Protective immune responses involved in host resistance to Brazilian isolates of Toxoplasma gondii: implications on vaccine development employing recombinant viral vectors
Ricardo T. Gazzinelli,*# Braulia Caetano,*# Blima
Fux,*# Marianne Garcia,*# Alexandre V. Machado,* Adriana M. Ferreira,£ Ricardo
W. A. Vitor, £ Maria Norma Melo,
£Oscar Bruna-Romero*#F
Departamento de Bioquímica e Imunologia,* Departamento de Microbiologia,F
Departamento de Parasitologia, £ Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais; and Laboratório de Imunopatologia, Centro
de Pesquisas René Rachou, Fundação Oswaldo Cruz#
Toxoplasma gondii is an intracellular
protozoan parasite that is widespread in nature, with a high prevalence in a
variety of warm-blooded animals, including humans. It is estimated that
toxoplasmosis exists in a chronic, asymptomatic form in between five hundred
million to one billion people (1). Toxoplasmosis establishes itself as a
lifelong, chronic infection, with transmission occurring in two ways: (i)
consumption or handling of uncooked meat bearing tissue cysts; and (ii)
ingestion of food or water contaminated with oocysts shed in cat feces (1).
Although infections are usually benign, high morbidity and even mortality can be
seen in immunocompromised patients, particularly in cancer and transplant
patients under immunosuppressive therapy or HIV infected individuals (2). In
addition, infection with T. gondii is also a major cause of economical loss in
veterinary medicine (3,4).
In humans, toxoplasmosis is a common cause of infectious retinitis in both
immunocompetent and immunosupressed persons, accounting for 30-50% of all cases
of posterior uveitis (5). Toxoplasmic retinochoroiditis is believed to result
mainly from periodic reactivation of latent cysts associated with congenital
infection. However, recent studies in France (6), Canada (7), Brazil (8-10) and
the United States (11) indicate that ocular disease after postnatal acquired
infection is also prevalent in healthy adults. Typical features of
retinochoroiditis include unilateral focal retinitis at the border of a
pre-existing pigmented retinochoroid lesion and an overlying vitritis. In some
cases, the lesions may be atypical, consisting of large areas of retinal
necrosis or retinochoroiditis without a preexisting scar (12).
Approximately, 50% of the adult population in Brazil is chronically infected
with the parasite, this prevalence is similar to other countries in Latin
America (8,10,13-15). However, in some areas of the country the prevalence is
significantly higher, reaching up 95% of the population, e.g. Erechim, Rio
Grande do Sul State (8,9). In regard to the ocular disease in the seropositive
individuals, a high frequency has been described in different areas of Brazil.
Studies performed in Erechim (8,9) show 17.7% of the infected population
developed ocular lesions, whereas in Melquiades, Minas Gerais State, where 49%
of prevalence was observed, 12.5% of the seropositive individuals presented
signs of ocular disease (10). We believe that the high frequency of ocular
toxoplasmosis in Brazilian populations may reflect the unique combination of
infective parasite strains, host genetic background, and/or epidemiological
aspects such as infection early in life as well as continuous exposure to
infective parasites.
The main objectives of the studies performed in our laboratories are: (i) to
define the genetic structure of T. gondii strains isolated in the Brazilian
territory; (ii) to define the immunological components involved in resistance of
the intermediary host to infection with Brazilian T. gondii isolates; (iii) to
elaborate subunit vaccines that protect the intermediary host against infection
with Brazilian T. gondii strains. Ultimately, we expect that this information
may contribute to the rational design of an effective vaccine to be used in
prophylaxis of T. gondii in humans and other intermediary hosts.
