Download Long-Term Protective Immunity Induced Against

HUMAN GENE THERAPY 17:1–11 (September 2006)
© Mary Ann Liebert, Inc.
Long-Term Protective Immunity Induced Against
Trypanosoma cruzi Infection After Vaccination with
Recombinant Adenoviruses Encoding Amastigote
Surface Protein-2 and Trans-Sialidase
ALEXANDRE V. MACHADO,1,2 JARBAS E. CARDOSO,3 CARLA CLASER,4,5
MAURICIO M. RODRIGUES,4,5 RICARDO T. GAZZINELLI,1,2 and OSCAR BRUNA-ROMERO1,2,6
ABSTRACT
Protection against protozoan parasite Trypanosoma cruzi has been shown to be dependent on the induction
of type 1 immune responses. Replication-deficient human type 5 recombinant adenoviruses have an unsurpassed ability to induce type 1 immune responses. Thus, we constructed two type 5 recombinant adenoviruses
encoding parasite antigens trans-sialidase (rAdTS) and amastigote surface protein-2 (rAdASP2). Both antigens were genetically engineered to secrete recombinant products in order to induce both optimal antibody
and T cell responses. Immunizations of mice with rAdASP2 and rAdTS induced high levels of serum antibodies specific for their recombinant products. In addition, both recombinant viruses were able to elicit a biased helper T cell type 1 (Th1) cellular immune response and a substantial CD8 T cell-mediated immune
response. Moreover, individual immunization with rAdASP2 or rAdTS induced high levels of protection
against a challenge with live parasites. CD8 T cells mediated, at least in part, such protection. Furthermore,
when combined in the same inoculum, rAdTS plus rAdASP2 induced complete protection in all animals tested,
even when challenges were performed 14 weeks after the last immunization. Taking together, these results
show that recombinant adenoviruses expressing TS and ASP-2 antigens of T. cruzi are interesting candidates
for the development of a vaccine against Chagas’ disease.
tially dependent on CD8 T cells. Moreover, simultaneous
immunization with rAdTS and rAdASP2 resulted in complete
protection of all animals, even when deadly challenges were
performed 14 weeks after the last immunization. Thus, these
results show the potential use of recombinant adenoviruses
expressing TS and ASP-2 antigens of T. cruzi for the development of a vaccine against Chagas’ disease.
OVERVIEW SUMMARY
Protection against the parasite Trypanosoma cruzi is highly
dependent on type 1 immune responses. Replication-deficient
human type 5 recombinant adenoviruses have an unsurpassed
ability to induce strong type 1 immune responses. Thus, in
our study, we attempted to immunize mice with type 5 recombinant adenoviruses encoding the parasite protective antigens trans-sialidase (rAdTS) or/and amastigote surface protein-2 (rAdASP2). Our results show that this strategy elicits
both optimal antibody and T cell responses and high levels of
protection against a challenge with live parasites. Further experiments demonstrated that such protection was at least par-
INTRODUCTION
I
NFECTION WITH THE PROTOZOAN PARASITE Trypanosoma cruzi
causes Chagas’ disease, a neglected disease that affects more
1Departamento
de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte 31270-910, Brazil.
de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte 30190-002, Brazil.
3Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte 31270-910, Brazil.
4Centro Interdisciplinar de Terapia Gênica (CINTERGEN), Universidade Federal de São Paulo, São Paulo 04044-010, Brazil.
5Departamento de Microbiologia, Imunologia, e Parasitologia, Universidade Federal de São Paulo, São Paulo 04044-010, Brazil.
6Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-910, Brazil.
2Centro
1
2
than 16 million inhabitants in Latin America. More than
300,000 new patients become infected with T. cruzi every year,
and approximately 21,000 humans die annually, despite the use
of drugs that have much diminished past morbidity and mortality rates. Drugs used for treatment are not effective in chronically infected individuals and parasites naturally resistant to
chemotherapy have been described in various regions of Latin
America (Urbina, 2001; Camandaroba et al., 2003). Thus, vaccination should be seriously considered as an alternative approach in therapy and prophylaxis of Chagas’ disease.
Protective immunity against this parasite seems to be highly
dependent on the induction of CD8 T cell responses and interferon (IFN)-, similarly to what was also found for the preerythrocytic stages of Plasmodium species, or for Toxoplasma gondii
infections. CD4 T cells with a helper T cell type 1 (Th1) cytokine secretion profile and antibodies have also been described as
mediators of protection against all these diseases (Brener and
Gazzinelli, 1997; Rodrigues et al., 2003). Therefore, the development of a vaccine against T. cruzi should aim at the induction
of broad immunity against parasitic antigens.
Several studies attempted to develop a vaccine using live attenuated (Menezes, 1968) or killed T. cruzi parasites (Basombrio, 1990). Even related trypanosomatids, such as the tomato
parasite Phytomonas serpens (Pinge-Filho et al., 2005), which
shares common antigens with T. cruzi, have been tested as inducers of cross-protection. Unfortunately, most of these immunizations were only able to delay manifestations of the disease or at best decrease the associated pathology. Long-term
protection could be achieved only when using Th1-driving adjuvants (Hoft and Eickhoff, 2005) and/or better characterized
DNA subunit vaccines encoding a few parasite antigens (Sepulveda et al., 2000; Schnapp et al., 2002; Fralish and Tarleton,
2003). When subunit plasmid DNA vaccines were used, two of
the most successful candidate antigens tested were trans-sialidase (TS) and amastigote surface protein-2 (ASP-2) (Costa et
al., 1998; Fujimura et al., 2001; Garg and Tarleton, 2002;
Boscardin et al., 2003; Fralish and Tarleton, 2003; Vasconcelos et al., 2004). TS is a trypomastigote-expressed antigen that
catalyzes the transfer of sialic acids from host glycoconjugates
to acceptor molecules on the plasma membrane of the parasite
(Schenkman et al., 1994); ASP-2 is a surface amastigote antigen (Low et al., 1998) with a yet undescribed function.
