As the initial aim of our study, we established an effective protocol of genetic vaccination with a heterologous prime-boost regimen using plasmid DNA and recombinant replication defective adenovirus both expressing the ASP-2 antigen of the human protozoan parasite Trypanosoma cruzi
. This protocol was an improvement over others described earlier when we used at least three doses of either plasmid DNA or recombinant protein to achieve significant protective immunity (6
). The heterologous prime-boost vaccination was more effective than two doses of plasmid DNA or a single dose of recombinant adenovirus.
There was no significant improvement noted in the comparison of the heterologous prime-boost regimen with two doses of recombinant adenovirus, since both were highly effective in our model. Nevertheless, this regimen may have a number of advantages for the long-term development of genetic recombinant vaccines providing a simple solution to the problem of widespread immunity to the human adenoviral vector type 5 (1
). Priming immunization with plasmid DNA seems to be sufficient for the subsequent expansion of the trans
-gene specific CD8+
T cells in recombinant adenovirus-boosted animals (9
). This expansion occurs even in animals with previous immunity to human adenovirus 5.
Hemocultures from these vaccinated mice were all negative denoting either very low or absent parasitemia. This result is an improvement compared to earlier studies in which we always observed parasites in hemocultures among a certain percentage of the vaccinated A/Sn mice (6
). Our results from this initial part of the study thus confirmed the findings and applied earlier studies of vaccination against experimental simian immunodeficiency virus and malaria infection to a human pathogen (2
Both CD4+ and CD8+ T cells accounted for nonredundant mechanisms of immunity since the depletion of each one prior to challenge renders the animals susceptible to infection. In vaccinated mice without CD8+ T cells, we observed a complete lack of control of parasitemia in both mouse strains (A/Sn and C57BL/6). Also, no delay in mouse mortality was seen in A/Sn mice, denoting a critical role for this subpopulation during immunity. In vaccinated C57BL/6 mice, we observed a slight delay in mouse mortality, suggesting the presence of effector non-CD8 immune cells. Whether they are in fact CD4+ remains to be investigated.
In CD4+ T-cell-depleted mice we still observed a significant delay in mouse mortality compared to nonimmune controls compatible with a CD8+ T-cel-mediated immunity. However, the control of parasitemia was significantly better in C57BL/6 mice. Finally, the fact that CD4+ and CD8+ T cells interact during the memory immune responses makes it more difficult to evaluate precisely the effector and/or helper function of CD4+ T cells during a complex process such as protective immunity in different experimental models. For that purpose more complex experimental systems will have to be developed.
In the second part of our study, we characterized the importance of perforin during protective immunity elicited by DNA prime adenovirus-boost vaccination. We observed that ASP-2-vaccinated perforin KO mice developed only limited immunity to the infection that did not prevent death.
Detailed analysis of the specific immune response revealed an impaired IFN-γ secretion by immune spleen cells. Although perforin deficiency did not impair the expansion of splenic specific CD8+ T cells, these cells had a significantly lower frequency of specific CD107a+/IFN-γ+ or IFN-γ+/TNF-α+ cells after in vitro restimulation. Also, the in vivo cytotoxicity was reproducibly reduced. Nevertheless, it is noteworthy that the in vivo cytotoxicity was present at certain levels in the perforin KO mice, indicating the presence of a perforin-independent mechanism(s) of lysis (TNF-α, FasL, etc.) yet to be identified. Finally, the pattern of expression of certain surface markers on CD8+ specific T cells from immune perforin KO mice were significantly different (CD44 and KLRG-1).
Earlier studies on the characteristics of specific CD8+
T cells in perforin-deficient mice indicated a dual function for this molecule during the homeostasis and effector phases of the immune response. In different reports (including ours), no significant modification was observed in the expansion of specific CD8+
T cells as estimated by the multimer staining (5
). In contrast, an increase in the number of peptide-specific cytokine-expressing CD8+
T cells was noted among these KO mice in certain studies (7
). Those authors provided evidence that perforin-mediated lyses of antigen-presenting cells could account for a restriction in the expansion of specific CD8+
T cells (64
). Equally important, it was also noted that during certain chronic viral infection, perforin-mediated downregulation of T-cell responses is critical to avoiding autoimmunity and immune-pathological damages (40
). To date, the most predictable immunological impairment of the perforin KO mice has been the reduced level of cytotoxicity (in vivo or in vitro) reported by distinct groups, including ours (8
Based on other experimental human parasitic infections, it was not possible to predict that perforin expression would be in fact critical to the protective immunity against T. cruzi
infection observed after DNA-prime adenovirus-boost vaccination. Compared to C57BL/6 WT mice, perforin KO mice are not more susceptible to infections with the intracellular protozoan parasites Toxoplasma gondii
or Plasmodium berghei
). The fact that perforin-deficient CD8+
T cells efficiently eliminate liver stages of malaria parasites is very important (46
). There is a single report stating that perforin deficiency abrogates protective immunity against Leishmania amazonensis
infection elicited by vaccination with a recombinant protein (18
). Therefore, we believe that the fact that perforin is critical to our system may help us to understand the role of this molecule in resistance to human parasitic infections.
