Studying the effect of chloroquine on sporozoite-induced protection and immune responses in Plasmodium berghei malaria

BACKGROUND Sporozoite immunization of animals and humans under a chemo-prophylactic cover of chloroquine (CPS-CQ) efficiently induces sterile protection against malaria. In humans, CPS-CQ is strikingly more efficient than immunization with radiation attenuated sporozoites (RAS), raising the hypothesis that this might be partially due to CQ. Chloroquine, an established anti-malarial drug, is also well known for its immune modulating properties including improvement of cross-presentation. The aim of this study was to investigate whether co-administration of CQ during sporozoite immunization improves cellular responses and protective efficacy in Plasmodium berghei models. METHODS A number of experiments in selected complimentary P. berghei murine models in Balb/cByJ and C57BL/6j mice was performed. First, the effect of CQ administration on the induction of protection and immune responses by RAS immunization was studied. Next, the effect of CQ on the induction of circumsporozoite (CS) protein-specific CD8(+) T cells by immunization with P. berghei parasites expressing a mutant CS protein was investigated. Finally, a direct comparison of CPS-CQ to CPS with mefloquine (MQ), an anti-malarial with little known immune modulating effects, was performed. RESULTS When CQ was co-administered during immunization with graded numbers of RAS, this did not lead to an increase in frequencies of total memory CD8(+) T cells or CS protein-specific CD8(+) T cells. Also parasite-specific cytokine production and protection remained unaltered. Replacement of CQ by MQ for CPS immunization resulted in significantly reduced percentages of IFNγ producing memory T cells in the liver (p = 0.01), but similar protection. CONCLUSIONS This study does not provide evidence for a direct beneficial effect of CQ on the induction of sporozoite-induced immune responses and protection in P. berghei malaria models. Alternatively, the higher efficiency of CPS compared to RAS might be explained by an indirect effect of CQ through limiting blood-stage exposure after immunization or to increased antigen exposure and, therefore, improved breadth of the immune response.EMB was supported by Top Institute Pharma (grant T4-102) and KN was supported by the NWO Mozaiek (grant no. 017.005.011)

Evaluation of immunity against malaria using luciferase-expressing Plasmodium berghei parasites

<p>Abstract</p> <p>Background</p> <p>Measurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic <it>Plasmodium berghei </it>parasite, <it>Pb</it>GFP-Luc_con, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques <it>in vivo </it>and <it>in vitro</it>.</p> <p>Methods</p> <p>Mice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with <it>Pb</it>GFP-Luc_con.The <it>in vitro </it>transgenic sporozoites neutralization assay (TSNA) was adapted by replacing <it>Pb</it>CS(Pf) parasites for <it>Pb</it>GFP-Luc_conparasites.</p> <p>Results</p> <p>Application of <it>Pb</it>GFP-Luc_contransgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses.</p> <p>Conclusions</p> <p><it>Pb</it>GFP-Luc_conparasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.</p

Bacterium-like particles as multi-epitope delivery platform for Plasmodium berghei circumsporozoite protein induce complete protection against malaria in mice

Contains fulltext : 110364.pdf (publisher's version ) (Open Access)BACKGROUND: Virus-like particles have been regularly used as an antigen delivery system for a number of Plasmodium peptides or proteins. The present study reports the immunogenicity and protective efficacy of bacterium-like particles (BLPs) generated from Lactococcus lactis and loaded with Plasmodium berghei circumsporozoite protein (PbCSP) peptides. METHODS: A panel of BLP-PbCSP formulations differing in composition and quantity of B-cell, CD4+ and CD8+ T-cell epitopes of PbCSP were tested in BALB/c mice. RESULTS: BLP-PbCSP1 induced specific humoral responses but no IFN-gamma ELISPOT response, protecting 30-40% of the immunized mice. BLP-PbCSP2, with reduced length of the non-immunogenic part of the T-cell-epitopes construct, increased induction of IFN-gamma responses as well as protection up to 60-70%. Compared to controls, lower parasitaemia was observed in unprotected mice immunized with BLP-PbCSP1 or 2, suggestive for partial immunity. Finally, further increase of the number of B-cell epitopes and codon optimization (BLP-PbCSP4) induced the highest anti-CSP antibody levels and number of IFN-gamma spots, resulting in sterile immunity in 100% of the immunized mice. CONCLUSION: Presentation of Plasmodium-derived antigens using BLPs as a delivery system induced complete protection in a murine malaria model. Eventually, BLPs have the potential to be used as a novel versatile delivery platform in malaria vaccine development

