001; van Binnendijk et al., 1992). Two overlapping A2-
restricted H epitopes were previously identified through use of a
predictive algorithm (H29-37) (Nanan et al., 1995) and elution
of peptides from MV-infected B cells (H30-38) (van Els et al.,
2000). These peptides were both identified in our predictions,
but their predicted binding scores were lower than those of the
peptides tested in this study.
All peptides predicted and tested, except for H10-250, in this
study lack amino acid residues associated with poor binding to
position 3;Arg,Lys,His,andAlaatposition 4;Proatposition5;
Arg, Lys, and His at position 7; Asp, Glu, Arg, Lys, and His at
position 8; and Arg, Lys, and His at position 9 (Rammensee et
al., 1995; Ruppert et al., 1993). H10-250 possesses Arg at
position 4, known tobeassociatedwithpoorHLA-A2.1 binding
at the secondary anchor positions. Although there were
discrepancies in rank order between the peptide-binding scores
Fig. 5. Protection against recombinant vaccinia virus expressing measles
hemagglutinin (H) can be provided by immunization with the peptide epitopes
with the peptide-adjuvant mixtures and boosted once as described in Fig. 1. For
the control, animals were immunized with the adjuvant mixture containing
i.p. with 5–6 × 106PFU of vaccinia virus expressing measles H. On day 5, virus
B were tested and the statistical significance was tested by Student's t test.
Fig. 6. Protection against recombinant vaccinia virus expressing measles fusion
(F) protein in the animals immunized with the peptide epitopes from measles F.
(A) Female A2Kband (B) HHD-2 mice were immunized with the peptide-
adjuvant mixtures and boosted once as described in Fig. 1. Control animals were
immunized with the adjuvant mixture containing cytokines and CD4 helper
epitope. Ten days after the boost, mice were challenged i.p. with 5–6 × 106PFU
of vaccinia virus expressing measles fusion protein. On day 5, virus titers in the
ovaries were determined. Five mice in panel A and three mice in panel B were
tested and the statistical significance was tested by Student's t test.
395 S. Oh et al. / Virology 352 (2006) 390–399
predicted by the algorithm and the actual binding affinities by
T2-binding assay, the peptides predicted by this algorithm
were relatively good binders to HLA-A2 molecules. Binding
affinity of peptide epitopes may correlate with the strength of
CD8 T-cell-mediated immunity. However, for CD8+T cell
responses to the peptides from H, such a correlation was not
apparent. This difficulty in seeing a correlation is probably
because their FI50values were in a similar range, 7.5–12.5
μM. Moreover, CD8+T cells specific for each peptide may
use different TCR repertoires.
In this study, we used HLA-A2 transgenic mice to test the
immunogenicity of predicted peptides. This animal model has
been widely used for determining human HLA-A2 epitopes and
has been proven to be a good predictor of human CD8+T cell
epitopes (Oh et al., 2004; Shirai et al., 1995; Tishon et al.,
2000). One concern is that the peptide/HLA-A2 complex
recognized by murine Tcells may differfrom that recognized by
human Tcells. However, MV CTL responses in transgenic mice
resembling those in humans have been demonstrated (Tishon et
al., 2000) and A2Kbtransgenic mice could generate anti-viral
CTL in the absence of human β2 m and human CD8. Possible
cross-reactions with mouse Kbwere eliminated by using HHD-
2 mice that express only human HLA-A2. Moreover, none of
the peptides tested in this study protected wild-type animals.
The better protection against vaccinia virus challenge (Figs. 5
and 6) in HHD-2 mice could be due to the fact that HHD-2 mice
express only one class I molecule, human HLA-A2. Indeed, a
greater number of peptide-specific CD8+CTL were observed in
HHD-2 mice compared to A2Kbtransgenic mice in our other
studies (data not shown). Data from this study are limited to
HLA-A2 and were tested in a transgenic animal model.
