The application of anti-Toso antibody enhances CD8(+) T cell responses in experimental malaria vaccination and disease.

Toso is a molecule highly expressed on B cells. It influences their survival and was identified as an IgM binding molecule. B cells and natural antibodies play a role in vaccination-induced CD8(+) T cell responses. We investigated the impact of an anti-Toso antibody on vaccination efficiency in a malaria vaccination model. In this model, CD8(+) T cells exert antiparasitic functions on infected hepatocytes in the liver stage of the disease. In vaccinated anti-Toso treated mice, more antigen-specific CD8(+) T cells were induced than in control mice and after infection with Plasmodium berghei ANKA (PbA) sporozoites, the liver parasite burden was lower. In B cell deficient mice, the anti-Toso antibody did not stimulate the CD8(+) T cell response, indicating that B cells were mediating this effect. Furthermore, we analyzed the influence of anti-Toso treatment on non-vaccinated mice in the PbA infection model, in which CD8(+) T cells cause brain pathology. Anti-Toso treatment increased cerebral pathology and the accumulation of CD8(+) T cells in the brain. Thus, anti-Toso treatment enhanced the CD8(+) T cell response against PbA in a vaccination and in an infection model. Our findings indicate that Toso may be a novel target to boost vaccine-induced CD8(+) T cell responses.

Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory.

Regulatory T cells (T(reg)) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+)CD25(+) T(reg) were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+)CD25(-) T(reg). To obtain more insights in the specific function of T(reg) during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg) are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine.

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

Nematode-induced interference with the anti-Plasmodium CD8+ T-cell response can be overcome by optimizing antigen administration.

Malaria is still responsible for up to 1 million deaths per year worldwide, highlighting the need for protective malaria vaccines. Helminth infections that are prevalent in malaria endemic areas can modulate immune responses of the host. Here we show that Strongy-Ioides ratti, a gut-dwelling nematode that causes transient infections, did not change the efficacy of vaccination against Plasmodium berghei. An ongoing infection with Litomosoides sigmodontis, a tissue-dwelling filaria that induces chronic infections in BALB/c mice, significantly interfered with vaccination efficacy. The induction of P. berghei circumspor-ozoite protein (CSP)-specific CD8(+) T cells, achieved by a single immunization with a CSP fusion protein, was diminished in L. sigmodontis-infected mice. This modulation was reflected by reduced frequencies of CSP-specific CD8(+) T cells, reduced CSP-specific IFN-y and TNF-a production, reduced CSP-specific cytotoxicity, and reduced protection against P. berghei challenge infection. Implementation of a more potent vaccine regime, by first priming with CSP-expressing recombinant live Salmonella prior to CSP fusion protein immunization, restored induction of CSP-specific CD8(+) T cells and conferred almost sterile immunity to P. berghei challenge infection also in L. sigmodontis-infected mice. In summary, we show that appropriate vaccination regimes can overcome helminth-induced interference with vaccination efficacy.

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid.

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

CTLA-4 blockade differentially influences the outcome of non-lethal and lethal Plasmodium yoelii infections.

An immune response against malaria has to be tightly controlled. The production of pro-inflammatory cytokines is required to control parasites but the same cytokines are also involved in severe malaria. We have shown that CTLA-4 expression during Plasmodium berghei malaria dampens the immune response. This strain provokes a pro-inflammatory immune response that is associated with the pathology of cerebral malaria. Accordingly a blockade of CTLA-4 during the blood-stage of P. berghei malaria leads to an exacerbation of disease. To analyze the effects of a CTLA-4 blockade in a malaria model which is not prone to immune pathology we employed P. yoelii infection. Blood-stage infection led to a rapid induction of CTLA-4 on T cells. Using the non-lethal P. yoelii strain Py17NL we found that a blockade of CTLA-4 resulted in an increased T cell activation and IFN-gamma production, which was accompanied by a lower peak parasitemia and earlier parasite clearance. In contrast, blockade of CTLA-4 during infection with a P. yoelii strain exhibiting a higher parasitemia induced markedly increased serum-levels of TNF-alpha, which was associated with severe inflammation and reduced survival.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria.

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.