Structural requirements for Yersinia YopJ inhibition of MAP kinase pathways.

MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition

Mechanisms of Stage-Transcending Protection Following Immunization of Mice with Late Liver Stage-Arresting Genetically Attenuated Malaria Parasites

<div><p>Malaria, caused by <i>Plasmodium</i> parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Development of an effective vaccine has been encumbered by the complex life cycle of the parasite that has distinct pre-erythrocytic and erythrocytic stages of infection in the mammalian host. Historically, malaria vaccine development efforts have targeted each stage in isolation. An ideal vaccine, however, would target multiple life cycle stages with multiple arms of the immune system and be capable of eliminating initial infection in the liver, the subsequent blood stage infection, and would prevent further parasite transmission. We have previously shown that immunization of mice with <i>Plasmodium yoelii</i> genetically attenuated parasites (GAP) that arrest late in liver stage development elicits stage-transcending protection against both a sporozoite challenge and a direct blood stage challenge. Here, we show that this immunization strategy engenders both T- and B-cell responses that are essential for stage-transcending protection, but the relative importance of each is determined by the host genetic background. Furthermore, potent anti-blood stage antibodies elicited after GAP immunization rely heavily on F_C-mediated functions including complement fixation and F_C receptor binding. These protective antibodies recognize the merozoite surface but do not appear to recognize the immunodominant merozoite surface protein-1. The antigen(s) targeted by stage-transcending immunity are present in both the late liver stages and blood stage parasites. The data clearly show that GAP-engendered protective immune responses can target shared antigens of pre-erythrocytic and erythrocytic parasite life cycle stages. As such, this model constitutes a powerful tool to identify novel, protective and stage-transcending T and B cell targets for incorporation into a multi-stage subunit vaccine.</p></div

Quantification of staining patterns of sera from LAGAP-immunized mice.

<p>Quantification of staining patterns of sera from LAGAP-immunized mice.</p

Antibodies elicited by a late liver stage-arresting but not early liver stage-arresting GAP recognize antigens of both late liver stages and blood stages.

<p>C57BL/6 mice were immunized twice with 50,000 LAGAP or EAGAP sporozoites. Serum was collected prior to immunization (“pre-immune”) and two weeks after the last immunization for use in immunofluorescent assays (IFA) against parasite life cycle stages. IFAs were performed using fixed salivary gland sporozoites (A), liver sections of infected mice obtained at 12, 24, 33 and 48 hours post-infection (B) and blood stages (C). Parasites were also visualized using antibodies recognizing binding immunoglobulin protein (BiP) and MSP1 (for 48h liver stages and BSs). DNA was visualized with 4',6-diamidino-2-phenylindole (DAPI). These data indicate that antibodies in LAGAP-immunized C57BL/6 mice recognize all parasite life stages whereas EAGAP immune serum only recognizes sporozoites/early liver stages. Scale bar: 10 μm.</p

LAGAP immunization elicits T cells and antibodies that can protect against blood stage infection.

<p>A) Blood stage parasitemia of BALB/cJ or C57BL/6 mice (n = 5 mice/group) immunized with 3 x 50,000 <i>Pyfabb/f</i>^<i>-</i> (LAGAP) sporozoites and challenged 3 weeks later with 10⁴ infected red blood cells (iRBC) of a lethal <i>Py</i> strain. Both mouse strains show stage-transcending protection (STP). B) Mice (n = 5–9 mice/group over 2 independent experiments) were immunized as in (A) but depleted of CD4 and CD8 T cells using monoclonal antibodies (mAb) 24 hours prior to iRBC challenge. BALB/cJ mice lose STP in the absence of T cells but C57BL/6 mice do not. C) Parasitemia of AID^<i>-/-</i> mice on the C57BL/6 background, deficient in antibody secretion, immunized and challenged as in (A). AID^<i>-/-</i> mice do not show significant protection, indicating the importance of antibodies. D) Passive transfer of immune sera confers protection against a lethal blood stage challenge. BALB/cJ mice received iv injections of 300μL serum from 3 x 50,000 <i>Pyfabb/f</i>^<i>—</i> sporozoite-immunized C57BL/6 mice on days 0, 3 and 5 following a lethal challenge with 10⁴<i>Py</i> iRBCs. Comparisons in (A) and (B) were performed by Student t test where significance is indicated by: *0.05≥p>0.01</p

Antibodies elicited by LAGAP in C57BL/6 but not BALB/cJ mice are of broad isotypes and recognize the merozoite surface.

<p>Serum was collected from LAGAP immunized C57BL/6 (top 3 rows) and BALB/cJ (bottom 3 rows) mice as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004855#ppat.1004855.g004" target="_blank">Fig 4</a> and used in a blood stage IFA. Secondary antibodies against total IgG, IgG1 and IgG2<u>b</u> were used to visualize isotype-specific anti-blood stage antibodies. Parasites were also visualized with antibodies recognizing MSP1 and DNA visualized with 4',6-diamidino-2-phenylindole (DAPI). Antibodies in LAGAP-immunized C57BL/6 and BALB/cJ mice do not differ in isotype but rather in antigen specificity with only C57BL/6 serum recognizing the merozoite surface.</p

Blood Stage Malaria Disrupts Humoral Immunity to the Pre-erythrocytic Stage Circumsporozoite Protein

Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria

Immunization of C57BL/6 and BALB/cJ mice with LAGAP elicits antibodies against both sporozoites and BS parasites.

<p>Serum from BALB/cJ and C57BL/6 mice (n = 10 mice per group, over two independent immunizations) immunized with 3 x 50,000 <i>Pyfabb/f-</i> sporozoites was collected 2 weeks after the final immunization and used in ELISA to detect total IgG against CSP (A) and blood stage lysate (B). C57BL/6 and BALB/cJ mice produce antibodies against both CSP and BS proteins with higher anti-BS titers in C57BL/6 mice. Comparisons were performed using one-way ANOVA with Tukey post-hoc analysis with significance indicated by: *0.05≥p>0.01; **0.01≥p>0.001; ***0.001≥p>0.0001; ****p≤0.0001; non-significant (NS) p≥0.05.</p