There are two main findings from this work: T cells are sufficient to provide vaccine protection against intestinal amebiasis, and IFN-γ is required for this protection.
The finding that CMI is protective in this model was somewhat surprising, since there was substantial anticipation that the presence of secretory IgA would be critical. In several human studies, fecal antilectin IgA to lectin or its subunits was associated with a decreased risk of amebic reinfection (1
). In vitro, antibodies to lectin diminish adherence to colonic mucins (9
), providing a putative mechanism for antibody-based protection. However, the vaccines tested here provided protection with no clear correlation between levels of antigen-specific fecal IgA or serum IgG and subsequent protection. Furthermore, passive transfer experiments using either immune serum or monoclonal antilectin IgA/IgG did not demonstrate protection.
The mouse model has increasingly shown that CMI responses can contribute to both protection and disease, in that CD4 cells can promote the severity of colitis in C3H mice (30
) and, conversely, can mediate clearance, in that anti-IL-4 treatment clears infection in CBA mice via an IFN-γ-dependent mechanism (21
). Human data also suggest that high parasite-specific IFN-γ production by PBMCs is associated with protection against invasive amebiasis (28
). In this context, it is not surprising that CMI is protective. Whether the mechanism of CMI protection is through outright prevention of the establishment of infection, or through rapid clearance of established infection, is unknown.
Though we have not established the cytokine requirements of the T cells for protection, all evidence points to IFN-γ. This is best argued from the IFN-γ depletion studies (Fig. ) and the correlation of protection with vaccine-induced IFN-γ+
CD4 T cells (Fig. ). Furthermore, several protective vaccines (EM014 plus LecA, CT-CFA plus lectin, and LecA alone), including the CT-CFA plus lectin vaccine that was documented to be CMI transferable, exhibited strong antigen-specific IFN-γ production. The Th1 polarity of synthetic lipid A-based adjuvant systems is well appreciated (34
). As for the downstream mechanism of IFN-γ-based protection, there are several possibilities. The simplest explanation is that IFN-γ activates macrophages and neutrophils for E. histolytica
killing, which has been shown in vitro (13
). Alternately, it is possible that IFN-γ exerts protective effects (e.g., inducing production of chemokines [15
] and mucosal defense molecules [16
]) on the epithelium, which bone marrow chimera experiments indicate is a critical cell type in the mouse strain-specific resistance to intestinal E. histolytica
It is surprising that IFN-γ was required for vaccine protection with alum-based LecA vaccines, since alum is a Th2 type adjuvant and only a marginal level of IFN-γ was detected in our alum-immunized PBMCs or splenocyte supernatants. However, there was a significant frequency of LecA-specific IFN-γ-positive CD4 cells in peripheral blood postvaccination, and this response correlated with vaccine-induced protection. Moreover there is precedent for IFN-γ in alum-based protection. For instance in an alum-based respiratory syncytial virus peptide vaccine, loss of protection was seen in mice treated with IFN-γ neutralizing antibody, and purified CD4 T cells from vaccinated IFN-γ knockout donors failed to transfer protection (41
). Other vaccine studies employing alum as an adjuvant against Toxoplasma gondii
) and Streptococcus pneumoniae
) infection also suggested a possible role of IFN-γ response in protection.
As for how the IFN-γ was elicited during alum vaccination, this response may be due to the LecA antigen itself. First, IFN-γ production seemed to be promoted by LecA, in that ConA-stimulated cells from our LecA-immunized mice produced more IFN-γ than cells from sham-immunized mice. Furthermore, the frequency of IFN-γ-secreting CD4 T cells in the LecA-only group was significantly higher than that observed for the control. There is a long precedent for the potential Th1 induction of Gal/GalNAc lectin, with several laboratories (8
) reporting that native lectin contains epitopes that stimulate IL-12 and TNF-α in macrophages and dendritic cells, particularly amino acids 596 to 1082, which are located within LecA. Such a combination of Th1 stimulus and alum has enhanced the efficacy of vaccines against human immunodeficiency virus (33
), Leishmania major
), malaria (49
), and Schistosoma mansoni
). Perhaps the LecA afforded a frequency of IFN-γ-secreting CD4 cells in the LecA-alum vaccine that was above a certain threshold for protection (~20%), even though the total quantity detected in the LecA-alum PBMC supernatants was marginal (and not predictive).
The IFN-γ-producing, or triple IFN-γ-, TNF-α-, and IL-2-producing, CD4 T-cell frequency served as the one meaningful marker for the protection observed. The so-called “multifunctional CD4” T cells that secrete IFN-γ, TNF-α, and IL-2 concordantly have been shown to correlate with protection from Leishmania major
) and Mycobacterium tuberculosis
) better than a single marker alone. We examined them in E. histolytica
because TNF-α and IFN-γ synergistically activate macrophages for trophozoite killing (36
) and IL-2 is associated with resistance to reinfection in amebic liver abscess models (7
). The additive value of TNF-α and IL-2 to IFN-γ is unclear, however, since the frequency of triple cytokine production was no more correlative than IFN-γ production alone.
In conclusion, we have demonstrated that protection by Gal/GalNAc lectin-derived vaccines was transferred to naive animals by the transfer of immune T cells and that IFN-γ was essential for protection. The frequency of IFN-γ-producing CD4 T cells in blood stands as a useful surrogate marker for alum-based LecA-mediated protection. The demonstration of efficacy by a vaccine utilizing alum and the delineation of the protective role of IFN-γ represent important steps toward a human amebiasis vaccine.