Recombinant viruses have been shown to be particularly effective for vaccine delivery purposes (Rocha et al., 2004). Clinical trials of new recombinant viral vaccine candidates for
malaria, tuberculosis, or acquired immunodeficiency syndrome
(AIDS) have shown unprecedented levels of cellular immunity
against the recombinant products expressed by viral vectors,
compared with preceding vaccine formulations based on purified recombinant proteins or plasmid DNA vaccines. An advantage of using some recombinant viruses is the universal adjuvant effect displayed by the viral particle itself, something
that would avoid the need of using adjuvants or cytokines such
as granulocyte-macrophage colony-stimulating factor (GMCSF) or interleukin (IL)-12. Thus, recombinant adenoviruses,
influenza viruses, or vaccinia viruses display profound immunemodulating properties (Molinier-Frenkel et al., 2003; Guillot et
al., 2005). Advantageously, replication-deficient viruses limit
these additional adjuvant effects to the first round of infection,
and are not expected to induce prolonged generalized systemic
MACHADO ET AL.
immune-stimulatory effects that could have deleterious consequences.
We report in this paper the construction of two replicationdeficient T. cruzi recombinant adenoviruses, encoding genetically modified TS and ASP-2 antigens. We tested their ability
to induce long-term immunity and protection against a lethal
challenge with T. cruzi parasites. Results indicate that these adenoviral vectors represent a promising strategy to develop a
vaccine for Chagas’ disease.
MATERIALS AND METHODS
Mice, parasites, and hemoculture
BALB/c, C57BL/6, and C57BL/6 CD8 knockout (CD8
KO) mice were housed and handled at the animal breeding facilities of the Federal University of Minas Gerais (Centro de
Bioterismo [CEBIO], Belo Horizonte, Brazil) or the Oswaldo
Cruz Foundation (FIOCRUZ-BIOTEX) according to approved
institutional guidelines. Vaccine challenges were performed
with 100 bloodstream trypomastigotes of the Y strain of T. cruzi
obtained from Swiss mice infected 7 days previously. Parasite
development was monitored in the blood according to standard
methods (Krettli and Brener, 1976). Mouse mortality was
recorded daily. For hemoculture, aliquots of 0.1 ml of blood
were collected and cultured in duplicate, at 28°C for 1 month
in axenic liver infusion tryptose medium. Parasite growth was
monitored weekly by microscopy.
Cell lines
The cell line 293 (ATCC CRL-1573) is transformed with the
adenoviral E1 genomic region that was used for generation, amplification, and characterization of all the E1 replication-deficient
recombinant adenoviruses (Shaw et al., 2002). Cells were grown
in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St.
Louis, MO) supplemented with 5% fetal bovine serum (FBS; Invitrogen GIBCO, Grand Island, NY) and antibiotics.
Recombinant adenoviruses and immunization
Construction of rAdlacZ, a recombinant adenovirus carrying
the Escherichia coli -galactosidase-coding sequence, was described previously, together with the detailed methodology followed in this study for generation and purification of all viruses
(Bruna-Romero et al., 1997). pAdCMV-TS is a transfer plasmid that contains a eukaryotic expression cassette formed by
the cytomegalovirus immediate-early promoter and the simian
virus 40 (SV40) RNA polyadenylation sequences. Inside this
cassette we cloned DNA sequences encoding the signal peptide
and catalytic domain of T. cruzi TS protein, obtained by restriction enzyme digestion of plasmid p154/13 (Costa et al.,
1998). Equivalently, pAdCMV-ASP2 encodes ASP-2 sequences obtained by restriction digestion of plasmid pIgSPclone9 (Boscardin et al., 2003).
Mice were inoculated subcutaneously in the tail base with
100 l of viral suspension consisting of RPMI supplemented
with 1% normal mouse serum containing 108 plaque-forming
units (PFU) of each adenovirus. Immunizations were performed
twice with a 6- to 8-week interval.
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VACCINATION WITH RECOMBINANT ADENOVIRUSES AGAINST T. cruzi
ELISA, Western blot, and lytic antibody detection
Recombinant TS or ASP-2 proteins were produced in Escherichia coli as previously described (Ribeirao et al., 1997;
Boscardin et al., 2003). Antibodies against those proteins were
detected by enzyme-linked immunosorbent assay (ELISA) as
previously described (Costa et al., 1998; Boscardin et al., 2003).
Each serum sample was analyzed in serial dilutions and individual titers were considered as the highest dilution of serum
that presented an optical density at 492 nm (OD492) higher than
0.1. Results are presented as mean logarithmic antibody titers standard deviation (SD) of six animals per group.
293 cells mock-infected or infected with rAdTS, rAdASP2,
or rAdlacZ at a multiplicity of infection (MOI) of 1 were collected 24 hr after infection and their extracts were analyzed by
Western blot. Membranes were incubated with a pool of mouse
sera specific for ASP-2 or TS followed by incubation with peroxidase-conjugated goat anti-mouse IgG antibody. Detection
was performed by membrane exposure to X-ray film after a
standard chemiluminescence reaction (ECL detection system;
GE Healthcare, Piscataway, NJ).
