Assessment of the Plasmodium falciparum Preerythrocytic Antigen UIS3 as a Potential Candidate for a Malaria Vaccine

Abstract

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei-P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced.

Trial registration: ClinicalTrials.gov NCT01465048.

Keywords: Plasmodium; UIS3; liver stage; malaria; sterile protection; synergy; vaccines.

Copyright © 2017 Longley et al.

Figures

FIG 1

PfUIS3 protects C57BL/6 mice against chimeric P. berghei-P. falciparum challenge. C57BL/6 mice (n = 8) were vaccinated i.m. with 1 × 108 inclusion-forming units (IFU) of ChAd63-PfUIS3 followed 8 weeks later by 6 × 106 PFU of MVA-PfUIS3. (A) Cellular immunogenicity was assessed in the blood at 7 days postboost by ICS after stimulation for 6 h with an overlapping peptide pool. Both medians and individual data points are shown. (B) At 10 days postboost, mice were challenged i.v. with 1,000 chimeric P. berghei-P. falciparum sporozoites expressing PfUIS3, as were eight naive control mice. The log-rank (Mantel-Cox) test was used to assess the difference between the survival curves (P < 0.0001).

FIG 2

PfUIS3 protects BALB/c mice against WT P. berghei challenge. BALB/c mice (n = 8) were vaccinated i.m. with 1 × 108 IFU of ChAd63-PfUIS3 followed 8 weeks later by 1 × 107 PFU of MVA-PfUIS3. (A) Cellular immunogenicity was assessed in the blood at 6 days postboost by ICS after stimulation for 6 h with an overlapping peptide pool. Both medians and individual data points are shown. (B) At 8 days postboost, mice were challenged i.v. with 1,000 WT P. berghei sporozoites, as were eight naive control mice. The log-rank (Mantel-Cox) test was used to assess the difference between the survival curves (P = 0.0048).

FIG 3

PfUIS3 requires CD8+ T cells to provide protection against chimeric P. berghei-P. falciparum challenge in BALB/c mice. BALB/c mice (n = 3 groups of 8) were vaccinated i.m. with 1 × 108 IFU of ChAd63-PfUIS3 followed 8 weeks later by 5 × 106 PFU of MVA-PfUIS3. Mice were then injected intraperitoneally (i.p.) with 100 μg of MAb to either CD4+ (GK1.5) or CD8+ (8.43) or with an IgG MAb control at days 8, 9, and 10 postboost. (A) At day 10, all mice were challenged i.v. with 1,000 chimeric P. berghei-P. falciparum sporozoites expressing PfUIS3, as were seven naive control mice. The log-rank (Mantel-Cox) test was used to assess differences between the survival curves: for CD8+ cell-depleted versus naive mice, P = 0.13; for CD8+ cell-depleted versus control IgG-treated mice, P < 0.0001; for CD4+ cell-depleted versus naive mice, P < 0.0001; and for CD4+ cell-depleted versus IgG control-treated mice, P = 0.0044. (B) Depletion of CD8+ or CD4+ T cells was confirmed in the blood at day 4 with respect to challenge. Cells were stained with anti-CD4–FITC clone RM4-5, 1/200 anti-CD8–PerCP-Cy5.5 clone 53-6.7, and 1/50 anti-CD3ε–APC, and the proportion of CD3+ cells that were CD4+ or CD8+ is shown.

FIG 4

PfUIS3 vaccination induces antigen-specific effector memory cells in the livers of both BALB/c and C57BL/c mice. BALB/c or C57BL/6 mice (n = 4 per strain) were vaccinated i.m. with 1 × 108 IFU of ChAd63-PfUIS3 followed 8 weeks later by 1 × 106 PFU of MVA-PfUIS3. Cellular immunogenicity was assessed in both the spleen and liver at 2 weeks postboost. (A) Percentages of CD8+ cells secreting IFN-γ in each organ. Closed circles, spleen; open squares, liver. (B) Percentages of antigen-specific cells in each organ that were of the effector memory subtype. Both medians and individual data points are shown. Differences between the spleen and liver for each mouse strain were assessed using two-way ANOVA with Sidak's multiple-comparison test. For panel A, the differences were not significant; for panel B, P < 0.0001 for BALB/c mice and P = 0.0027 for C57BL/6 mice.

FIG 5

Coadministration of ChAd63-MVA PfUIS3 with the vaccine ChAd63-MVA TRIP is not detrimental to immunogenicity and increases efficacy in BALB/c mice. BALB/c mice (n = 3 to 6 per group) were vaccinated i.m. with 1 × 108 IFU of ChAd63 followed 8 weeks later by 1 × 107 PFU of MVA, with the antigens indicated on the x axis. When two vaccines were given, mice were vaccinated with a full dose of each vaccine administered in separate legs (separate), or the vaccines were mixed prior to administration in both legs (mixed). Cellular immunogenicity in the blood and IgG responses in plasma were assessed at 1 week postboost. Cells or plasma was incubated with a peptide pool or protein lysate for either PfTRAP (A) or PfUIS3 (B). Both medians and individual data points are shown. The Kruskal-Wallis test with Dunn's multiple-comparison test was used to assess statistical differences between coadministered vaccines and those given alone; no significant differences were found. (C) The same mice were then challenged at 10 days postboost with 1,000 double chimeric P. berghei-P. falciparum sporozoites that expressed both PfTRAP and PfUIS3. The log-rank (Mantel-Cox) test was used to determine the statistical difference between survival curves: for the PfTRIP versus naive groups, the difference was not significant (P = 0.5); for the PfUIS3 versus naive groups, P = 0.0005; for the PfUIS3 versus separate groups, the difference was not significant (P = 0.26); and for the PfUIS3 versus mixed groups, P = 0.0005.

FIG 6

PfUIS3-specific memory responses were induced in human volunteers who had recently undergone CHMI with cryopreserved P. falciparum sporozoites. Frozen PBMCs from 18 volunteers 35 days after CHMI were stimulated for 10 days with a pool of overlapping peptides to one of five preerythrocytic antigens (PfTRAP, PfUIS3, PFI0580c, PFE1590w, and PfLSA1) and recombinant human IL-2. For some volunteers, there were not enough PBMCs available to perform stimulations with all five antigens. The stimulated cells were then used in a standard IFN-γ ELISpot assay, and samples were tested in quadruplicate. Responses to antigens are expressed as numbers of spot-forming cells (SFC) per million original PBMCs. The positivity cutoff (167 SFC) was set using PBMCs from four volunteers from the day before CHMI, as described in Materials and Methods. Medians and individual data points are shown.