I - Genetic analysis of Brazilian T.
gondii isolates by multilocus PCR-RFLP
T. gondii strains can be divided in three main
lineages based on various genetic markers (16). Studies in mice have shown that
infection with each of the three lineages of T. gondii results in different
outcomes: Type I strains are highly virulent, whereas types II and III strains
are relatively avirulent (16). Type I differ genetically by 1% or less from type
II and type III (17). However, the main determinants that dramatically affect
the virulence of different T. gondii strains in the host and pathogenesis of
toxoplasmosis are poorly understood. Type II strains of T. gondii appear to be
dominant in the US territory and are frequently isolated from AIDS patients with
toxoplasmic encephalitis (17). Interestingly, different studies suggest the
involvement of Type I and Type I/III strains in the development of ocular
disease. Consistently, Type I strain was identified as responsible for a
toxoplasmosis epidemic outbreak associated with a high rate of development of
acquired ocular disease (18). In addition, a recent study indicates a high
frequency of Type I as well as Type I/III recombinant isolates from ocular
lesions in patients from US (19).
Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP)
at eight independent loci was used to determine the type (I, II or III) lineage
of 20 Toxoplasma gondii strains isolated from humans and animals in Brazil. RH
(type I, highly virulent), ME49 (type II, avirulent) and VEG (type III,
avirulent) were used as reference strains. Differently from expected
frequencies, all Brazilian strains showed to have recombinant genotypes, with
typical alleles of types I, II or III at almost all loci assessed. The cB21-4
locus, a microsatellite marker, showed a higher allelic polymorphism with seven
alleles among strains under analysis. Data have also shown that many Brazilian
T. gondii strains presented a new haplotype at the L363 locus. When results of
the eight loci were combined, 14 schizodemes were characterized out of the 20 T.
gondii strains isolated in Brazil. The phenogram representing PCR-RFLP data
separated Brazilian strains into two distinct genetic groups associated with
murine virulence phenotype, termed group I-A and group I-B. Strains from group
I-A (AS28, BV and N) that were highly virulent in BALB/c mice, were clustered
with RH reference strain. Only those strains presented the haplotype I at the
L363 locus, suggesting that this could be a possible marker of highly virulent
strains. Strains from group I-B (cystogenic strains) showed a more heterogeneous
behavior regarding virulence: a few of them (EGS, RAR, SAF, D5 and D6) were
virulent, others (C4, P and D8) avirulent and most of them (D1, D2, D3, D4, D7,
EFP, CH1, CH2 and CH3) intermediate virulent in mice. A significant linkage
disequilibrium was observed in the population surveyed. However, the role of
sexual recombination in the population structure of T. gondii in Brazil seems to
be more central than in Europe and North America, where most studies have been
performed.
The fact that all Brazilian T. gondii strains are more closely related to the
type I lineage is noteworthy. From the studied strains, 85% showed a certain
degree of virulence (highly virulent, virulent and intermediate virulence) and
only 15% of the strains were avirulent (20). These findings contrasts with
studies performed in the US and Europe, where most strains are avirulent of type
II or type III (17). Although speculative at this point, we would like to
suggest that the high frequency of virulent strains closely related to type I
lineage may be in part responsible for the high frequency of acquire ocular
toxoplasmosis commonly found in Brazil (8-10).
It seems that sexual reproduction may plays a central role in the population
structure of T. gondii in Brazil, although we have detected a significant
correlation between PCR-RFLP, RAPD-PCR and SSR-PCR genetic distances, suggesting
linkage disequilibrium in this population. It is also possible that the
epidemiology of toxoplasmosis may be different in Brazil, with ingestion of
oocysts being the main mechanism of T. gondii transmission in our country, which
could explain the high percentage (100%) of recombinant strains observed in our
study. Circumstantial evidence of the potential importance of oocyst
transmission in Brazil was the finding that the consumption of untreated or
unfiltered water was implicated as a source of T. gondii infection for human
populations in north Rio de Janeiro State, a disease endemic area (15).
Moreover, an outbreak of clinical toxoplasmosis in humans was epidemiologically
linked to drinking water from a municipal water reservoir in Santa Isabel do
Ivai, Paraná State (21). Whether asexual or sexual stage transmission is
responsible for the majority of Toxoplasma infections, it should be possible to
identify an oocyst-specific immunogenic peptide or whole protein that would
identify the stage of the parasite responsible for causing infection. The
identification of factors that contribute to the high percentage of genetic
recombination in Brazilian T. gondii population would be of clinical and
epidemiological importance and should be explored in the future.