Immunofluorescence reactions were performed by incubating sera of mice immunized with rAdlacZ, rAdTS, rAdASP2,
or a mixture of the latter two with T. cruzi trypomastigotes and
amastigotes air dried onto glass slides. Secondary fluorescein
isothiocyanate (FITC)-conjugated anti-mouse antibodies were
used to reveal the results of the reactions. Samples were analyzed by ultraviolet (UV) light microscopy. The presence of
lytic antibodies in the sera of immunized mice was determined
essentially as described (Krettli and Brener, 1976).
Determination of IFN- in spleen cell supernatants
BALB/c or C57BL/6 mice were immunized as described
above. Two weeks after the last immunization, splenocytes were
isolated and restimulated with 2.5-g/ml (TS) or 1-g/ml
(ASP-2) protein solutions. After 3 days, supernatants were collected for cytokine determination. IFN- concentration in supernatants was estimated by capture ELISA, using antibodies
and recombinant cytokine purchased from BD Biosciences
Pharmingen (San Diego, CA) exactly as described previously
(Rodrigues et al., 1999). Supernatants were diluted up to 10
times for a precise estimate of cytokine concentration. Cytokine concentration in each sample was determined from standard curves performed in parallel with known concentrations
of recombinant mouse IFN-. The detection limit of the assays
was 0.2 ng/ml.
Enzyme-linked immunospot assays
Enzyme-linked immunospot (ELISPOT) assays were performed as previously described (Bruna-Romero et al., 2001).
Briefly, 1.5 105 splenocytes from normal BALB/c or
C57BL/6 mice were used as antigen-presenting cells (APCs)
after preincubation with H-2Kd TS peptide IYNVGQVSI
(amino acids 359–367) or H-2Kb ASP-2 peptide VNHRFTLV
(amino acids 552–559). APCs were added to Multiscreen HA
nitrocellulose plates (Millipore, Bedford, MA) coated with antiIFN- antibody R4-6A2 (BD Biosciences Pharmingen). Responder cells, isolated from immunized or control mice 2 weeks
after the last immunization, were added to the same wells at
various dilutions. After incubation, plates were extensively
washed and incubated with a biotinylated anti-mouse IFN- antibody (XMG1.2; BD Biosciences Pharmingen) followed by
peroxidase-labeled streptavidin. Reactions were developed by
adding a peroxidase substrate to the wells (50 mM Tris-HCl
[pH 7.5], containing 3,3-diaminobenzidine tetrahydrochloride
[1 mg/ml] and 30% hydrogen peroxide solution [1 l/ml]) and
stopped under running water before the spots were counted with
a stereomicroscope.
In vivo cytotoxicity assays and flow
cytometric analyses
In vivo cytotoxicity assays were performed according to the
technique described by Oehen and Brduscha-Riem (1998).
Briefly, BALB/c or C57BL/6 spleen cells were divided into two
populations and labeled with the fluorogenic dye carboxyfluorescein diacetate succinimidyl diester (CFSE) at a concentration of 10 M (CFSEhigh) or 1.0 M (CFSElow). BALB/c and
C57BL/6 CFSEhigh cells were pulsed for 40 min at 37°C with
the H-2Kd TS peptide (IYNVGQVSI) or with the H-2Kb ASP2 peptide (VNHRFTLV), respectively. CFSElow cells remained
unpulsed. Subsequently, CFSEhigh cells were washed and mixed
with equal numbers of CFSElow cells before intravenously injecting 2 107 total cells per mouse. Recipient mice were
BALB/c, C57BL/6, or CD8 knockout C57BL/6 mice, 2 weeks
after the second immunization with rAdlacZ, rAdTS (BALB/c),
or rAdASP2 (C57BL/6 and CD8 KO). Spleen cells of recipient mice were collected 20 hr after transfer, fixed, and analyzed by fluorescence-activated cell sorting (FACS), using a
FACSCalibur cytometer (BD Biosciences Immunocytometry
Systems, Mountain View, CA). The percentage of specific lysis for each peptide was determined with the following formula:
1 [(%CFSEhigh infected %CFSElow infected)/(%CFSEhigh
naive %CFSElow naive)] 100.
Statistical analyses
Differences in parasitemia among the different groups of
mice were determined by Mann–Whitney U test (two-tailed)
for nonparametric samples. Mortality rates were compared by
log-rank test.
RESULTS
Generation and characterization of recombinant
adenoviruses expressing T. cruzi antigens
Amastigote surface protein-2 (rAdASP2) and the enzyme
trans-sialidase (rAdTS) were selected for generation of recombinant adenoviruses on the basis of earlier studies in which successful vaccination was achieved with plasmid DNA as vectors
(Costa et al., 1998; Boscardin et al., 2003). rAdASP-2 encodes
the mouse immunoglobulin chain signal peptide (MQVQIQSLFLLLLWVPGSRG) fused to amino acids 1–694 of ASP2. rAdTS was engineered to encode the functional native protein signal peptide (amino acids 1–33) and the entire catalytic
domain of TS (amino acids 34–678) (Costa et al., 1998;
Boscardin et al., 2003) (Table 1). The resulting recombinant
ASP-2 or TS proteins do not contain glycosylphosphatidyl-
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MACHADO ET AL.
TABLE 1. ADENOVIRUSES USED
Virus
rAdlacZ
rAdASF2
rAdTS
FOR IMMUNIZATION
Insert size
(bp)
Antigen sequence details
(aa)
Ref.
