Plasmodium infection of erythrocytes induces clinical malaria. Parasite-specific CD4+ T cells correlate with reduced parasite burdens and severity of human malaria, and are required to control blood-stage infection in mice. However, the characteristics of CD4+ T cells that determine protection or parasite persistence remain unknown. Here we show that P. falciparum infection of humans increased expression of an inhibitory receptor (PD-1) associated with T cell dysfunction. In vivo blockade of PD-L1 and LAG-3 restored CD4+ T cell function, amplified T follicular helper cell and germinal center B cell and plasmablast numbers, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, which can be rescued to enhance parasite control using inhibitory therapies.

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Summary While subunit vaccines have shown partial efficacy in clinical trials, radiation-attenuated-sporozoites (RAS) remain the “gold-standard” for sterilizing protection against Plasmodium-infection in human vaccinees. The variability in immunogenicity and replication introduced by the extensive, random DNA damage necessary to generate RAS could be overcome by genetically attenuated parasites (GAP) designed via gene deletion to arrest at defined points during liver-stage development. Here, we demonstrate the principle that late-liver-stage arresting GAP induce larger and broader CD8 T cell responses that provide superior protection in inbred and outbred mice compared to RAS or early-arresting GAP immunizations. Late-liver-stage arresting GAP also engender high-levels of cross-stage and cross-species protection and complete protection when administered by translationally-relevant intradermal or subcutaneous routes. Collectively, our results underscore the potential utility of late-liver stage arresting GAP as broadly protective next-generation live-attenuated malaria vaccines and support their potential as a powerful model for identifying antigens to generate cross-stage protection.

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum (Pf) exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear if the “atypical” memory B-cells (MBCs) and “exhausted” CD4 T-cells described in humans exposed to endemic malaria are driven by Pf per se or by other factors commonly associated with malaria, such as co-infections and malnutrition. To address this critical question we took advantage of a ‘natural’ experiment near Kilifi, Kenya, and compared profiles of B and T-cells of children living in a rural community where Pf-transmission is on-going, to the profiles of age-matched children living under similar conditions in a nearby community where Pf transmission ceased 5 years prior to this study. We found that continuous exposure to Pf drives the expansion of “atypical” MBCs. Persistent Pf-exposure was associated with an increased frequency of CD4 T-cells expressing phenotypic markers of exhaustion, both PD-1 alone and PD-1 in combination with LAG-3. This expansion of PD-1 expressing and PD-1/LAG-3 co-expressing CD4 T-cells was largely confined to CD45RA+ CD4 T-cells. The percentage of CD45RA+CD27+ CD4 T-cells co-expressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Together, these results suggest that Pf infection per se drives the expansion of atypical MBCs and phenotypically “exhausted” CD4 T-cells, which has been reported in other endemic areas.