II – Role of cytokines and MHC
haplotype in mouse resistance to infection with natural recombinant (Type I/III)
strains of T. gondii.
Different studies performed in murine experimental
show the important role of cytokines such as IL-12, TNF-a and IFN-y and
generation of reactive nitrogen intermediates (RNI) as mediators of host
resistance to early T. gondii infection (22). Thus, animals deficient in IL-12,
IFN-g, inducible nitric oxide synthase (iNOS), treated with neutralizing
antibodies anti-cytokines or specific inhibitors of iNOS are more susceptible to
infection with T. gondii (23-27). Acquired immunity to T. gondii is associated
with a Th1-type response (24). During chronic infection, neutralization of
either IFN-g or TNF-a results in the reactivation of disease and the development
of toxoplasmic encephalitis (TE) (28,29). Further, other host genetic factors,
including MHC alleles are important determinants of host resistance and
susceptibility to early infection, as well as controlling cyst numbers and
encephalitis at later stages of infection with T. gondii in mice (30,31).
Consistently, both CD4+ T as well as CD8+ T lymphocytes are important components
in host resistance to this parasite (28,30).
Considering the immunological studies described above, we decided to
characterize the importance of such immunological mechanisms in host resistance
to three natural recombinant T. gondii strains (Type I/ III) isolated in Brazil,
named P-Br (32), D8 and C4, which are cystogenic in mice. The IL-12/IFN-y axis
and iNOS were defined as main determinants of resistance during the acute
infection with the Brazilian strains. Different from the Type II strain of T.
gondii (ME-49), per-oral infection with the Type I/III strains led only to a
light inflammatory infiltrate and no major lesions in the intestine of the
C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6
(susceptible to ME-49) mice were shown, respectively, to be more susceptible and
resistant to cyst formation and toxoplasmic encephalitis, when infected with
Type I/III strains. Consistently, a congenic BALB/c strain containing MHC
haplotype “b” was highly susceptible to ME-49 (Type II strain), but not to
the recombinant Type I/III strains. Together, our results indicate that MHC
haplotype “b” is a major determinant of susceptibility to cyst formation and
toxoplasmic encephalitis induced during infection with Type II, but not with
Type I/III strains of T. gondii.
III – Protection against challenge
with a natural recombinant (Type I/III) strain of T. gondii in mice vaccinated
with recombinant viruses coding the main tachyzoite surface antigens.
Vaccine development is based on the observation that
exposure to T. gondii can elicit a life-long immune response, capable of
protecting the host against a secondary challenge with the parasite (22). In
early stages of infection, this response is characterized by activation of
innate mechanisms mediated by macrophages, that internalize free tachyzoites and
produce IL-12, and by NK cells, which secrete IFN-g (22). This last cytokine
controls the replication of the parasite and, in conjunction with IL-12, drives
the differentiation of CD4+ T lymphocytes specific for parasite antigens to a
Th1 cytokine profile (24). In addition, CD8+ T cells are crucial to control
parasite replication in the chronic phase of the disease, preventing
reactivation of infection. Both CD4+ and CD8+ T cells secrete IFN-g, and CD8+ T
cells develop cytotoxic activity against infected cells (30,31).
Initial attempts to induce protection against toxoplasmosis involved the use of
live attenuated tachyzoites from mutant strains of T. gondii, like the heat
sensitive ts-4 (33). Despite the high efficiency in terms of activation of CD4+
and CD8+ T cells and the efficacy of protection, vaccines based on live
tachyzoites are not applicable to humans, due to the risk of pathogenic side
effects. Thus, most recent protocols focus on development of recombinant
vaccines. As target antigens, there is a great interest in tachyzoite surface
proteins, particularly SAG1 (34-38). In addition, a few studies have also been
performed with SAG2 and SAG3 (39). The SAG proteins, the most abundant on the
tachyzoite surface, are anchored to the parasite membrane by
glicosylphosphatidylinositol (GPI) structures and are believed to be involved in
the process of host cell invasion. Further, their sequences are highly conserved
among different strains of T. gondii, sharing high degree of homology between
type I (pathogenic and lethal to mice) and type II/III (cystogenic) strains
(32,40).