3045
2154
2034
Complete E. coli -galactosidase (1015 aa)
Mouse Ig SP(24 aa) complete ASP-2 (694 aa)
TS signal peptide (33 aa) catalytic domain (645 aa)
Bruna-Romero et al., 1997
Boscardin et al., 2003
Fujimura et al., 2001
inositol (GPI)-anchoring motifs, impeding any GPI anchor
modification of the nascent polypeptides (Fig. 1A). In addition
to these viruses, rAdlacZ was used as control in all experiments.
The capacity of infected cells to express -galactosidase as well
as the viral infectivity of rAdlacZ had been previously determined (Bruna-Romero et al., 1997). The resulting replicationdeficient viruses were first used to infect cells in vitro. Extracts
of those cells were tested in Western blot assays. As shown in
Fig. 1B, ASP-2 or TS proteins were abundantly produced in
cells infected with rAdASP2 or rAdTS, respectively. It is worth
noting that four peptide products from rAdTS-infected cells reacted with anti-TS antibodies, the highest molecular weight
band displaying the predicted molecular weight for the fulllength recombinant product (66 kDa). Lower molecular weight
bands that react with the same TS-specific serum must represent proteolytic products that keep native antibody epitopes. Because we have previously shown that the absence of GPI-anchoring motifs increases proteolytic processing and favors
antigen-loading onto MHC molecules for presentation to T cells
(Bruna-Romero et al., 2004), the proteolytic products observed
for the GPI-deleted form of TS used in our study could favor
its presentation to T cells.
strains analyzed. Importantly, animals immunized with a mixture
of both viruses simultaneously (rAdTS rAdASP2) mounted
antigen-specific responses of similar magnitude against each of
the proteins, compared with those detected when recombinant
viruses were administered individually.
Type 1 immune response after vaccination with
rAdASP2 or rAdTS
Because protection against T. cruzi has been repeatedly
shown to be mediated by type 1 immune responses, mediated
by CD4 and CD8 T cells, we evaluated in vitro IFN- production by splenic cells from BALB/c and C57BL/6 mice im-
Antibody immune responses after vaccination with
rAdASP2 or rAdTS
To determine the capacity of rAdTS or rAdASP2 to elicit
antibody responses against T. cruzi antigens, mice of two different genetic backgrounds were immunized with these recombinant viruses. Sera from these mice were initially analyzed by
immunofluorescence to test their specificity to react with T.
cruzi parasites. Figure 2A shows that sera from mice immunized with rAdTS specifically reacted with T. cruzi trypomastigotes and sera from AdASP2-immunized mice specifically reacted with amastigote forms of the parasite.
Specific IgG antibody titers in the sera of immunized mice
were also determined by ELISA, using recombinant TS or ASP2 as antigen. Antibody titers in the sera of BALB/c (H-2d) and
C57BL/6 (H-2b) mice immunized with either rAdTS or
rAdASP2, or a mixture of both, are shown in Fig. 2B and C, respectively. Immunization with either rAdTS or rAdASP2, or a
mixture of both, resulted in IgG antibodies specific to the corresponding antigen. In contrast, control animals immunized with
rAdlacZ had negligible antibody immune responses against either ASP-2 or TS (titers less than 102). No significant complement-mediated lytic activity was detected in the sera of immunized mice compared with mice infected with T. cruzi (Fig. 2D).
Comparison of serum IgG levels revealed that antibody titers
against TS in BALB/c mice were higher than those detected in
C57BL/6 mice. The levels of antibodies to ASP-2 were similar
in mice of different genetic backgrounds. Antibody titers to TS
were significantly higher than the titers to ASP-2 in both mouse
FIG. 1. Trypanosoma cruzi ASP-2 and TS sequences expressed by recombinant adenoviruses. (A) Scheme of ASP-2and TS-coding sequences cloned in the genomes of both recombinant viruses. (B) Protein expression in 293 cells infected
with one of the following recombinant adenoviruses: rAdASP2,
rAdTS, or the control rAdlacZ assessed by Western blot, using
a pool of mouse sera specific for ASP-2 (left) or TS (right) as
primary antibodies.
VACCINATION WITH RECOMBINANT ADENOVIRUSES AGAINST T. cruzi
munized with rAdTS and rAdASP2, respectively. Initially, we
determined the levels of IFN- secreted by spleen cells after in
vitro restimulation with recombinant proteins. As previously reported by one of us, IFN- secreted by spleen cells in this in
vitro assay is dependent on the activation of CD4 but not
CD8 T cells (Rodrigues et al., 1999; Boscardin et al., 2003;
Vasconcelos et al., 2004). As shown in Fig. 3A, when restimulated in vitro with the corresponding recombinant protein,
splenocytes from mice immunized with either rAdTS or
rAdASP2 secreted IFN-. This IFN- production suggests that
these cells must have expanded after immunization with the recombinant viruses because splenocytes from control mice im-
munized with rAdlacZ secreted little or no IFN- when restimulated in vitro with recombinant TS or ASP-2.
The presence of type 1 CD8 T cells in mice immunized
with rAdTS or rAdASP2 was determined by ex vivo ELISPOT
to detect IFN--producing splenocytes. BALB/c and C57BL/6
splenic cells were restimulated in vitro with the TS-derived
H-2d synthetic peptide IYNVGQVSI or with the ASP-2-derived
H-2b peptide VNHRFTLV, respectively. Previous studies established that only CD8 T cells of those mouse strains (Low
et al., 1998; Rodrigues et al., 1999) recognized these peptides.
