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

Abstract

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.

Keywords: antibodies; antigen specific B cells; attenuated parasites; humoral immunity; malaria; memory B cells; plasmodium; vaccines.

Conflict of interest statement

All authors in these manuscript have no conflict of interest.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Infection with P. yoelii blood stages abrogates antibody response to pre-erythrocytic antigen

C57BL/6 mice (n=6–9 mice) were infected with 50,000 Pyfabb/f− (open circles) or PyWT (closed circles) sporozoites. Serum was collected 6, 14, and 30 days after infection and was analyzed for the presence of (A) anti-PyCSP IgM (left panel), PyCSP IgG (right panel), (B) PyCSP IgG1(left panel), and PyCSP IgG2c (right panel) and (C) anti-PyMSP1 IgM (left panel) and IgG (right panel). (D) Serum collected from memory mice (n=3–4) on day 90 was also analyzed for the presence of IgM and IgG against PyCSP (left panel) and PyMSP1 (right panel). See also figure S1.

Figure 2. Kinetics of PyCSP-specific B cells

C57BL/6 mice were infected with Pyfabb/f− or PyWT. Spleens were isolated and PyCSP-specific B cells were enriched after 6,14, 30 or 90 days post-infection. (A) Representative flow cytometry plots of spleen B cells from Pyfabb/f− or PyWT infected mice after staining CSP tetramer enrichment with anti-PE magnetic beads and staining for flow cytometry as described in Figure S2. PyCSP specific B cells were identified as B220+CD138+ Decoy−CSP+. (B) Kinetics of CSP specific B cells isolated from spleens of Pyfabb/f− (open circles) or PyWT infected mice.(black circles). Data were compiled from two to three separate experiments with 3–6 mice per time point. Error bars represent standard deviation (SD). See also figure S2.

Figure 3. Blood stage infection alters differentiation of CSP-specific B cells

(A, left) Splenocytes were isolated from Pyfabb/f− or PyWT infected mice harvested on day 6, 14, 30 and 90 and analyzed by flow cytometry for CSP+CD38low CD138hi plasmablasts. (A, right) Absolute number of CSP-specific CD38low CD138hi cells. (B, left) Splenocytes from Pyfabb/f− or PyWT infected mice were analyzed for CSP+CD38low CD138− GL7hi germinal center B cells, on days 6, 14, 30 and 90 by flow cytometry. (B, right) Absolute numbers of CSP+CD38low CD138− GL7hi cells. Results are representative of 2–3 independent experiments with 3–6 mice in each. Error bars represent SD. See also figure S3.

Figure 4. Blood stage infection compromises the development of CSP-specific memory B cells

(A) Representative plots of CSP-specific memory B cells (PyCSP+CD138−GL7−CD38high) stained with CD73 and CD80 30 days post-infection. (B) Summary of data presented in A. (C) Representative plots of PyCSP+ memory B cells stained with IgD+ and IgM+ 30 days post infection (D) Percent of PyCSP-specific memory B cells that are IgM+ or IgM−IgD−. Results represent data from at least 2–4 independent experiments with 4–8 mice total. Error bars represent SD.

Figure 5. Blood stage infection impedes secondary response to liver stage antigens

Naïve or Memory mice (day 30) infected with Pyfabb/f− or PyWT sporozoites mice were challenged with 50,000 Pyfabb/f− sporozoites and PyCSP B cells were enriched and analyzed by flow cytometry 3 days later. (A) Representative plots identifying PyCSP+ B cells before and after challenge (B) A summary of total numbers of PyCSP+ B cells presented in (A). (C) Representative plots of plasmablasts (PyCSP+B220+CD38lowCD138+) before and after challenge in memory mice (D) Total number of plasmablasts presented in (C). Data are from two independent experiments with 5 mice per group. Error bars represent SD.

Figure 6. Drug treatment of WT restores CSP specific responses

(A) Parasitemia of mice infected with 50,000 PyWT-GFPluc sporozoites with or without treatment with atovoquone from days 3–13, as measured by flow cytometry. (B) Serum was collected from naive or 14 days after infection with or without treatment and anti-PyCSP total IgG was measured by ELISA. (C) Splenocytes from PyWT infected mice with or without atovoquone treatment were isolated, enriched and analyzed for CSP+CD138− CD38-GL7hi germinal center B cells on day 14 by flow cytometry. (D) Graph representing a summary of absolute numbers of CSP+CD138− CD38−GL7hi cells presented in C. Results are representative of 2 independent experiments with 4 mice per group. Error bars represent SD. See also figure S2.

Figure 7. Blood stages interfere with chemokine expression

(A) Immunofluorescence analysis of spleen sections from mice 6 and 14 days after infection with Pyfabb/f− or PyWT sporozoites. Left panel, B cell follicles were identified with anti-IgD (green), T cells with anti-CD4 (red) and germinal center B cells with anti-GL7 (blue). Yellow dashed lines denote PALS at day 6, white dashed lines denote GCs at day 14. Right panel, quantification of CD4 expression in PALS and GC, expressed as percent of CD4 in PALS or GC to total CD4 signal per field. Images are representative from at least 3 independent experiments. (B and C): Mice were infected with Pyfabb/f− or PyWT sporozoites and spleens isolated 3, 4 and 6 days later. mRNA levels were analyzed by qRT-PCR. All samples were standardized to the housekeeping gene hprt and fold changes were calculated relative to naïve controls (B) Gene expression levels of the chemokines ccl21, ccl19, cxcl13, cxcl9 and cxcl10. (C) Gene expression of regulatory cytokines; lymphotoxin α, lymphotoxin β and ifn-γ. Data represent 3–6 mice with 2–3 independent experiments. D) Splenocytes from C57BL/6 mice and mice that lack IFNγ signaling (IFNGr−/−) mice were infected with PyWT sporozoites, splenocytes were isolated, enriched and analyzed for CSP+CD38low CD138− GL7hi germinal center B cells on day 14 by flow cytometry. Results are representative of 2 independent experiments with 5 mice per group. Error bars represent SD.