Genes coding for T. gondii surface antigens SAG1 and SAG2 have been cloned and
expressed in recombinant procariotic and eucaryotic systems, and both
recombinant protein and plasmid have been used to immunize mice and other
species. It was observed that recombinant proteins generally induce high titers
of IgG1 antibodies, which have poor effect against challenge with pathogenic
strains of T. gondii (38), and that improvement of the response depends on
combination with proper adjuvants. On the other hand, vaccines based on plasmids
coding for SAG1 and SAG2 were able in many cases, depending on administration
route and formulation, to induce cellular immune response with Th1 profile,
inducing better protection (34).
In this context, viral vectors such as adenoviruses can improve the immune
responses in comparison to naked plasmid vaccines, since they are more efficient
in transferring the genetic sequences and inducing protein expression (41).
Adenoviruses infect a great variety of cells, including key immune cells, like
dendritic cells, leading to an efficient activation of CD4+ and CD8+ T cells
(42) and are capable of driving the immune response to a desired Th1 type (43).
We generated recombinant adenoviruses encoding three genetically modified
surface antigens (SAG) of the parasite Toxoplasma gondii, i.e. AdSAG1, AdSAG2
and AdSAG3 (44). Modifications included the removal of their GPI-anchoring
motifs and, in some cases, the exchange of the native signal peptide by
influenza virus haemaglutinin signal sequence (HASS). Adenovirus immunization of
BALB/c mice elicited potent antibody responses against each protein, displaying
a characteristic bias to a Th1 profile when the IgG2a/IgG1 antibody ratio was
determined. Furthermore, the presence of parasite-specific IFN-g producing CD8+
T cells was analyzed in the same animals by ELISPOT. Splenocytes from immunized
mice secreted IFN-g after recognition of antigen-presenting cells infected with
each recombinant adenovirus. However, only AdSAG2 was able to induce in vivo
sufficient numbers of antigen-specific lymphocytes to be readily distinguished
from the control animals vaccinated with an irrelevant virus. We finally tested
the capacity of the immune responses detected to protect mice against a
challenge with live T. gondii parasites. Although no major protection was
observed against tachyzoites of the highly virulent RH strain, a significant
reduction in brain cyst loads was observed in animals challenged with cysts of
the P-Br strain. Thus, up to 80% of the parasitic forms were eliminated from
animals vaccinated with a mix of the three recombinant viruses. Since
adenoviruses seemed capable of inducing broad Th1-biased protective immune
responses against T. gondii antigens, other parasite antigens should be tested
alone or in combination with the ones described here to further develop a
protective vaccine against toxoplasmosis.
Considering that the regular route of infection with T. gondii is through the
mucosa of the digestive tract, we decided to direct our studies towards a
vaccine that induces a strong mucosal immunity. For that we are considering the
use a heterologous prime-boost protocol with SAG2 encoding adenovirus and
influenza virus. It is noteworthy that both viruses infect cells from the
digestive tract and can be use to elicit mucosal immunity. Thus, we have also
constructed influenza vector (45) expressing the T. gondii surface antigens, and
the experiments with heterologous prime-boost with recombinant virus are being
performed.
IV – Conclusions
We conclude that the great majority of the T. gondii
Brazilian isolates are natural recombinants generated from lineages Type I and
III. Importantly, the majority of these isolates are virulent in mice. Whereas
the IL-12/IFN-g and iNOS were critical for host resistance to these isolates, we
found that the mouse MHC haplotype “b” is not a determinant of
susceptibility to infection, as previously determined by parasites of Type II
lineage. Finally, adenovirus coding modified genes of SAG1, SAG2 and SAG3 led to
induction of protective immunity, as indicated by 80% reduction of cyst numbers
in mice challenged with a natural recombinant Type I/III Brazilian isolate.
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