As can be observed in Fig. 3B, significant numbers of TS-specific or ASP-2-specific T cells were present in the spleen of
4C
5
FIG. 2. Antibodies generated in mice of different genetic backgrounds immunized with recombinant adenoviruses expressing
ASP-2 or TS proteins and their lytic activity. (A) Right: Immunofluorescence staining of mixtures of T. cruzi trypomastigotes
and amastigotes, using sera of rAdlacZ-, rAdTS-, and/or rAdASP2-immunized mice as primary antibodies. Left: Side-by-side
comparison with corresponding phase-contrast light microscopy images to identify the different parasite forms (original magnification, 400). (B and C) Anti-TS (left) and anti-ASP-2 (right) IgG titers in the sera of BALB/c (B) and C57BL/6 (C) mice
immunized twice with recombinant adenoviruses rAdTS (TS), rAdASP2 (ASP-2), or rAdlacZ (C, control) as described in Materials and Methods. Results are presented as mean log antibody titers SD of six animals per group and are representative of
two equivalent experiments. (D) Complement-mediated lytic activity against T. cruzi parasites of sera from mice immunized with
rAdlacZ (C, control), rAdTS (TS), rAdASP2 (ASP-2), or rAdTS plus rAdASP2 (TS ASP-2) compared with sera from nonimmunized mice (NMS) or from mice infected with T. cruzi parasites (IMS).
6
mice immunized with the corresponding recombinant viruses.
On the other hand, in control mice immunized with rAdlacZ
no peptide-specific IFN--secreting splenic cells were detected
ex vivo by ELISPOT assay (data not shown).
In addition to the ex vivo ELISPOT assay, we determined
the presence of antigen-specific cytotoxic cells, using an in vivo
assay. For this purpose, splenocytes from normal mice were labeled with various concentrations of the vital dye CFSE. The
population of cells containing the highest amount of fluorescent label (CFSEhigh) was then incubated with the TS-derived
H-2d synthetic peptide IYNVGQVSI or with the ASP-2-derived
H-2b peptide VNHRFTLV to be used as target cells during the
assay. Equivalent amounts of CFSElow and CFSEhigh cells, that
is, control or target cells preincubated with each relevant peptide, were mixed before being inoculated in groups of BALB/c
mice immunized with rAdTS or rAdlacZ (control) or in groups
of C57BL/6 mice immunized with rAdASP2 or rAdlacZ (control). The numbers of CFSElow and CFSEhigh fluorescent cells
that remained in the spleens of the animals 20 hr after injection
MACHADO ET AL.
were determined by FACS analysis, and the percentage of specific cell lysis was determined for each group of animals.
Figure 3C shows a representative result of the in vivo analyses of cytotoxic activity against target cells presenting the TSderived peptide. As can be observed in Fig. 3C, the frequencies of CFSElow and CFSEhigh cells were equivalent in mice
immunized with a control virus (top left). Conversely, mice immunized with rAdTS (Fig. 3C, middle left) displayed a much
lower frequency of CFSEhigh cells when compared with frequency number of CFSElow cells, indicating that the cytotoxic
activity of the lymphocytes present in the rAdTS-immunized
mice was eliminating those cells presenting the TS-derived peptide. The lytic activity displayed in vivo by the lymphocytes of
the rAdTS-immunized mice (Fig. 3C, bottom left) represented
40%. The panels on the right in Fig. 3C show representative
results of the equivalent analysis performed against target cells
incubated with the ASP-2 peptide injected into mice immunized
with the control virus (top right) or with rAdASP2 (middle
right). In this case, C57BL/6 mice immunized with rAdASP2
FIG. 3. Trypanosoma cruzi-specific cellular immune responses in
mice immunized with recombinant adenoviruses expressing ASP-2 or
TS. (A) Interferon (IFN)- production by spleen cells of BALB/c and
C57BL/6 mice. Two weeks after the last immunization, animals were
sacrificed and their splenocytes were restimulated in vitro with recombinant TS (BALB/c) or recombinant ASP-2 (C57BL/6) protein. IFN-
production in supernatants was estimated by ELISA 3 days later. Results are expressed as an average of at least six individual mice SD
and are representative of two equivalent experiments. (B) Alternatively,
the numbers of IFN--secreting cells in spleens of immunized mice were
determined by ELISPOT, using synthetic peptides representing TS (H2Kd peptide IYNVGQVSI for BALB/c) or ASP-2 (H-2Kb peptide
VNHRFTLV for C57BL/6) epitopes recognized by CD8 T cells. Results represent an average of three mice per group SD
and are representative of two equivalent experiments. (C) In vivo cytotoxicity assay of BALB/c (left) or C57BL/6 (right) mice
immunized with recombinant adenoviruses rAdlacZ (assay control), rAdTS, or rAdASP2. Two weeks after the last immunization, normal splenocytes labeled with two concentrations of CFSE (CFSEhigh and CFSElow) were inoculated intravenously into
the tail vein of immunized mice. CFSEhigh cells were pulsed with peptides representing either the CD8 T cell epitope of TS
(BALB/c) or ASP-2 (C57BL/6). CFSElow cells were unpulsed and served as internal controls. Percentage of specific cell lysis
was measured 20 hr later by FACS and is expressed (bottom) as the mean SD of three animals per group.
VACCINATION WITH RECOMBINANT ADENOVIRUSES AGAINST T. cruzi
displayed stronger cytotoxic activity against cells presenting the
ASP2-derived peptide. The lytic activity displayed by the lymphocytes of these mice reached 90% (bottom right).