Long Term Protection after Immunization with P. berghei Sporozoites Correlates with Sustained IFNγ Responses of Hepatic CD8+ Memory T Cells

Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (TEM) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ TEM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r2 = 0.60, p<0.0001). The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells

Ex Vivo Blockade of the PD-1 Pathway Improves Recall IFNγ Responses of HIV-Infected Persons on Antiretroviral Therapy

Despite antiretroviral therapy (ART), immune exhaustion persists in HIV infection and limits T cell responses to HIV or other pathogens. Moreover, HIV infection results in the loss of pre-existing immunity. Here, we investigated the effect of blocking the PD-1 pathway on recall IFNγ responses to tetanus toxoid (TT) and measles virus (MV) antigens in HIV-infected persons on ART with prior TT and MV immunity. The ex vivo treatment of lymphocytes with anti-PD-1 and anti-PD-L1 antibodies significantly increased TT- and MV-specific IFNγ responses. The responses to TT and MV antigens alone or in combination with antibodies blocking the PD-1 pathway positively correlated with CD4 T cell levels. Furthermore, T cell PD-1 expression levels inversely correlated with recall IFNγ responses in combination with antibodies blocking the PD-1 pathway but not with IFNγ responses to antigens only. Our study suggested that targeting the PD-1 pathway may boost vaccine-induced pre-existing immunity in HIV-infected persons on ART depending on the degree of immune exhaustion

CD4+ and CD8+ T_EM cells in response to challenge.

<p>Changes in the effector memory CD8+ and CD4+ T-cell compartment from the liver, spleen and PBMC of mice immunized with low dose of RAS (n = 15) or CPS (n = 12) were evaluated at various time-points around challenge (C-1, C+6, C+21). Results are from 3 independent experiments (n_naïve = 18) and cells from individual mice assayed. Error bars represent standard error of the mean (SEM). * = p<0.05, *** = p<0.0001.</p

Phenotypic analyses CD44hi T-cells.

<p>(A) Gating strategy. After lymphocytes gating based on forward-scatter (FSC) and side-scatter (SSC) properties, CD4+ and CD8+ T cells were selected. Total memory T cells were gated based on high CD44 expression. T cells with effector memory (T_EM) and central memory (T_CM) phenotype were identified based on CD62L expression. (B) CD8+CD44hi and CD4+CD44hi T-cell pool at day 40 post-immunization with high or lose dose. Composition of the CD8+CD44hi and CD4+CD44hi T-cell pool was assessed in the liver (left panel), spleen (central panel) or PBMC (right panel) of mice immunized by high and low dose of RAS or CPS. Results are from 2 independent experiments (n_RAS = 10; n_CPS = 10; n_naïve = 13) and cells from individual mice assayed. Error bars represent standard error of the mean (SEM). * = p<0.05, ** = p<0.005, *** = p<0.0001.</p

Long-term RAS and CPS^a protection following <i>P. berghei</i> sporozoite challenge^b.

a<p>CPS mice received 24-days chloroquine treatment. Three of the six naïve mice challenged at t = 3months receive the same chloroquine treatment.</p>b<p>Mice were challenged by i.v. injection of 10.000 WT sporozoites. Protection was defined as negative blood-smears at day 21 after challenge.</p

Sporozoite and blood-stage specific IgG.

<p>Plasma were collected from mice immunized by RAS or CPS before (C-1) and 6 to 21 days after (C+6; C+21) challenge. Levels of anti-sporozoite or anti-blood-stage IgG antibodies were determined by ELISA (n_RAS = 5; n_CPS = 5; n_naïve = 9). Error bars represent standard error of the mean (SEM). ** = p<0.005, *** = p<0.0001.</p