Vaccines against exoerythrocytic stages of malaria remain ineffective in the face of limited knowledge on processing and presentation of parasite antigens in the liver. Here, we show that vaccine candidate circumsporozoite protein (CSP) is deposited and retained in human hepatocytes traversed by P. falciparum sporozoites for an extended time. Therefore, two reservoirs of hepatocytes contain malarial antigens: invaded and traversed cells. We demonstrate that CSP in traversed cells is long-lived, resides within several dynamic cellular compartments, and its turnover is regulated by distinct proteolytic systems, including the proteasome, lysosome and autophagy. We provide the first demonstration of endogenous processing of a malaria liver stage antigen by non-infected, non-professional antigen-presenting cells (APCs) through multiple proteolytic systems. We propose that these processes are likely to generate a variety of antigenic products from CSP, as well as other sporozoite antigens shed into traversed cells, able to affect the effector phase of anti-malarial immunity.Development of Plasmodium parasites in the liver can be controlled by cytotoxic T lymphocytes (CTLs) recognizing malarial antigenic peptides presented by the major histocompatibility complex (MHC) I on the surface of hepatocytes. It is unknown if breaching of the plasma membrane during sporozoite traversal affects the ability of traversed hepatocytes to stimulate CTL responses, and if traversal affects the immunogenicity of nearby ;;non-traversed” hepatocytes in the infected liver. We show that traversed cells remain viable and competent to present endogenously processed antigen to CD8+ T cells.Stimulated CTLs undergo the complete program of antigen-specific activation, including cytokine secretion, cytotoxic granule release, and proliferation. Soluble factors released by either traversed hepatocytes or by T cells activated on traversed hepatocytes do not impose traversal-specific in-trans effects on CTL activation. Finally, traversed hepatocytes endogenously presented a C-terminal epitope of CSP to specific CTLs, stimulating IFNγ production. Our data propose a novel view of traversed cells as a potential site of parasite-specific immune activation.Since protective immune responses achieved in experimental vaccination differ dramatically from the partial protection naturally acquired in malaria-endemic regions, we next investigated the repertoire of HLA-A*02-restricted CSP-specific CD8+ memory T cells in PBMCs from adults living in malaria-endemic Mali. While plasma of 28/45 of Malian adults included in the study had detectable CSP antibodies, PBMCs from only 10/45 had functional ex vivo recall responses to CSP. Compared to malaria-naïve US volunteers, PBMC samples from Malian adults exhibited altered composition of the T cell compartment and increased expression of T cell exhaustion marker, PD-1. Our preliminary data demonstrate that T cell activation induced via TCR triggering could be facilitated by PD-1-specific, neutralizing monoclonal antibodies. However, it still needs to be discovered if malaria-specific memory T cell responses can be enhanced upon PD-1 blockade in PBMCs from donors living in malaria endemic areas.Together, we propose a role for sporozoite traversal in CD8+ T cell responses against liver stage infection, and that novel vaccination strategies are needed to overcome immune dysregulation in T cell compartment found in populations from malaria-endemic regions.
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Plasmodium falciparum circumsporozoite protein: target of cellular immunity against traversed hepatocytes and naturally induced memory responses
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