Protective immunity induced by vaccination with
adenoviral vectors
The most definitive assays with which to test the efficiency
of a vaccine are those that determine protective immunity after
challenge with infective parasites. To gain insights concerning
the real capacity of our recombinant vaccines to induce
protective immunity against experimental Chagas’ disease,
BALB/c mice were immunized with control or relevant viruses.
Vaccinated mice were challenged 9 weeks after administration
of the last immunizing dose with 100 T. cruzi Y strain bloodstream trypomastigotes. Figure 4A shows that mice immunized
with rAdlacZ displayed a high number of parasites in their
blood at the time of peak parasitemia, that is, 8 days after the
challenge, whereas mice that received rAdTS or rAdASP2 displayed significantly lower parasitemia (p 0.01). Moreover,
parasites were almost undetectable in mice immunized with
rAdTS plus rAdASP2 (p 0.001). The parasitemia of the various groups of mice were monitored until day 86, and no increase was observed during that period (data not shown).
Survival rates were also determined in the various groups of
immunized BALB/c mice after the challenge (Fig. 4B). In clear
correlation with the results of parasitemia shown above, animals injected with rAdlacZ (control virus) died quickly, before
day 20 after the challenge. In contrast, approximately 50% of
the animals immunized with rAdTS (p 0.01) and 80% of
those immunized with rAdASP2 (p 0.01) survived the challenge. No statistically significant differences in survival rates
were found between these two groups. However, inoculation
with a mixture of both recombinant viruses simultaneously induced protective immune responses that seemed complementary, providing resistance to 100% (p 0.001) of all immunized animals against a challenge with T. cruzi.
Hemoculture analyses were performed in surviving mice 6
months after the challenge. All mice (100%) immunized with
rAdASP2 were found to harbor live parasites. However, we
found that 60% of mice immunized with rAdTS and 40% of
mice immunized with rAdTS plus rAdASP2 had no parasites
in their blood. These results showed that the immunity elicited
by vaccination was sterilizing in a significant number of animals vaccinated.
Protective vaccination can also be induced in
C57BL/6 mice and is dependent on CD8 T cells
In earlier studies, genetic immunization with ts or asp-2
genes elicited protective immunity against T. cruzi infection
(Costa et al., 1998; Garg and Tarleton, 2002; Boscardin et al.,
2003; Fralish and Tarleton, 2003). Evaluation of the possible
mechanisms of protection failed to correlate protective immunity
with the levels of specific antibodies (Fujimura et al., 2001;
Boscardin et al., 2003; Vasconcelos et al., 2004). As opposed to
antibodies, the type 1 immune response mediated by IFN--producing CD4 and CD8 T lymphocytes has been linked to protection against T. cruzi (Boscardin et al., 2003; Vasconcelos et
al., 2004). To discriminate the importance of CD8 T cells after vaccination with rAdTS and rAdASP2, we immunized
7
C57BL/6 and C57BL/6-isogeneic CD8 KO mice with rAdTS
and rAdASP2 before challenge with blood forms of T. cruzi.
The numbers of parasites in the blood detected at the time
of peak parasitemia in C57BL/6 mice are shown in Fig. 5A.
Those numbers were lower in samples collected in C57BL/6
mice injected with either rAdTS or rAdASP2. Interestingly,
mice immunized with both viruses displayed significantly lower
parasitemia than did mice immunized with rAdTS alone (p 0.01), similar to what was observed in BALB/c mice. However,
the difference was not found to be statistically significant in
this mouse strain when we compared rAdASP2 with rAdTS
plus rAdASP2. Figure 5 also shows the levels of parasitemia
in CD8 KO mice immunized with either rAdTS or rAdASP2.
As can be observed, immunized CD8 KO animals displayed
significantly higher levels of parasites in their blood when com-
FIG. 4. Parasitemia and mortality in BALB/c mice after challenge with live trypomastigotes. BALB/c mice were immunized
with recombinant adenoviruses rAdASP2, rAdTS, or rAdlacZ
(C, control), or with a mixture of rAdASP2 and rAdTS, as described in Materials and Methods. Four weeks after the last immunization, mice were challenged intraperitoneally with 100
bloodstream trypomastigotes of T. cruzi Y strain. (A) Number of
trypomastigotes per milliliter of blood measured in individual
mice on the day of peak parasitemia (8 days after challenge). Results are expressed as means SD of numbers obtained for each
of eight animals per group and are representative of three equivalent independent experiments. (B) Kaplan–Meier curves representing percentages of surviving mice. Results are expressed as
percentages of surviving mice in groups of seven mice and are
representative of three equivalent independent experiments.
8
MACHADO ET AL.
FIG. 5. Parasitemia and mortality in C57BL/6 and
CD8 KO mice after challenge with live trypomastigotes. C57BL/6 and CD8 KO mice were immunized
with recombinant adenoviruses rAdASP2, rAdTS,
rAdlacZ (C), or a mixture of rAdASP2 plus rAdTS as
described in Materials and Methods. Six weeks after
the last immunization, mice were challenged intraperitoneally with 100 bloodstream trypomastigotes
of T. cruzi Y strain. (A) Number of trypomastigotes
per milliliter of blood, measured in individual mice on
the day of peak parasitemia (8 days after challenge).
