Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites

Abstract

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.

Figure 1. P. yoelii fabb/f⁻ vaccination of outbred Swiss Webster mice generates a larger and less variable CD8 T cell response than P. yoelii RAS or P. yoelii sap1⁻ vaccination

(A) Representative plots showing the percent of circulating CD8 T cells that exhibit the antigen-experienced CD8α^loCD11a^hi phenotype before and after immunization with 2×10⁴ RAS, sap1⁻, or fabb/f⁻ sporozoites. Mice were given a homologous boost of 2×10⁴ sporozoites on day 91. (B) Cumulative data showing the percent of circulating CD8 T cells that are CD8α^loCD11a^hi. Data (mean±S.E.M.) are from 40 to 52 mice/group from two independent experiments. Data were analyzed by One-Way ANOVA († = P<0.01; ¶ = P<0.001). (C) The fraction of circulating CD8 T cells that exhibit the CD8α^loCD11a^hi phenotype from individual mice at the peak of the primary or secondary response. Symbols represent each individual mouse examined daily from day 5 to 9 following primary or booster immunization. The absolute peak CD8 T cell response for each individual mouse (which may have occurred on a different day due to genetic variability in outbred mice) was plotted. Numbers to the right indicate the fold difference between the highest and lowest responses within each group. See also Figure S1.

Figure 2. Late-liver-stage arresting P. yoelii GAP (fabb/f⁻) vaccinated C57BL/6 mice have a larger CD8 T cell response that exhibits a more effector memory-like phenotype compared to RAS or early-liver-stage arresting GAP (sap1⁻) vaccination

C57BL/6 mice were vaccinated with 2×10⁴ RAS, sap1⁻, or fabb/f⁻ sporozoites and given a homologous boost (2×10⁴ sporozoites) on day 111. (A) Fraction of mice that exhibited complete, sterilizing immunity following single (1° memory) or prime-boost (2° memory) vaccination. Mice were challenged with 1000 virulent Py sporozoites and protection was evaluated as described in Experimental Procedures. Numbers refer to no. mice protected/no. mice challenged in each group. Data are cumulative results from two challenges (RAS and fabb/f⁻) or a single challenge (sap1⁻). Results were analyzed by Fisher’s Exact Test. P<0.0001 for 2° memory RAS versus fabb/f⁻. P<0.0001 for 2° memory sap1⁻ versus fabb/f⁻ (n.d. = not determined) (B) Cumulative data showing the percent of circulating CD8 T cells that are CD8α^loCD11a^hi. Data (mean±S.E.M.) are from 10–40 mice per group from three independent experiments analyzed by One-Way ANOVA († = P<0.01; ¶ = P<0.001). (C) Frequency of CD8α^loCD11a^hi T cells expressing CD27, CD43^glyco, CD62L or CD127. Data (mean±S.E.M.) are from 3–9 pooled samples from two independent experiments analyzed by One-Way ANOVA followed by Tukey’s Multiple Comparison Test. (n.s. = not significant.) (D) Frequency of CD8α^loCD11a^hi secondary memory T cells positive for the indicated marker. Data (mean±S.E.M.) are from 3–6 pooled samples from two independent experiments analyzed by One-Way ANOVA followed by Tukey’s Multiple Comparison Test. See also Figure S2.

Figure 3. Vaccination with P. yoelii fabb/f⁻ sporozoites diversifies the CD8 T cell response compared to P. yoelii RAS vaccination

(A) Thy1.1+ CS280 TCR Tg CD8 T cells (1000) were transferred to naïve Thy1.2+ BALB/c mice one day before vaccination with 2×10⁴ RAS or fabb/f⁻ sporozoites. Representative plots show the fraction of CD8 T cells that are CD8α^loCD11a^hi and the fraction of those that are Thy1.1+ CS280 TCR Tg CD8 T cells on day six post-vaccination. Cumulative data showing the number of CD8α^loCD11a^hi T cells (B) or Thy1.1+ CS280 TCR Tg CD8 T cells (C) per spleen. Data in B and C (mean±S.D.) are from 3 mice per group analyzed by unpaired student’s t-test. Data are representative of two independent experiments. (D) RAS or fabb/f⁻ specific memory CD8 T cells (Thy1.2+), or naïve (Thy1.2+) CS280 TCR Tg CD8 T cells, were CFSE labeled and transferred into separate naïve Thy1.1+ BALB/c mice. Recipient mice were immunized with 2×10⁴ RAS or fabb/f⁻ sporozoites. Seven days later naïve or memory CD8 T cells were assayed for dilution of CFSE. (E) Representative plots show the gating strategy. Numbers in histograms are the percent of RAS, fabb/f⁻ or CS280 TCR Tg CD8 T cells that remained undivided (CFSE^hi) following vaccination with RAS or fabb/f⁻ sporozoites. (F) Cumulative results showing percent of RAS-specific (left bars) or fabb/f⁻ specific memory CD8 T cells (middle bars), or CS280 TCR Tg CD8 T cells (right bars) that remained undivided (CFSE^hi) following vaccination with RAS or fabb/f⁻ sporozoites. Data (mean±S.D.) are from 3 mice per group analyzed by unpaired student’s t-test. Data are representative of three independent experiments. See also Figure S3.

Figure 4. Vaccination of mice with late-liver-stage-arresting fabb/f⁻ sporozoites protects against challenge with blood stage parasites

BALB/c mice were immunized with 1×10⁵ fabb/f⁻ sporozoites on three occasions at two-week intervals. Naïve and immunized mice were challenged one month later with 100 Py XNL (non-lethal, A) or 100 Py YM (lethal, B) blood-stage parasites. Parasitemia was measured daily. Mice in B were euthanized on day eight when parasitemia reached >60%. Data (mean±S.D.) in A and B are from 5 mice per group. See also Figure S4.

Figure 5. Model for enhanced protective immunity and diversification of antigenic targets by parasite-specific CD8 T cells induced following late-liver-stage arresting GAP vaccination

(A) Schematic depiction of liver-stage developmental progression by early (RAS or sap1⁻) and late (fabb/f⁻) arresting attenuated parasites in rodent malaria models. Early arresting RAS and sap1⁻ parasites fail to undergo schizogony, exhibit smaller exoerythrocytic liver stage forms (small parasite biomass) and limited replication as indicated by few parasite nuclei relative to late-liver-stage arresting fabb/f⁻ parasites. Colors shown within early and late arresting liver-stage forms depict both overlapping and non-overlapping expression of parasite-derived antigenic targets. (B) Schematic depiction of changes in parasite gene expression as a function of liver-stage developmental progression. Yellow, blue and green primary colors indicate expression of developmental stage-specific, parasite-derived antigenic targets, whereas gradients of green and violet secondary colors represent putative antigens that are coordinately expressed during multiple developmental stages or the transition between stages. (C) Relative number and altered antigenic specificity of parasite-specific CD8 T cells induced following vaccination with early- (RAS or sap1⁻) versus late- (fabb/f⁻) arresting attenuated sporozoites. Colors in C correspond to the developmental stages in B and represent the relative breadth of antigens targeted by parasite-specific CD8 T cells following vaccination with early- versus late-liver-stage arresting sporozoites. Vaccination with late-arresting fabb/f⁻ sporozoites induces a larger population of CD8 T cells whose antigenic specificity is only partially overlapping with CD8 T cells that arise in response to vaccination with early-arresting sporozoites.