However, studies in progress will reveal the peptide epitopes
presented on human cells. Furthermore, it is important to map
epitopes for other HLA types to broaden the coverage of the
human population. Nearly half of most populations express
HLA-A2, and it has been estimated that only 5 class I HLA
alleles are sufficient to cover 90% of almost any given
population (Gulukota and DeLisi, 1996).
In conclusion, identification and evaluation of CD8+T cell
epitopes are necessary for selecting vaccine components of new
MV vaccines as well as for evaluating cellular immune
responses. In this study, we have identified and characterized
four endogenously processed and presented HLA-A2-restricted
epitopes, one from F and three from H. These peptides also
induced protective immunity in animals challenged with
recombinant vaccinia viruses expressing MV proteins. These
studies may contribute to the development and evaluation of
new vaccines against measles.
Materials and methods
A2Kbtransgenic mice express a chimeric HLA-A2.1
transgene with the α1 and α2 domains from HLA-A2.1 and the
α3 domain from H-2Kbto allow binding to mouse CD8 (Vitiello
et al., 1991). HHD-2 (Firat et al., 2001; Harrer et al., 1996;
Pascolo et al., 1997) mice have the murine β2-microglobulin and
H-2Dbgenes knocked out and are transgenic for a chimeric
human HLA-A2.1 expressing the α1 and α2 domains of HLA-
A2.1 and a murine Db-derived α3 domain to allow interaction
with mouse CD8 and also have a covalently linked human β2-
microglobulin to compensate for lack of free β2-microglobulin.
the H-2Kbgene is not knocked out, the only class I MHC
molecule expressed is the chimeric human HLA-A2.1 with the
covalent human β2-microglobulin.
These mice were bred and housed in appropriate animal care
facilities. All procedures with animals were conducted in
accordance with institutionally approved protocols.
The Jurkat-A2Kbcell line, a gift from Dr. L. Sherman
(Scripps Research Institute, La Jolla, CA), is transfected with
the HLA chimeric molecule containing the α1 and α2 domains
from human HLA-A2.1 and α3 from mouse H-2Kb. The T2 cell
line is deficient in TAP1 and TAP2 transporter proteins and
expresses low levels of HLA-A2 (Nijman et al., 1993; Oh et al.,
2004). BSC-1 cells, monkey kidney cells, were acquired from
ATCC. Cells were maintained in 10% FCS-RPMI containing 1
mM sodium pyruvate, nonessential amino acids (Biofluids,
Rockville, MD), 4 mM glutamine, 100 U/ml penicillin, 100 μg/
ml streptomycin, and 50 μM 2-ME.
Peptides were prepared in an automated multiple peptide
synthesizer (Symphony; Protein Technologies, Tucson, AZ)
using fluorenylmethoxycarbonyl chemistry. They were purified
by reverse phase HPLC, and sequences were confirmed where
necessary on an automated sequencer (477A; Applied Biosys-
tems, Foster City, CA).
Peptide binding to HLA-A2 molecules was measured by
using the T2 cell line (Nijman et al., 1993; Oh et al., 2004). T2
cells (3 × 105/well) were incubated overnight in 96-well plates
with culture medium (a 1:1 mixture of RPMI 1640-Eagle-
Hank's amino acid (EHAA) media containing 2.5% FCS, 100
U/ml penicillin, and 100 μg/ml streptomycin) with 10 μg/ml
human β2-microglobulin (Sigma-Aldrich, St. Louis, MO) and
peptides. Cells were washed twice with cold PBS containing
2% FCS and then incubated for 30 min at 4 °C with anti-HLA-
A2.1 BB7.2 mAb (1/100 dilution of hybridoma supernatant).
After washing, cells were stained with 5 μg/ml FITC-labeled
goat anti-mouse Ig (BD PharMingen, San Diego, CA) and
expression levels of HLA-A2.1 were measured by flow
cytometry (FACSCaliber; BD Biosciences, Mountain View,
CA). HLA-A2.1 expression was quantified as fluorescence
index (FI) according to the formula: FI = [(mean fluorescence
with peptide − mean fluorescence without peptide) / mean
fluorescence without peptide]. Background fluorescence
396S. Oh et al. / Virology 352 (2006) 390–399
without BB7.2 was subtracted for each individual value. To
compare the different peptides, FI50, the peptide concentration,
μM, that increases HLA-A2.1 expression by 50% over no
peptide control background, was calculated from the titration
curve for each peptide.