Results are expressed as means SD of numbers obtained for each one of eight (C57BL/6) or four (CD8
KO) animals per group and are representative of two
equivalent independent experiments. (B) Kaplan–
Meier curves representing percentages of surviving
mice. Results are expressed as percentages of surviving mice in groups of eight (C57BL/6) or seven (CD8
KO) mice and are representative of two equivalent independent experiments.
pared with their immune-competent counterparts. These results
confirmed that CD8 immune T cells are involved in protective
immunity after immunization with recombinant virus rAdTS or
rAdASP2 and support our previous observations about an additive protective effect conferred by simultaneous immunization
with adenoviruses encoding two different antigens.
Analysis of survival rates of the different groups of mice revealed that approximately half of C57BL/6 mice immunized with
rAdlacZ (control virus) survive a challenge with live parasites
whereas, significantly (p 0.05), 90% of animals die in the absence of CD8 cells. Immunization with rAdTS and rAdASP2
conferred a high degree of protection, that is, 84 and 100%, respectively. Complete protection (100% survival) was also
achieved when both viruses were administered in the same inoculum. These results confirmed that the antigens used in our
study could also induce protection in animals of the C57BL/6
background. Moreover, protection seemed highly dependent on
the presence of CD8 cells in both cases, because CD8 KO animals immunized with either of these two viruses were significantly less protected that their immune-competent counterparts.
Long-term immunity is induced after immunization
with recombinant adenoviruses
In the development of a vaccine, it is of key importance to
determine its capacity to induce long-term protective immunity
against infection. Complementary to the data presented above
are the percentages of protection determined in mice of different genetic backgrounds challenged at different times after the
last immunizing dose with the recombinant adenoviruses. As
shown in Table 2, death after a challenge was prevented in
100% of the mice vaccinated with rAdASP2 plus rAdTS when
tested 6, 9, or even 14 weeks after administration of the last
immunizing dose of the recombinant adenoviruses. Lower, although maintained throughout the study, protective immunity
could also be observed in mice immunized with rAdTS or
rAdASP2.
DISCUSSION
It is now widely accepted that generation of vaccines against
parasites with intracellular life stages such as Leishmania
species, Toxoplasma gondii, Plasmodium species, or T. cruzi
will require the induction of potent type 1 T cell immune responses to elicit complete and sustained protection (Brener and
Gazzinelli, 1997; Denkers and Gazzinelli, 1998; Rodrigues et
al., 2003). Research on these new vaccines has been hampered
by the lack of efficient means to induce cell-mediated immunity. Our results provide evidence that vaccination of mice with
recombinant adenoviruses expressing two distinct T. cruzi antigens stimulates not only specific antibody-secreting B cells but
also type 1 IFN--producing and/or cytotoxic T cells. Also, vaccination with recombinant adenoviruses led to a significant de-
9
VACCINATION WITH RECOMBINANT ADENOVIRUSES AGAINST T. cruzi
TABLE 2. LONG-TERM IMMUNITY INDUCED
Time after boost
(wk)
Immunogen
rAdTS
rAdASP2
rAdASP2 rAdTS
aSurvival
06
09
14
06
09
14
06
09
14
BY IMMUNIZATION WITH
rAdASP2rAdTS
Percentage of survivala
075%
050%
100%
100%
080%
100%
100%
100%
100%
(12/16)
(5/10)
(8/8)
(16/16)
(4/5)
(8/8)
(16/16)
(8/8)
(8/8)
Mouse strain
C57BL/6
BALB/c
C57BL/6
C57BL/6
BALB/c
C57BL/6
C57BL/6
BALB/c
C57BL/6
of challenged animals was monitored for at least 150 days after challenge.
crease in peak parasitemia and increase in mouse survival after a lethal challenge with trypomastigotes. Protective immunity was observed in both mouse strains tested (BALB/c and
C57BL/6) and could be detected for as much as 14 weeks after the last immunizing dose. The most relevant observation of
our study was the fact that in mice immunized simultaneously
with both recombinant viruses, we observed complete and longlasting protection against experimental infection.
Our results confirm and extend a number of studies showing
that human type 5 recombinant adenoviruses are excellent antigen-encoding gene transfer vectors, capable of inducing both cellular and humoral protective immune responses (Bruna-Romero
et al., 2001; Shiver et al., 2002; Rocha et al., 2004; Tatsis and
Ertl, 2004). Also, they confirmed and extended previous studies
showing that vaccination with asp-2 and ts genes elicits protective immunity against T. cruzi infection in different mouse strains
against different parasite strains (Costa et al., 1998; Fujimura et
al., 2001; Garg and Tarleton, 2002; Boscardin et al., 2003; Fralish and Tarleton, 2003; Vasconcelos et al., 2004).
Miyahira and colleagues (2005) reported protection against T.
cruzi infection after vaccination with a recombinant adenovirus
encoding a previously described amino acid nonamer of transsialidase surface antigen (TSSA) that represents a CD8 epitope
in C57BL/6 mice, followed by a second immunization with a recombinant vaccinia virus encoding the same antigen. The levels
of protection induced by their viral constructs were clear but modest. In the present study we went several steps forward by using
two different long polypeptide antigens for immunization. As already observed in other parasitic infections such as malaria
(Wang et al., 2004), the combined use of antigens seems to be
an advantage when fighting parasitic diseases, especially when
those parasites have different stages in their life cycle.
Our previous (Vasconcelos et al., 2004) and present results
suggest that pronounced protective immunity against T. cruzi
can be achieved by using a combination of a trypomastigotederived antigen such as TS and an amastigote-derived antigen
such as ASP-2. It is noteworthy that similar levels of antibodies were elicited against each protein after individual or combined immunizations (see Fig. 2B and C). Consequently, it is
feasible to believe that the augmented levels of protection observed after simultaneous administration of both viruses (see,
e.g., Fig. 5A and B) result from the presence of the two antigens of interest and are not due to an increased adjuvant effect
of a double dose of adenoviral vector.