The fact that perforin can be expressed in other cell types of the adaptive or innate immune system does not allow us to conclude that its expression on CD8+
T cells is the single restricting factor in our system. Other types of specific lymphocytes that may express perforin are CD4+
T cells (61
). We observed that in our vaccination strategy CD4+
T cells were important for T. cruzi
immunity after infection of A/Sn and C57BL/6. CD8 KO mice were more susceptible than perforin KO mice, indicating the presence of a non-perforin-mediated mechanisms mediated by these cells.
Other cell types may play important roles in mice immunity against T. cruzi
infection. Natural killer (NK) cells have been described as mediators of natural resistance to T. cruzi
experimental infection. The depletion of NK cells by treatment with polyclonal anti-asialo GM1 renders animals more susceptible to infection (24
). However, these cells are thought to act early, secreting IFN-γ (11
). A role for cytolysis mediated by perforin has been discarded during NK direct contact-mediated lysis of T. cruzi
or T. cruzi
-infected cells (35
). The role of NK cells in our system remains to be studied.
CD1d-restricted NKT cells also have been described as capable of improving the resistance to T. cruzi
). Immunization of CD1d KO mice that fail to express NKT cells allowed us to test whether NKT cells are involved in immunity in our vaccination regimen. We found that following heterologous prime-boost immunization, these mice developed protective immunity similar to C57BL/6 WT mice (B. C. G. de Alencar and M. M. Rodrigues, unpublished data). Based on this result, these cells are clearly not involved in the development of protective immunity after vaccination. Finally, γδ T cells are also not clearly associated with protective immunity during experimental T. cruzi
Based on these observations and the fact that CD8+ T cells are critical for mouse survival after experimental vaccination and infection, we consider it plausible that these cells represent a major, but not the single, source of perforin in our system. Perforin mediates this function, allowing the full maturation of effector CD8+ T cells. The correlation between protective immunity and the presence of specific CD8+ T cells expressing CD107a+/IFN-γ+ or IFN-γ+/TNF-α+ simultaneously is important to defining the type of CD8+ T cells that should be generated during vaccination protocols. Up to now, vaccine studies aimed at determining immunity have relied heavily on the detection of the number of CD8+ T-cell using multimer staining or IFN-γ production (ELISPOT or ICS). However, as we established, these may not be the best criteria for determining the presence of immune protective CD8+ T cells. Although we can observe reproducible differences in the number of peptide-specific IFN-γ-producing cells by ELISPOT assay, we considered that the presence of double positive for IFN-γ/TNF-α or IFN-γ/CD107a was more accurate to estimate the protective immunity in our model of vaccination.
The final goal of our study was to define whether the in vivo cytotoxic activity prior to challenge would ensure protective immunity in the absence of IFN-γ, an important mediator of adaptive immunity during experimental T. cruzi infection. We concluded from experiments using IFN-γ KO mice that this cytokine is critical even in the presence of high levels of in vivo cytotoxicity. IFN-γ KO C57BL/6 mice were more susceptible than CD4-depleted or CD8 KO animals, indicating that IFN-γ most likely come from both sources.
A possible role for antibodies during the protective immunity response we observed is highly unlikely. ASP-2 is expressed only by intracellular amastigotes or it is not accessible to antibodies in the other forms of the parasite (10
). Also, immune sera or MAbs incubated with parasites are not able to neutralize their infectivity in vivo (M. M. Rodrigues, unpublished results).
In summary, we provide evidences that CD4+ and CD8+ T-cell mediated immunity elicited by the DNA-prime recombinant adenovirus-boost vaccination requires both perforin and IFN-γ. The implications are that in the case of T. cruzi infection, in addition to the number of specific cells (multimer staining) and IFN-γ production (ELISPOT assay), other parameters, such as the presence of double-positive IFN-γ/TNF-α or IFN-γ/CD107a or perforin-dependent cytotoxicity, might be crucial to determining whether immune protective T cells are present during infection in preclinical or clinical vaccination trials.