Mice were immunized with syngeneic peptide-loaded
dendritic cells (DC), plasmid DNAs expressing measles H or
F protein, or the mixture of peptide and cytokine in incomplete
Freund's adjuvant (IFA). DC were prepared from bone marrow
as previously described (Celluzzi et al., 1996). On day 7 of the
culture, DC were pulsed with 10 μM peptide in serum-free
RPMI for 2 h at 37 °C, and then mice were immunized s.c. with
1–3 × 105peptide-pulsed DC. Three weeks after the primary
immunization, mice were boosted with the same peptide-pulsed
DC. Alternatively, mice were immunized intramuscularly with
100 μg plasmid DNA, SINCP/H, or SINCP/F (Polo et al., 2000;
Song et al., 2005). Mice were boosted with 100 μg plasmids 3
weeks after the primary immunization. Mice were immunized s.
c. in the base of the tail with 100 μl of an emulsion containing
1:1 IFA and PBS with peptide antigens and cytokines (50 nmol
CTL epitope, 50 nmol HBV core 128–140 helper epitope, 3 μg
of IL-12, and 5 μg of granulocyte macrophage colony
stimulating factor; GM-CSF). Mice were boosted with the
emulsion 3–4 weeks after the primary immunization. IFA and
cytokines were purchased from Sigma and Peprotech (Rocky
Hill, NJ), respectively.
CD8+T cells from the immunized mice were restimulated
with peptide-loaded splenocytes for 1 week as previously
described (Oh et al., 2003) and then tested in 5 h51Cr release
assays. Target cells were labeled with51Cr and washed twice.
Cells were then pulsed with peptides for 2 h and used as
target cells without further washing. Control cells were not
pulsed with peptides. The mean of triplicate samples was
calculated, and the percentage of specific lysis was deter-
mined by using the following formula: Percentage of specific
lysis = 100 × (experimental51Cr release − spontaneous51Cr
release) / (maximum
release). The maximum release refers to counts from targets
in 2.5% Triton X-100.
51Cr release − spontaneous
Viral challenge and protection assay
Female mice, A2Kband HHD-2, were immunized with the
peptide-adjuvant mixture as described above, boosted 3–4
weeks after the primary immunization. Ten days after the boost,
animals were challenged i.p. with recombinant vaccinia virus
(6 × 106PFU/mouse) expressing measles H or F protein (Tamin
et al., 1994; Zhu et al., 2000), kindly provided by Dr. Paul Rota
(Center for Disease Control, Atlanta GA). On day 5, virus titers
in the ovaries of individual mice were determined on BSC-1
cells as previously described (Ahlers et al., 2001).
Antibodies and flow cytometry
FITC-labeled anti-mouse CD8 (53-6.7), CD11c, CD80 (B7-
1), and CD54 (ICAM-1) were used for staining of cell surface
molecules. Intracellular IFN-γ staining followed the manufac-
ture's protocol (Pharmingen). All Abs and reagents were
purchased from Pharmingen. For flow cytometric analysis of
cell surface antigens, 5 × 105cells were washed and
resuspended in PBS containing 0.2% BSA and 0.1% sodium
azide. Cells were incubated on ice with the appropriate antibody
for 30 min and then washed. Samples were analyzed on a
FACSCaliber (BD Biosciences, Mountain View, CA). Back-
ground staining was assessed by using isotype control
We thank Dr. Linda Sherman (Scripps) and Dr. François
Lemonnier (Institut Pasteur for kind gifts of breeding pairs of
the A2Kb and HHD-2 mouse strains to be bred in our facility).
This work was supported in part by a grant from the Bill and
Melinda Gates Foundation and by intramural research funds of
the CCR, NCI, and NIH.
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