The development of human vaccines based on a single epitope, that is, those constructed by Miyahira and colleagues,
would plausibly be constrained by the marked degree of genetic
heterogeneity of human beings. In sharp contrast, the use of
antigenic proteins harboring a more complete array of B and T
cell epitopes, and belonging to two distinct parasitic life stages,
would allow a more efficient elimination of the parasite from
entire human populations as well as confer more effective crossprotection against diverse strains of T. cruzi.
Several authors have demonstrated the key importance of
CD8 T lymphocytes for protection against T. cruzi (Hoft et
al., 2000; Rodrigues et al., 2003; Martin and Tarleton, 2004).
Protection mediated by CD8 T cells takes place via two different antiparasitic mechanisms: (1) secretion of large amounts of
cytokines (e.g., IFN-) that act on the infected host cell by activating protective molecular mechanisms such as inducible nitric
oxide synthase (iNOS)/nitric oxide (Holscher et al., 1998; Hoft
and Eickhoff, 2005), and (2) activation of cytolytic activities that
can eliminate infected cells by releasing molecules (e.g., granzyme
B or perforin) with the ability to form pores in the cell or parasite membranes (Nickell and Sharma, 2000). We show in this paper that immunization with the recombinant viruses constructed
induces high levels of T. cruzi TS- and ASP-2-specific CD8
IFN--producing T lymphocytes. Furthermore, those cells displayed potent cytolytic activity, being able to lyse, in vivo, cells
coated with T. cruzi peptides. In addition, immunizations with TS
plus ASP-2 antigens elicited solid protection levels, both in
BALB/c and C57BL/6 mice. Thus, 100% of BALB/c mice immunized with a mixture of rAdASP2 and rAdTS survived a deadly
challenge with live T. cruzi parasites for more than 5 months of
study and 100% of C57BL/6 mice equivalently immunized survived a similar challenge for more than 3 months of study.
It is worth noting that these experiments provide the proofof-concept that vaccination against T. cruzi may be possible,
because most of the challenges were performed 2 and 3 months
after receiving the last immunogen, something never performed
previously and that confirms the presence of parasite-specific
long-lived immune memory cells in those animals. Using CD8
KO mice, we could confirm that protection was mediated at
least in part by CD8 T cells. Whereas 100% of common
C57BL/6 mice immunized with rAdASP2 survived the challenge, only 30% of the CD8 KO isogeneic mice did. CD8
T cells also partially mediated protection induced by immunization with TS. Both the results of parasitemia and survival
10
MACHADO ET AL.
postchallenge showed that CD8 T cells are profoundly involved but not acting alone, because knockout mice were just
slightly different from their immune-competent counterparts (in
parasitemia, p 0.05 for both rAdTS and rAdASP2), suggesting that other complementary immune mechanisms may be also
acting, although these are not mediated by lytic antibodies, because no T. cruzi-specific complement-mediated lytic activity
was detected in the sera of immunized animals.
Many authors have detected CD8 T cell responses during
natural T. cruzi infections. However, these CD8 T cell-mediated immune responses were unable to completely eliminate the
parasites. One of the possible explanations why the CD8 responses detected in vivo against T. cruzi do not protect against
natural infection is the dispersion of CD8 responses to multiple epitopes. As suggested by Martin and Tarleton (2004) for
the case of T. cruzi and by many other authors for other diseases, flooding the MHC presentation pathway with all the parasite antigens (more than 10,000 in the case of T. cruzi) may
imply that the density of any individual peptide–MHC complex
on the host cell surface will be below the threshold necessary
to efficiently activate CD8 T cells specific for that epitope. In
our case we may have been successful in vaccination because
we have used only a few CD8 epitopes, something that has
probably focused the immune response. Focusing the immune
response may be a better choice because the expansion of a few
clones of naive cells that can respond to high levels of the corresponding antigens can be achieved faster that the expansion
of many clones with lower levels of expression of their multiple corresponding antigens.
In conclusion, our present work shows that only two inoculations of recombinant adenoviruses encoding a few carefully
selected protective antigens are sufficient to induce high and
sustained levels of immunity against T. cruzi. Our results open
the possibility for testing our protocol in an animal model closer
to humans that is already available (i.e., rhesus monkeys; Carvalho et al., 2003) and, because previous works (Dumonteil et
al., 2004; Zapata-Estrella et al., 2006) have demonstrated the
feasibility of using DNA vaccines for therapeutic purposes, future studies on the beneficial effect of this kind of vaccines for
chronically infected animals are also being considered.
ACKNOWLEDGMENTS
This work was supported by grants from PRONEX,
FIOCRUZ/PDTIS-Vacinas, and the Millennium Institute for Vaccine Development and Technology (CNPq-420067/2005-1), and
by FAPEMIG and FAPESP. CNPq provided fellowships to
A.V.M., M.M.R., R.T.G., and O.B.R., and FAPESP to C.C.
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Address reprint requests to:
Dr. Oscar Bruna-Romero
Departamento de Microbiologia
Instituto de Ciências Biológicas
Universidade Federal de Minas Gerais
Belo Horizonte 31270-910, MG, Brazil
E-mail: [email protected]
Received for publication January 30, 2006; accepted after revision July 26, 2006.
Published online: August 24, 2006.